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Bacteria Engineered to Treat Diseases that Benefit from Reduced Gut
Inflammation
and/or Tightened Gut Mucosal Barrier
RELATED APPLICATIONS
[01] This application is a continuation-in-part of PCT Application No.
PCT/US2016/020530, filed March 2, 2016; PCT Application No. PCT/US2016/050836,
filed September 8, 2016, and U.S. Application No. 15/260,319, filed September
8, 2016;
and claims the benefit of U.S. Provisional Application No. 62/291,461 filed
February 4,
2016; U.S. Provisional Application No. 62/291,468 filed February 4, 2016; U.S.
Provisional Application No. 62/291,470 filed February 4, 2016; U.S.
Provisional
Application No. 62/347,508, filed June 8, 2016; U.S. Provisional Application
No.
62/354,682, filed June 24, 2016; U.S. Provisional Application No. 62/362,954,
filed July
15, 2016; U.S. Provisional Application No. 62/385,235, filed September 8,
2016; U.S.
Provisional Application No. 62/423,170, filed November 16, 2016; U.S.
Provisional
Application No. 62/439,871, filed December 28, 2016; PCT Application No.
PCT/U52016/032565, filed May 13, 2016; U.S. Provisional Application No.
62/347,576,
filed June 8, 2016; U.S. Provisional Application No. 62/348,620, filed June
10, 2016; PCT
Application No. PCT/U52016/039444, filed June 24, 2016; and PCT Application
No.
PCT/U52016/069052, filed December 28, 2016. The entire contents of each of the
foregoing applications are expressly incorporated herein by reference in their
entireties to
provide continuity of disclosure.
BACKGROUND OF THE INVENTION
[02] This disclosure relates to compositions and therapeutic methods for
inhibiting inflammatory mechanisms in the gut, restoring and tightening gut
mucosal
barrier function, and/or treating and preventing autoimmune disorders. In
certain aspects,
the disclosure relates to genetically engineered bacteria that are capable of
reducing
inflammation in the gut and/or enhancing gut barrier function. In some
embodiments, the
genetically engineered bacteria are capable of reducing gut inflammation
and/or enhancing
gut barrier function, thereby ameliorating or preventing an autoimmune
disorder. In some
aspects, the compositions and methods disclosed herein may be used for
treating or
preventing autoimmune disorders as well as diseases and conditions associated
with gut
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inflammation and/or compromised gut barrier function, e.g., diarrheal
diseases,
inflammatory bowel diseases, and related diseases.
[03] Inflammatory bowel diseases (IBDs) are a group of diseases characterized
by significant local inflammation in the gastrointestinal tract typically
driven by T cells
and activated macrophages and by compromised function of the epithelial
barrier that
separates the luminal contents of the gut from the host circulatory system
(Ghishan et al.,
2014). IBD pathogenesis is linked to both genetic and environmental factors
and may be
caused by altered interactions between gut microbes and the intestinal immune
system.
Current approaches to treat IBD are focused on therapeutics that modulate the
immune
system and suppress inflammation. These therapies include steroids, such as
prednisone,
and tumor necrosis factor (TNF) inhibitors, such as Humira (Cohen et al.,
2014).
Drawbacks from this approach are associated with systemic immunosuppression,
which
includes greater susceptibility to infectious disease and cancer.
[04] Other approaches have focused on treating compromised barrier function
by supplying the short-chain fatty acid butyrate via enemas. Recently, several
groups have
demonstrated the importance of short-chain fatty acid production by commensal
bacteria
in regulating the immune system in the gut (Smith et al., 2013), showing that
butyrate
plays a direct role in inducing the differentiation of regulatory T cells and
suppressing
immune responses associated with inflammation in IBD (Atarashi et al., 2011;
Furusawa
et al., 2013). Butyrate is normally produced by microbial fermentation of
dietary fiber and
plays a central role in maintaining colonic epithelial cell homeostasis and
barrier function
(Hamer et al., 2008). Studies with butyrate enemas have shown some benefit to
patients,
but this treatment is not practical for long term therapy. More recently,
patients with IBD
have been treated with fecal transfer from healthy patients with some success
(Ianiro et al.,
2014). This success illustrates the central role that gut microbes play in
disease pathology
and suggests that certain microbial functions are associated with ameliorating
the IBD
disease process. However, this approach raises safety concerns over the
transmission of
infectious disease from the donor to the recipient. Moreover, the nature of
this treatment
has a negative stigma and thus is unlikely to be widely accepted.
[05] Compromised gut barrier function also plays a central role in autoimmune
diseases pathogenesis (Lerner et al., 2015a; Lerner et al., 2015b; Fasano et
al., 2005;
Fasano, 2012). A single layer of epithelial cells separates the gut lumen from
the immune
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cells in the body. The epithelium is regulated by intercellular tight
junctions and controls
the equilibrium between tolerance and immunity to nonself-antigens (Fasano et
al., 2005).
Disrupting the epithelial layer can lead to pathological exposure of the
highly
immunoreactive subepithelium to the vast number of foreign antigens in the
lumen (Lerner
et al., 2015a) resulting in increased susceptibility to and both intestinal
and extraintestinal
autoimmune disorders can occur" (Fasano et al., 2005). Some foreign antigens
are
postulated to resemble self-antigens and can induce epitope-specific cross-
reactivity that
accelerates the progression of a pre-existing autoimmune disease or initiates
an
autoimmune disease (Fasano, 2012). Rheumatoid arthritis and celiac disease,
for example,
are autoimmune disorders that are thought to involve increased intestinal
permeability
(Lerner et al., 2015b). In individuals who are genetically susceptible to
autoimmune
disorders, dysregulation of intercellular tight junctions can lead to disease
onset (Fasano,
2012). In fact, the loss of protective function of mucosal barriers that
interact with the
environment is necessary for autoimmunity to develop (Lerner et al., 2015a).
[06] Changes in gut microbes can alter the host immune response (Paun et al.,
2015; Sanz et al., 2014; Sanz et al., 2015; Wen et al., 2008). For example, in
children with
high genetic risk for type 1 diabetes, there are significant differences in
the gut
microbiome between children who develop autoimmunity for the disease and those
who
remain healthy (Richardson et al., 2015). Others have shown that gut bacteria
are a
potential therapeutic target in the prevention of asthma and exhibit strong
immunomodulatory capacity... in lung inflammation (Arrieta et al., 2015).
Thus,
enhancing barrier function and reducing inflammation in the gastrointestinal
tract are
potential therapeutic mechanisms for the treatment or prevention of autoimmune
disorders.
[07] Recently there has been an effort to engineer microbes that produce anti-
inflammatory molecules, such as IL-10, and administer them orally to a patient
in order to
deliver the therapeutic directly to the site of inflammation in the gut. The
advantage of
this approach is that it avoids systemic administration of immunosuppressive
drugs and
delivers the therapeutic directly to the gastrointestinal tract. However,
while these
engineered microbes have shown efficacy in some pre-clinical models, efficacy
in patients
has not been observed. One reason for the lack of success in treating patients
is that the
viability and stability of the microbes are compromised due to the
constitutive production
of large amounts of non-native proteins, e.g., human interleukin. Thus, there
remains a
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great need for additional therapies to reduce gut inflammation, enhance gut
barrier
function, and/or treat autoimmune disorders, and that avoid undesirable side
effects.
Summary
[08] The genetically engineered bacteria disclosed herein are capable of
producing therapeutic anti-inflammation and/or gut barrier enhancer molecules.
In some
embodiments, the genetically engineered bacteria are functionally silent until
they reach
an inducing environment, e.g., a mammalian gut, wherein expression of the
therapeutic
molecule is induced. In certain embodiments, the genetically engineered
bacteria are
naturally non-pathogenic and may be introduced into the gut in order to reduce
gut
inflammation and/or enhance gut barrier function and may thereby further
ameliorate or
prevent an autoimmune disorder. In certain embodiments, the anti-inflammation
and/or
gut barrier enhancer molecule is stably produced by the genetically engineered
bacteria,
and/or the genetically engineered bacteria are stably maintained in vivo
and/or in vitro.
The invention also provides pharmaceutical compositions comprising the
genetically
engineered bacteria, and methods of treating diseases that benefit from
reduced gut
inflammation and/or tightened gut mucosal barrier function, e.g., an
inflammatory bowel
disease or an autoimmune disorder.
[09] In some embodiments, the genetically engineered bacteria of the invention
produce one or more therapeutic molecule(s) under the control of one or more
promoters
induced by an environmental condition, e.g., an environmental condition found
in the
mammalian gut, such as an inflammatory condition or a low oxygen condition. In
on-
limiting exemplary embodiments, the genetically engineered bacteria produce
one or more
therapeutic molecule(s) under the control of an oxygen level-dependent
promoter, a
reactive oxygen species (ROS)-dependent promoter, or a reactive nitrogen
species (RNS)-
dependent promoter, and a corresponding transcription factor. In some
embodiments, the
therapeutic molecule is butyrate; in an inducing environment, the butyrate
biosynthetic
gene cassette is activated, and butyrate is produced. Local production of
butyrate induces
the differentiation of regulatory T cells in the gut and/or promotes the
barrier function of
colonic epithelial cells. In some embodiments, the genetically engineered
bacteria
produce their therapeutic effect only in inducing environments such as the
gut, thereby
lowering the safety issues associated with systemic exposure.
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[010] Disclosed herein is a butyrate-producing bacterium comprising at least
one
gene or gene cassette encoding one or more non-native biosynthetic pathways
for
producing butyrate, wherein the bacteria produces acetyl CoA and wherein the
bacterium
has at least one mutation in or deletion of an endogenous pta gene. Such
bacterium is
capable of producing butyrate, but does not produce acetate. In some
embodiments, the
bacterium further has at least one mutation in or deletion of an endogenous
adhE gene. In
some embodiments, the bacterium further has at least one mutation in or
deletion of an
endogenous ldhA gene. In some embodiments, the bacterium further has at least
one
mutation in or deletion of an endogenous frd gene. In some embodiments, the
bacterium
further has at least one mutation in or deletion of an endogenous adhE gene
and an
endogenous ldhA gene. In some embodiments, the bacterium further has at least
one
mutation in or deletion of an endogenous adhE gene and an endogenous frd gene.
In
some embodiments, the bacterium further has at least one mutation in or
deletion of an
endogenous ldhA gene and an endogenous frd gene. In some embodiments, the
bacterium further has at least one mutation in or deletion of an endogenous
adhE gene, an
endogenous frd gene, and an endogenous ldhA gene. In certain specific
embodiments, the
butyrate-producing bacterium comprises at least one gene or gene cassette
encoding one or
more non-native biosynthetic pathways for producing butyrate, wherein the
bacteria
produces acetyl CoA and wherein the bacterium has at least one mutation in or
deletion of
an endogenous pta gene and at least one mutation in or deletion of an
endogenous gene
selected from adhE gene and/or ldhA gene and/or frd gene.
[011] In any of the above described embodiments of butyrate-producing
bacteria,
the at least one gene or gene cassette for producing butyrate is operably
linked to a
directly or indirectly inducible promoter that is not associated with the gene
or gene
cassette in nature. In any of the above described embodiments of butyrate-
producing
bacteria, the at least one gene or gene cassette for producing butyrate is
operably linked to
a directly or indirectly inducible promoter that is not associated with the
gene or gene
cassette in nature and is induced by exogenous environmental conditions found
in a
mammalian gut.
[012] In some embodiments, the butyrate-producing bacterium may produce an
increased level of butyrate as compared to a bacterium which produces butyrate
naturally
or which comprises a gene or gene cassette for producing butyrate, but does
not comprise
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at least one mutation in or deletion of an endogenous ldhA gene. In some
embodiments,
the butyrate-producing bacterium may produce an increased level of butyrate as
compared
to a bacterium which produces butyrate naturally or which comprises a gene or
gene
cassette for producing butyrate, but does not comprise at least one mutation
in or deletion
of an endogenous adhE gene. In some embodiments, the butyrate-producing
bacterium
may produce an increased level of butyrate as compared to a bacterium which
produces
butyrate naturally or which comprises a gene or gene cassette for producing
butyrate, but
does not comprise at least one mutation in or deletion of an endogenous frd
gene. In some
embodiments, the butyrate-producing bacterium may produce an increased level
of
butyrate as compared to a bacterium which produces butyrate naturally or which
comprises a gene or gene cassette for producing butyrate, but does not
comprise at least
one mutation in or deletion of an endogenous pta gene. In some embodiments,
the
butyrate-producing bacterium may produce an increased level of butyrate as
compared to a
bacterium which produces butyrate naturally or which comprises a gene or gene
cassette
for producing butyrate, but does not comprise at least one mutation in or
deletion of an
endogenous gene selected from frd and/or ldhA and/or adhE and/or pta. In some
embodiments, the butyrate-producing bacterium may produce an increased level
of
butyrate as compared to a bacterium which produces butyrate naturally or which
comprises a gene or gene cassette for producing butyrate, but does not
comprise at least
one mutation in or deletion of an endogenous ldhA gene, frd gene, adhE gene,
and pta
gene.
[013] In some embodiments, the bacterium described above comprises an
endogenous pta gene and produces acetate. In these embodiments, the bacterium
comprises at least one gene or gene cassette encoding one or more non-native
biosynthetic
pathways for producing butyrate, wherein the bacteria produces acetyl CoA and
wherein
the bacterium has an endogenous pta gene. Such bacterium is capable of
producing
butyrate and acetate. In some embodiments of this bacterium, the bacterium
further has at
least one mutation in or deletion of an endogenous adhE gene. In some
embodiments, the
bacterium further has at least one mutation in or deletion of an endogenous
ldhA gene. In
some embodiments, the bacterium further has at least one mutation in or
deletion of an
endogenous frd gene. In some embodiments, the bacterium further has at least
one
mutation in or deletion of an endogenous adhE gene and an endogenous ldhA
gene. In
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some embodiments, the bacterium further has at least one mutation in or
deletion of an
endogenous adhE gene and an endogenous frd gene. In some embodiments, the
bacterium further has at least one mutation in or deletion of an endogenous
ldhA gene and
an endogenous frd gene. In some embodiments, the bacterium further has at
least one
mutation in or deletion of an endogenous adhE gene, an endogenous frd gene,
and an
endogenous ldhA gene. In certain specific embodiments, the butyrate-producing
bacterium comprises at least one gene or gene cassette encoding one or more
non-native
biosynthetic pathways for producing butyrate, wherein the bacteria produces
acetyl CoA
and wherein the bacterium has an endogenous pta gene and at least one mutation
in or
deletion of an endogenous gene selected from adhE gene and/or ldhA gene and/or
frd
gene.
[014] In any of the above-described embodiments of butyrate-producing
bacterium, the at least one gene or gene cassette for producing butyrate may
comprise ter,
thiAl, hbd, crt2, pbt, and buk genes. In any of the above-described
embodiments of
butyrate-producing bacterium, the at least one gene or gene cassette for
producing butyrate
may comprise ter, thiAl, hbd, crt2, and tesB genes.
[015] In any of the above described embodiments of butyrate- and acetate-
producing bacteria, the at least one gene or gene cassette for producing
butyrate is
operably linked to a directly or indirectly inducible promoter that is not
associated with the
gene or gene cassette in nature. In any of the above described embodiments of
butyrate-
and acetate-producing bacteria, the at least one gene or gene cassette for
producing
butyrate is operably linked to a directly or indirectly inducible promoter
that is not
associated with the gene or gene cassette in nature and is induced by
exogenous
environmental conditions found in a mammalian gut.
[016] In another aspect, disclosed herein is an acetate-producing bacterium
that
produces acetate but not butyrate. In any of these embodiments, the acetate-
producing
bacterium produces acetyl CoA and comprises a wild-type pta gene. In some
embodiments, the acetate-producing bacterium comprises at least one mutation
in or
deletion of a ldhA gene. In some embodiments, the acetate-producing bacterium
comprises at least one mutation in or deletion of an adhE gene. In some
embodiments, the
acetate-producing bacterium comprises at least one mutation in or deletion of
a frd gene.
In some embodiments, the acetate-producing bacterium comprises at least one
mutation in
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or deletion of an ldhA gene and at least one mutation in or deletion of an
adhE gene. In
some embodiments, the acetate-producing bacterium comprises at least one
mutation in or
deletion of a ldhA gene and at least one mutation in or deletion of an frd
gene. In some
embodiments, the acetate-producing bacterium comprises at least one mutation
in or
deletion of an adhA gene and at least one mutation in or deletion of an frd
gene. In some
embodiments, the acetate-producing bacterium comprises at least one mutation
in or
deletion of an adhA gene, at least one mutation in or deletion of an frd gene,
and at least
one mutation in or deletion of an ldhA gene.
[017] The bacterium may produce an increased level of acetate as compared to a
bacterium which produces Acetyl CoA and comprises an endogenous pta gene, and
has an
endogenous frd gene and/or endogenous ldhA gene and/or endogenous adhA gene.
The
bacterium may produce an increased level of acetate as compared to a bacterium
which
produces Acetyl CoA and comprises an endogenous pta gene, and does not
comprise at
least one mutation in or deletion of an ldhA gene, an adhE gene, and/or a frd
gene.
[018] In any of the above-described embodiments comprising a gene or gene
cassette for producing butyrate in which the gene or gene cassette is operably
linked to a
directly or indirectly inducible promoter, the promoter may be induced under
low-oxygen
or anaerobic conditions. In some embodiments, the promoter is selected from an
FNR-
responsive promoter, an ANR-responsive promoter, and a DNR-responsive
promoter. In
some embodiments, the promoter is an FNR-responsive promoter. In some
embodiments,
the promoter may be induced by the presence of reactive nitrogen species. In
some
embodiments, the promoter is selected from an NsrR-responsive promoter, NorR-
responsive promoter, and a DNR-responsive promoter. In some embodiments, the
promoter may be induced by the presence of reactive oxygen species. In some
embodiments, the promoter is selected from an OxyR-responsive promoter, PerR-
responsive promoter, OhrR-responsive promoter, SoxR-responsive promoter, or a
RosR-
responsive promoter.
[019] In some embodiments, the gene and/or gene cassette is located on a
chromosome in the bacterium. In some embodiments, the at least one gene and/or
gene
cassette is located on a plasmid in the bacterium.
[020] In some embodiments, the bacterium is a probiotic bacterium. In some
embodiments, the bacterium is selected from the group consisting of
Bacteroides,
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Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus. In
some
embodiments, thebacterium is Escherichia coli strain Nissle.
[021] In some embodiments, the bacterium is an an auxotroph in a gene that is
complemented when the bacterium is present in a mammalian gut. The bacterium
may be
an auxotroph in diaminopimelic acid or an enzyme in the thymine biosynthetic
pathway.
[022] Disclosed herein is a pharmaceutically acceptable composition comprising
one or more of any of the bacterium disclosed herein; and a pharmaceutically
acceptable
carrier. In some embodiments, the composition is formulated for oral or rectal
administration.
[023] Disclosed herein is a method of treating or preventing an autoimmune
disorder, comprising the step of administering to a patient in need thereof, a
composition
disclosed herein.
[024] Disclosed herein is a method of treating a disease or condition
associated
with gut inflammation and/or compromised gut barrier function comprising the
step of
administering to a patient in need thereof, a composition.
[025] The autoimmune disorder may be selected from the group consisting of
acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic
leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata,
amyloidosis,
ankylo sing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid
syndrome (APS),
autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia,
autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia,
autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune
myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune
retinopathy,
autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease,
autoimmune
urticarial, Axonal & neuronal neuropathies, Balo disease, Behcet's disease,
Bullous
pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease,
Chronic
inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal
ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign
muco sal
pemphigoid, Crohn's disease, Cogan syndrome, Cold agglutinin disease,
Congenital heart
block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia,
Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's
disease
(neuromyelitis optica), Discoid lupus, Dressler's syndrome, Endometriosis,
Eosinophilic
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esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic
encephalomyelitis, Evans syndrome, Fibrosing alveolitis, Giant cell arteritis
(temporal
arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture's
syndrome,
Granulomatosis with Polyangiitis (GPA), Graves' disease, Guillain-Barre
syndrome,
Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic anemia, Henoch-
Schonlein
purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic
thrombocytopenic
purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease,
Immunoregulatory
lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile
arthritis, Juvenile
idiopathic arthritis, Juvenile myositis, Kawasaki syndrome, Lambert-Eaton
syndrome,
Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous
conjunctivitis,
Linear IgA disease (LAD), Lupus (Systemic Lupus Erythematosus), chronic Lyme
disease, Meniere's disease, Microscopic polyangiitis, Mixed connective tissue
disease
(MCTD), Mooren's ulcer, Mucha-Habermann disease, Multiple sclerosis,
Myasthenia
gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's), Neutropenia,
Ocular
cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS
(Pediatric
autoimmune Neuropsychiatric Disorders Associated with Streptococcus),
Paraneoplastic
cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry
Romberg
syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis),
Pemphigus,
Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS
syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular
syndromes,
Polymyalgia rheumatic, Polymyositis, Postmyocardial infarction syndrome,
Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary
cirrhosis, Primary
sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary
fibrosis,
Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, reactive
arthritis,
reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis,
restless legs
syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis,
sarcoidosis,
Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm &
testicular
autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE),
Susac's
syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal
arteritis/giant cell
arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse
myelitis,
type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective
tissue disease
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(UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and
Wegener's
granulomatosis.
[026] The autoimmune disorder may be selected from the group consisting of
type 1 diabetes, lupus, rheumatoid arthritis, ulcerative colitis, juvenile
arthritis, psoriasis,
psoriatic arthritis, celiac disease, and ankylosing spondylitis.
[027] The disease or condition may be selected from an inflammatory bowel
disease, including Crohn's disease and ulcerative colitis, and a diarrheal
disease.
Brief Description of the Figures
[028] FIG. 1A, FIG. 1B, FIG 1C, FIG. 1D, FIG. 1E, FIG. 1F, FIG. 1G, FIG.
1H, FIG. 11, FIG. 1J, and FIG. 1K depict schematics of E. coli that are
genetically
engineered to express a propionate biosynthesis cassette (FIG. 1A), a butyrate
biosynthesis cassette (FIG. 1B), an acetate biosynthesis cassette (FIG. 1C), a
cassette for
the expression of GLP-2 (FIG. 1D), a cassette for the expression of human IL-
10 (FIG.
1E) or v-IL-22 or hIL-22 (FIG. 1F) under the control of a FNR-responsive
promoter. The
genetically engineered E. coli depicted in FIG. 1D, FIG. 1E, and FIG. 1F may
further
comprise a secretion system for secretion of the expressed polypeptide out of
the cell.
FIG. 1Gdepicts bacteria overexpressing butyrate (and not expressing acetate)
by
expressing a butyrate biosynthesis cassette in combination with deletions in
adhE and pta
(FIG. 1G), FIG. 1H depicts bacteria overexpressing butyrate by expressing a
butyrate
biosynthesis cassette in combination with deletions in ldhA, FIG. 11 depicts
bacteria
overexpressing butyrate by expressing a butyrate biosynthesis cassette in
combination
with deletions in adhE and frdA (FIG. 1I). FIG. 1J depicts bacteria
overexpressing
acetate by deletion in ldhA. FIG. 1K depicts bacteria overexpressing GLP-2 in
combination with a deletion in adhE and pta.
[029] FIG. 2A, FIG. 2B, FIG. 2C, and FIG.2D depict schematics of a butyrate
production pathway and schematics of different butyrate producing circuits.
FIG. 2A
depicts a metabolic pathway for butyrate production. FIG. 2B and FIG. 2C
depict
schematics of two different exemplary butyrate producing circuits, both under
the control
of a tetracycline inducible promoter. FIG. 2B depicts a bdc2 butyrate cassette
under
control of tet promoter on a plasmid. A "bdc2 cassette" or "bdc2 butyrate
cassette" refres
to a butyrate producing cassette that comprises at least the following genes:
bcd2, etfB3,
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etfA3, hbd, crt2, pbt, and buk genes. FIG. 2C depicts a ter butyrate cassette
(ter gene
replaces the bcd2, etfB3, and etfA3 genes) under control of tet promoter on a
plasmid. A
"ter cassette" or "ter butyrate cassette" refers to a butyrate producing
cassete that
comprises at least the following genes: ter, thiAl, hbd, crt2, pbt, buk. FIG.
2D depicts a
schematic of a third exemplary butyrate gene cassette under the control of a
tetracycline
inducible promoter, specifically, a tesB butyrate cassette (ter gene is
present and tesB gene
replaces the pbt gene and the buk gene) under control of tet promoter on a
plasmid. A "tes
or tesB cassette or "tes or tesB butyrate cassette" refers to a butyrate
producing cassette
that comprises at least ter, thiAl, hbd, crt2, and tesB genes. An alternative
butyrate
cassette of the disclosure comprises at least bcd2, etfB3, etfA3, thiAl, hbd,
crt2, and tesB
genes. In some embodiments, the tes or tesB cassette is under control of an
inducible
promoter other than tetracycline. Exemplary inducible promoters which may
control the
expression of the tesB cassette include oxygen level-dependent promoters
(e.g., FNR-
inducible promoter), promoters induced by inflammation or an inflammatory
response
(RNS, ROS promoters), and promoters induced by a metabolite that may or may
not be
naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose
and
tetracycline.
[030] FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, and FIG. 3F depict
schematics of the gene organization of exemplary bacteria of the disclosure.
FIG. 3A and
FIG. 3B depict the gene organization of an exemplary engineered bacterium of
the
invention and its induction of butyrate production under low-oxygen
conditions. FIG. 3A
depicts relatively low butyrate production under aerobic conditions in which
oxygen (02)
prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and activating
the FNR-
responsive promoter ("FNR promoter"). Therefore, none of the butyrate
biosynthesis
enzymes (bcd2, e033, e03, thiAl, hbd, crt2, pbt, and buk; white boxes) is
expressed.
FIG. 3B depicts increased butyrate production under low-oxygen or anaerobic
conditions
due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-responsive
promoter,
and inducing expression of the butyrate biosynthesis enzymes, which leads to
the
production of butyrate. FIG. 3C and FIG. 3D depict the gene organization of an
exemplary recombinant bacterium of the invention and its derepression in the
presence of
nitric oxide (NO). In FIG. 3C, in the absence of NO, the NsrR transcription
factor (circle,
"NsrR") binds to and represses a corresponding regulatory region. Therefore,
none of the
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butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, buk)
is
expressed. In FIG. 3D, in the presence of NO, the NsrR transcription factor
interacts with
NO, and no longer binds to or represses the regulatory sequence. This leads to
expression
of the butyrate biosynthesis enzymes (indicated by black arrows and black
squiggles) and
ultimately to the production of butyrate.
[031] FIG. 3E and FIG. 3F depict the gene organization of an exemplary
recombinant bacterium of the invention and its induction in the presence of
H202. In
FIG. 3E, in the absence of H202, the OxyR transcription factor (circle,
"OxyR") binds to,
but does not induce, the oxyS promoter. Therefore, none of the butyrate
biosynthesis
enzymes (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, buk) is expressed. In FIG.
3F, in the
presence of H202, the OxyR transcription factor interacts with H202 and is
then capable
of inducing the oxyS promoter. This leads to expression of the butyrate
biosynthesis
enzymes (indicated by black arrows and black squiggles) and ultimately to the
production
of butyrate.
[032] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, and FIG. 4F depict
schematics of the gene organization of exemplary bacteria of the disclosure.
FIG. 4A and
FIG. 4B depict the gene organization of another exemplary engineered bacterium
of the
invention and its induction of butyrate production under low-oxygen conditions
using a
different butyrate circuit from that shown in FIG. 3A, FIG 3B, FIG. 3C, FIG.
3D, FIG.
3E, and FIG. 3F. FIG. 4A depicts relatively low butyrate production under
aerobic
conditions in which oxygen (02) prevents (indicated by "X") FNR (boxed "FNR")
from
dimerizing and activating the FNR-responsive promoter ("FNR promoter").
Therefore,
none of the butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, pbt, and
buk; white
boxes) is expressed. FIG. 4B depicts increased butyrate production under low-
oxygen or
anaerobic conditions due to FNR dimerizing (two boxed "FNR"s), binding to the
FNR-
responsive promoter, and inducing expression of the butyrate biosynthesis
enzymes, which
leads to the production of butyrate. FIG. 4C and FIG. 4D depict the gene
organization of
another exemplary recombinant bacterium of the invention and its derepression
in the
presence of NO. In FIG. 4C, in the absence of NO, the NsrR transcription
factor (circle,
"NsrR") binds to and represses a corresponding regulatory region. Therefore,
none of the
butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, pbt, buk) is expressed.
In FIG. 4D,
in the presence of NO, the NsrR transcription factor interacts with NO, and no
longer
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binds to or represses the regulatory sequence. This leads to expression of the
butyrate
biosynthesis enzymes (indicated by black arrows and black squiggles) and
ultimately to
the production of butyrate. FIG. 4E and FIG. 4F depict the gene organization
of another
exemplary recombinant bacterium of the invention and its induction in the
presence of
H202. In FIG. 4E, in the absence of H202, the OxyR transcription factor
(circle, "OxyR")
binds to, but does not induce, the oxyS promoter. Therefore, none of the
butyrate
biosynthesis enzymes (ter, thiAl, hbd, crt2, pbt, buk) is expressed. In FIG.
4F, in the
presence of H202, the OxyR transcription factor interacts with H202 and is
then capable of
inducing the oxyS promoter. This leads to expression of the butyrate
biosynthesis
enzymes (indicated by black arrows and black squiggles) and ultimately to the
production
of butyrate.
[033] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F depict
schematics of the gene organization of exemplary bacteria of the disclosure.
FIG. 5A and
FIG. 5B depict the gene organization of an exemplary recombinant bacterium of
the
invention and its induction under low-oxygen conditions. FIG. 5A depicts
relatively low
butyrate production under aerobic conditions in which oxygen (02) prevents
(indicated by
"X") FNR (boxed "FNR") from dimerizing and activating the FNR-responsive
promoter
("FNR promoter"). Therefore, none of the butyrate biosynthesis enzymes (ter,
thiAl, hbd,
crt2, and tesB) is expressed. FIG. 5B depicts increased butyrate production
under low-
oxygen conditions due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-
responsive promoter, and inducing expression of the butyrate biosynthesis
enzymes, which
leads to the production of butyrate. FIG. 5C and FIG. 5D depict the gene
organization of
another exemplary recombinant bacterium of the invention and its derepression
in the
presence of NO. In FIG. 5C, in the absence of NO, the NsrR transcription
factor (
"NsrR") binds to and represses a corresponding regulatory region. Therefore,
none of the
butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, tesB) is expressed. In
FIG. 5D, in
the presence of NO, the NsrR transcription factor interacts with NO, and no
longer binds
to or represses the regulatory sequence. This leads to expression of the
butyrate
biosynthesis enzymes (indicated by black arrows and black squiggles) and
ultimately to
the production of butyrate. FIG. 5E and FIG. 5F depict the gene organization
of another
exemplary recombinant bacterium of the invention and its induction in the
presence of
H202. In FIG. 5E, in the absence of H202, the OxyR transcription factor
(circle, "OxyR")
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binds to, but does not induce, the oxyS promoter. Therefore, none of the
butyrate
biosynthesis enzymes (ter, thiAl, hbd, crt2, tesB) is expressed. In Figs. 6F,
in the
presence of H202, the OxyR transcription factor interacts with H202 and is
then capable of
inducing the oxyS promoter. This leads to expression of the butyrate
biosynthesis
enzymes (indicated by black arrows and black squiggles) and ultimately to the
production
of butyrate.
[034] FIG. 6A and FIG. 6B depict schematics of the gene organization of
exemplary bacteria of the disclosure for inducible propionate production. FIG.
6A depicts
relatively low propionate production under aerobic conditions in which oxygen
(02)
prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and activating
the FNR-
responsive promoter ("FNR promoter"). Therefore, none of the propionate
biosynthesis
enzymes (pct, lcdA, lcdB, lcdC, e0, acrB, acrC) is expressed. FIG. 6B depicts
increased
propionate production under low-oxygen or anaerobic conditions due to FNR
dimerizing
(two boxed "FNR"s), binding to the FNR-responsive promoter, and inducing
expression of
the propionate biosynthesis enzymes, which leads to the production of
propionate. In
other embodiments, propionate production is induced by NO or H202 as depicted
and
described for the butyrate cassette(s) in the preceding FIG. 3C-3F, FIG. 4C-
4F, FIG. 5C-
5F.
[035] FIG. 7 depicts an exemplary propionate biosynthesis gene cassette.
[036] FIG. 8A, FIG. 8B, and FIG. 8C depict schematics of the gene
organization of exemplary bacteria of the disclosure for inducible propionate
production.
FIG. 8A depicts relatively low propionate production under aerobic conditions
in which
oxygen (02) prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and
activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of
the
propionate biosynthesis enzymes (thrA, thrB, thrC, ilvA, aceE, aceF, 1pc1) is
expressed.
FIG. 8B depicts increased propionate production under low-oxygen or anaerobic
conditions due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-
responsive
promoter, and inducing expression of the propionate biosynthesis enzymes,
which leads to
the production of propionate. FIG. 8C depicts an exemplary propionate
biosynthesis gene
cassette. In other embodiments, propionate production is induced by NO or H202
as
depicted and described for the butyrate cassette(s) in the preceding FIG. 3C-
3F, FIG. 4C-
4F, FIG. 5C-5F.
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[037] FIG. 9A and FIG. 9B depict schematics of the gene organization of
exemplary bacteria of the disclosure for inducible propionate production. FIG.
9A depicts
relatively low propionate production under aerobic conditions in which oxygen
(02)
prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and activating
the FNR-
responsive promoter ("FNR promoter"). Therefore, none of the propionate
biosynthesis
enzymes (thrA, thrB, thrC, ilvA, aceE, aceF, 1pd, tesB) is expressed. FIG. 9B
depicts
increased propionate production under low-oxygen or anaerobic conditions due
to FNR
dimerizing (two boxed "FNR"s), binding to the FNR-responsive promoter, and
inducing
expression of the propionate biosynthesis enzymes, which leads to the
production of
propionate. In other embodiments, propionate production is induced by NO or
H202 as
depicted and described for the butyrate cassette(s) in the preceding FIG. 3C-
3F, FIG. 4C-
4F, FIG. 5C-5F.
[038] FIG. 10A, FIG. 10B, and FIG. 10C depict schematics of the sleeping
beauty pathway and the gene organization of an exemplary bacterium of the
disclosure.
FIG. 10A depicts a schematic of a genetically engineered sleeping beauty
metabolic
pathway from E. coli for propionate production. The SBM pathway is cyclical
and
composed of a series of biochemical conversions forming propionate as a
fermentative
product while regenerating the starting molecule of succinyl-CoA. FIG. 10B and
FIG.
10C depict schematics of the gene organization of another exemplary engineered
bacterium of the invention and its induction of propionate production under
low-oxygen
conditions. FIG. 10B depicts relatively low propionate production under
aerobic
conditions in which oxygen (02) prevents (indicated by "X") FNR (boxed "FNR")
from
dimerizing and activating the FNR-responsive promoter ("FNR promoter").
Therefore,
none of the propionate biosynthesis enzymes (sbm, ygfD, ygfG, ygfH) is
expressed. FIG.
10C depicts increased propionate production under low-oxygen or anaerobic
conditions
due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-responsive
promoter,
and inducing expression of the propionate biosynthesis enzymes, which leads to
the
production of propionate. In other embodiments, propionate production is
induced by NO
or H202 as depicted and described for the butyrate cassette(s) in the
preceding FIG. 3C-
3F, FIG. 4C-4F, FIG. 5C-5F.
[039] FIG. 11 depicts a bar graph showing butyrate production of butyrate
producing strains of the disclosure. FIG. 11 shows butyrate production in
strains
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pLOGIC031 and pLOGIC046 in the presence and absence of oxygen, in which there
is no
significant difference in butyrate production. Enhanced butyrate production
was shown in
Nissle in low copy plasmid expressing pLOGIC046 which contain a deletion of
the final
two genes (ptb-buk) and their replacement with the endogenous E. Coli tesB
gene (a
thioesterase that cleaves off the butyrate portion from butyryl CoA).
Overnight cultures of
cells were diluted 1:100 in Lb and grown for 1.5 hours until early log phase
was reached at
which point anhydrous tet was added at a final concentration of 100ng/m1 to
induce
plasmid expression. After 2 hours induction, cells were washed and resuspended
in M9
minimal media containing 0.5% glucose at 0D600=0.5. Samples were removed at
indicated times and cells spun down. The supernatant was tested for butyrate
production
using LC-MS.
[040] FIG. 12 depicts a bar graph showing butyrate production of butyrate
producing strains of the disclosure. FIG. 12 shows butyrate production in
strains
comprising a tet¨butyrate cassette having ter substitution (pLOGIC046) or the
tesB
substitution (ptb-buk deletion), demonstrating that the tesB substituted
strain has greater
butyrate production.
[041] FIG. 13 depicts a graph of butyrate production using different butyrate-
producing circuits comprising a nuoB gene deletion. Strains depicted are
BW25113
comprising a bcd-butyrate cassette, with or without a nuoB deletion, and
BW25113
comprising a ter-butyrate cassette, with or without a nuoB deletion. Strains
with deletion
are labeled with nuoB. The NuoB gene deletion results in greater levels of
butyrate
production as compared to a wild-type parent control in butyrate producing
strains. NuoB
is a main protein complex involved in the oxidation of NADH during respiratory
growth.
In some embodiments, preventing the coupling of NADH oxidation to electron
transport
increases the amount of NADH being used to support butyrate production.
[042] FIG. 14A, FIG. 14B, FIG.14C, and FIG. 14D depict schematics and
graphs showing butyrate or biomarker production of a butyrate producing
circuit under the
control of an FNR promoter. FIG. 14A depicts a schematic showing a butyrate
producing
circuit under the control of an FNR promoter. FIG. 14B depicts a bar graph of
anaerobic
induction of butyrate production. FNR-responsive promoters were fused to
butyrate
cassettes containing either the bcd or ter circuits. Transformed cells were
grown in LB to
early log and placed in anaerobic chamber for 4 hours to induce expression of
butyrate
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genes. Cells were washed and resuspended in minimal media w/ 0.5% glucose and
incubated microaerobically to monitor butyrate production over time. SYN-501
led to
significant butyrate production under anaerobic conditions. FIG. 14C depicts
SYN-501 in
the presence and absence of glucose and oxygen in vitro. SYN-501 comprises
pSC101
PydfZ-ter butyrate plasmid; SYN-500 comprises pSC101 PydfZ-bcd butyrate
plasmid;
SYN-506 comprises pSC101 nirB-bcd butyrate plasmid. FIG. 14D depict levels of
mouse
lipocalin 2 (left) and calprotectin (right) quantified by ELISA using the
fecal samples in an
in vivo model. SYN-501 reduces inflammation and/or protects gut barrier
function as
compared to wild type Nissle control.
[043] FIG. 15 depicts a graph measuring gut-barrier function in dextran sodium
sulfate (DSS)-induced mouse models of IBD. The amount of FITC dextran found in
the
plasma of mice administered different concentrations of DSS was measured as an
indicator of gut barrier function.
[044] FIG. 16 depicts serum levels of FITC-dextran analyzed by
spectrophotometry. FITC-dextran is a readout for gut barrier function in the
DSS-induced
mouse model of IBD.
[045] FIG. 17 depicts a scatter graph of butyrate concentrations in the feces
of
mice gavaged with either H20, 100 mM butyrate in H20, streptomycin resistant
Nissle
control or SYN501 comprising a PydfZ-ter ->pbt-buk butyrate plasmid.
Significantly
greater levels of butyrate were detected in the feces of the mice gavaged with
SYN501 as
compared mice gavaged with the Nissle control or those given water only.
Levels are close
to 2 mM and higher than the levels seen in the mice fed with H20 (+) 200 mM
butyrate.
[046] FIG. 18 depicts a bar graph comparing butyrate concentrations produced
in
vitro by the butyrate cassette plasmid strain SYN501 as compared to Clostridia
butyricum
MIYARISAN (a Japanese probiotic strain), Clostridium tyrobutyricum VPI 5392
(Type
Strain), and Clostridium butyricum NCTC 7423 (Type Strain) under aerobic and
anaerobic
conditions at the indicated timepoints. The Nissle strain comprising the
butyrate cassette
produces butyrate levels comparable to Clostridium spp. in RCM media.
[047] FIG. 19A depicts a bar graph showing butyrate concentrations produced in
vitro by strains comprising chromsolmally integrated butyrate copies as
compared to
plasmid copies. Integrated butyrate strains, SYN1001 and SYN1002 (both
integrated at the
agaI/rsml locus) gave comparable butyrate production to the plasmid strain
SYN501.
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[048] FIG. 19B and FIG. 19C depict bar graphs showing the effect of the
supernatants from the engineered butyrate-producing strain, SYN1001, on
alkaline
phosphatase activity in HT-29 cells represented in bar (FIG. 19B) and
nonlinear fit (FIG.
19C) graphical formats.
[049] FIG. 20A and FIG. 20B depicts the construction and gene organization of
an exemplary plasmids. FIG. 20A depicts the construction and gene organization
of an
exemplary plasmids comprising a gene encoding NsrR, a regulatory sequence from
norB,
and a butyrogenic gene cassette (pLogic031-nsrR-norB-butyrate construct). FIG.
20B
depicts the construction and gene organization of another exemplary plasmid
comprising a
gene encoding NsrR, a regulatory sequence from norB, and a butyrogenic gene
cassette
(pLogic046- nsrR-norB-butyrogenic gene cassette).
[050] FIG. 21 depicts butyrate production using SYN001 + tet (control wild-
type
Nissle comprising no plasmid), SYN067 + tet (Nissle comprising the pLOGIC031
ATC-
inducible butyrate plasmid), and SYN080 + tet (Nissle comprising the pLOGIC046
ATC-
inducible butyrate plasmid).
[051] FIG. 22 depicts butyrate production by genetically engineered Nissle
comprising the pLogic031-nsrR-norB-butyrate construct (SYN133) or the
pLogic046-
nsrR-norB-butyrate construct (SYN145), which produce more butyrate as compared
to
wild-type Nissle (SYN001).
[052] FIG. 23 depicts the construction and gene organization of an exemplary
plasmid comprising an oxyS promoter and butyrogenic gene cassette (pLogic031-
oxyS-
butyrogenic gene cassette).
[053] FIG. 24 depicts the construction and gene organization of another
exemplary plasmid comprising an oxyS promoter and butyrogenic gene cassette
(pLogic046-oxyS- butyrogenic gene cassette).
[054] FIG. 25 depicts a schematic illustrating a strategy for increasing
butyrate
and acetate production in engineered bacteria. Aerobic metabolism through the
citric acid
cycle (TCA cycle) (crossed out) is inactive in the anaerobic environment of
the colon. E.
coli makes high levels of acetate as an end production of fermentation. To
improve
acetate production, while still maintaining highlevels of butyrate production,
targeted
deletion can be introduced to prevent the production of unnecessary metabolic
fermentative byproducts (thereby simultaneously increasing butyrate and
acetate
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production). Non-limiting examples of competing routes (shown in in rounded
boxes) are
frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to
lactate) and
adhE (converts Acetyl-CoA to Ethanol). Deletions of interest therefore include
deletion of
adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered
bacteria
further comprise mutations and/or deletions in one or more of frdA, ldhA, and
adhE.
[055] FIG. 26A and FIG. 26B depict line graphs showing acetate production
over a 6 hour time course post-induction in 0.5% glucose MOPS (pH6.8) (FIG.
26A) and
in 0.5% glucuronic acid MOPS (pH6.3) (FIG. 26B). Acetate production of an
engineered
E. coli Nissle strain comprising a deletion in the endenous ldh gene (SYN2001)
was
compared with streptomycin resistant Nissle (5YN94).
[056] FIG. 26C and FIG. 26D depict bar graphs showing acetate and butyrate
production in 0.5% glucose MOPS (pH6.8) (FIG. 26C) and acetate and butyrate
production in 0.5% glucuronic acid MOPS (pH6.3) (FIG. 26D). Deletions in
endogenous
adhE (Aldehyde-alcohol dehydrogenase) and ldh (lactate dehydrogenase) were
introduced
into Nissle strains with either integrated FNRS ter-tesB or FNRS-ter-pbt-buk
butyrate
cassettes. SYN2006 comprises a FNRS ter-tesB cassette integrated at the HA1/2
locus and
a deletion in the endogenous adhE gene. SYN2007 comprises a FNRS ter-tesB
cassette
integrated at the HA1/2 locus and a deletion in the endogenous ldhA gene.
5YN2008
comprises a FNRS-ter-pbt-buk butyrate cassette and a deletion in the
endogenous adhE
gene. 5YN2003 comprises a FNRS-ter-pbt-buk butyrate cassette and a deletion in
the
endogenous ldhA gene.
[057] FIG. 26E depicts a bar graph showing acetate and butyrate production at
the indicated time points post induction in 0.5% glucose MOPS (pH6.8). A
strain
comprising a FNRS-ter-tesB butyrate cassette integrated at the HA1/2 locus of
the
chromosome (SYN1004) was compared with a strain comprising the same integrated
cassette and additionally a deletion in the endogenous frd gene (5YN2005).
[058] FIG. 26F depicts a bar graph showing acetate and butyrate production at
18
hours in 0.5% glucose MOPS (pH6.8), comparing three strains engineered to
produce
short chain fatty acids. SYN2001 comprises a deletion in the endenous ldh
gene;
5YN2002 comprises a FNRS-ter-tesB butyrate cassette integrated at the HA1/2
locus and
deletions in the endogenous adhE and pta genes. 5YN2003 comprises FNRS-ter-pbt-
buk
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butyrate cassette integrated at the HA1/2 locus and a deletion in the
endogenous ldhA
gene.
[059] FIG. 26G and FIG. 26H depict line graphs showing the effect of
supernatants from the engineered acetate-producing strain, SYN2001, on LPS-
induced
IFN7 secretion in primary human PBMC cells from donor 1 (D1) (Fig. 26G) and
donor 2
(D2) (FIG. 26H).
[060] FIG. 27 depicts a schematic of an exemplary propionate biosynthesis gene
cassette.
[061] FIG. 28 depicts a schematic of a construct comprising the sleeping
beauty
mutase operon from E. coli under the control of a heterologous FnrS promoter.
[062] FIG. 29 depicts a bar graph of proprionate concentrations produced in
vitro
by the wild type E coli BW25113 strain and a BW25113 strain which comprises
the
endogenous SBM operon under the control of the FnrS promoter, as depicted in
the
schematic in FIG. 28.
[063] FIG. 30A, FIG. 30B, and FIG. 30C depict schematics of the gene
organization of exemplary circuits of the disclosure for the expression of
therapeutic
polypeptides, which are secreted using components of the flagellar type III
secretion
system. A therapeutic polypeptide of interest, such as, GLP-2, IL-10, and IL-
22, is
assembled behind a fliC-5'UTR, and is driven by the native fliC and/or fliD
promoter
(FIG. 30A and FIG. 30B) or a tet-inducible promoter (FIG. 30C). In alternate
embodiments, an inducible promoter such as oxygen level-dependent promoters
(e.g.,
FNR-inducible promoter), promoters induced by IBD specific molecules or
promoters
induced by inflammation or an inflammatory response (RNS, ROS promoters), and
promoters induced by a metabolite that may or may not be naturally present
(e.g., can be
exogenously added) in the gut, e.g., arabinose can be used. The therapeutic
polypeptide of
interest is either expressed from a plasmid (e.g., a medium copy plasmid) or
integrated
into fliC loci (thereby deleting all or a portion of fliC and/or fliD).
Optionally, an N
terminal part of FliC is included in the construct, as shown in FIG. 30B and
FIG. 30D.
[064] FIG. 31A and FIG. 31B depict schematics of the gene organization of
exemplary circuits of the disclosure for the expression of therapeutic
polypeptides, which
are secreted via a diffusible outer membrane (DOM) system. The therapeutic
polypeptide
of interest is fused to a prototypical N-terminal Sec-dependent secretion
signal or Tat-
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dependent secretion signal, which is is cleaved upon secretion into the
periplasmic space.
Exemplary secretion tags include sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-
dependent tags (TorA, FdnG, DmsA). In certain embodiments, the genetically
engineered
bacteria comprise deletions in one or more of 1pp, pal, tolA, and/or nlpI.
Optionally,
periplasmic proteases are also deleted, including, but not limited to, degP
and ompT, e.g.,
to increase stability of the polypeptide in the periplasm. A FRT-KanR-FRT
cassette is
used for downstream integration. Expression is driven by a tet promoter (FIG.
31A) or an
inducible promoter, such as oxygen level-dependent promoters (e.g., FNR-
inducible
promoter, FIG. 31B), promoters induced by IBD specific molecules or promoters
induced
by inflammation or an inflammatory response (RNS, ROS promoters), and
promoters
induced by a metabolite that may or may not be naturally present (e.g., can be
exogenously added) in the gut, e.g., arabinose.
[065] FIG. 32A, FIG. 32B, FIG. 32C, FIG. 32D, and FIG. 32E depict
schematics of non-limiting examples of constructs for the expression of GLP2
for bacterial
secretion. FIG. 32A depicts a schematic of a human GLP2 construct inserted
into the FliC
locus, under the control of the native FliC promoter. FIG. 32B depicts a
schematic of a
human GLP2 construct, including the N terminal 20 amino acids of FliC,
inserted into the
FliC locus under the control of the native FliC promoter. FIG. 32C depicts a
schematic of
a human GLP2 construct, including the N-terminal 20 amino acids of FliC,
inserted into
the FliC locus under the control of a tet inducible promoter. FIG. 32D depicts
a schematic
of a human GLP2 construct with a N terminal OmpF secretion tag (sec-dependent
secretion system) under the control of a tet inducible promoter. FIG. 32E
depicts a
schematic of a human GLP2 construct with a N terminal TorA secretion tag (tat
secretion
system) under the control of a tet inducible promoter.
[066] FIG. 33A and FIG. 33B depict line graphs of ELISA results. FIG. 33A
depicts a line graph, showing an phopho-STAT3 (Tyr705) ELISA conducted on
extracts
from serum-starved Co10205 cells treated with supernatants from engineered
bacteria
comprising a PAL deletion and an integrated construct encoding hIL-22 with a
phoA
secretion tag. The data demonstrate that hIL-22 secreted from the engineered
bacteria is
functionally active. FIG. 33B depicts a line graph, showing an phopho-STAT3
(Tyr705)
ELISA showing a antibody completion assay. Extracts from Co10205 cells were
treated
with the bacterial supernatants from the IL-22 overexpres sing strain
preincubated with
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increasing concentrations of neutralizing anti-IL-22 antibody. The data
demonstrated that
phospho-Stat3 signal induced by the secreted hIL-22 is competed away by the
hIL-22
antibody MAB7821.
[067] FIG. 33C depicts a line graph showing SYN3001 (PhoA-IL-22 in pal
mutant chassi), but not SYN3000 (pal mutant chassi) supernatant induces STAT3
activation.
[068] FIG. 33D depicts a line graph showing that anti IL-22 neutralizing
antibody inhibits SYN3001-induced STAT3 activation (n=3).
[069] FIG. 33E depicts a Western blot analysis of bacterial supernatants from
strain SYN2980 and SYN2982, using IL-10 antibody (IL-10 (D13A11) XP Rabbit
mAb
#12163, Cell Signaling Technology). The secreted polypepetide has the same
molecular
weight as the standards, indicating that the signal sequence is cleaved from
the native
peptide.
[070] FIG. 34 depicts a schematic of tryptophan metabolism along the
kynurenine and the serotonin arms in humans. The abbreviations for the enzymes
are as
follows: 3-HAO: 3-hydroxyl-anthranilate 3,4-dioxidase; AAAD: aromatic ¨amino
acid
decarboxylase; ACMSD, alpha-amino-beta-carboxymuconate-epsilon-semialdehyde
decarboxylase; HIOMT, hydroxyl-0-methyltransferase; IDO, indoleamine 2,3-
dioxygenase; KAT, kynurenine amino transferases I-III; KMO: kynurenine 3-
monooxygenase; KYNU, kynureninase; NAT, N-acetyltransferase; TDO, tryptophan
2,3-
dioxygenase; TPH, tryptophan hydroxylase; QPRT, quinolinic acid phosphoribosyl
transferase.
[071] FIG. 35 depicts a schematic of bacterial tryptophan catabolism
machinery,
which is genetically and functionally homologous to IDO1 enzymatic activity,
as
described in Vujkovic-Cvijin et al., Dysbiosis of the gut microbiota is
associated with HIV
disease progression and tryptophan catabolism; Sci Transl Med. 2013 July 10;
5(193):
193ra91, the contents of which is herein incorporated by reference in its
entirety. In certain
embodiments of the disclosure, the genetically engineered bacteria comprise
gene
cassettes comprising one or more of the bacterial tryptophan metabolism
enzymes
depicted in FIG. 35. In certain embodiments, the genetically engineered
bacteria comprise
one or more gene cassettes which produce one or more of the metabolites
depicted in FIG.
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35, including but not limited to, kynurenine, indole-3-aldehyde, indole-3-
acetic acid,
and/or indole-3 acetaldehyde.
[072] FIG. 36A and FIG. 36B depict schematics of indole metabolite mode of
action (FIG.36A) and indole biosynthesis (FIG. 36B). FIG.36A depicts a
schematic of
molecular mechanisms of action of indole and its metabolites on host
physiology and
disease. Tryptophan catabolized by bacteria to yield indole and other indole
metabolites,
e.g., Indole-3-propionate (IPA) and Indole-3-aldehyde (I3A), in the gut lumen.
IPA acts on
intestinal cells via pregnane X receptors (PXR) to maintain mucosal
homeostasis and
barrier function. I3A acts on the aryl hydrocarbon receptor (AhR) found on
intestinal
immune cells and promotes IL-22 production. Activation of AhR plays a crucial
role in
gut immunity, such as in maintaining the epithelial barrier function and
promoting
immune tolerance to promote microbial commensalism while protecting against
pathogenic infections. Indole has a number of roles, such as a signaling
molecule to
intestinal L cells to produce glucagon-like protein 1 (GLP-1) or as a ligand
for AhR
(Zhang et al. Genome Med. 2016; 8: 46). FIG. 36B depicts a schematic of the
trypophan
catabolic pathway/indole biosynthesis pathways. Host and microbiota
metabolites with
AhR agonistic activity are in in diamond and circled, respectively (see, e.g.,
Lamas et al.,
CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into
aryl
hydrocarbon receptor ligands; Nature Medicine 22, 598-605 (2016). In certain
embodiments of the disclosure, the genetically engineered bacteria comprise
gene
cassettes comprising one or more of the bacterial tryptophan metabolism
enzymes which
catalyze the reactions shown in FIGs. 36A and 36B. In certain embodiments, the
genetically engineered bacteria comprise one or more gene cassettes which
produce one or
more of the metabolites depicted in FIGs. 36A and 36B, including but not
limited to,
kynurenine, indole-3-aldehyde, indole-3-acetic acid, and/or indole-3
acetaldehyde.
[073] FIG. 37A and FIG. 37B depict diagrams of bacterial tryptophan
metabolism pathways. FIG. 37A depicts a schematic of the bacterial tryptophan
metabolism, as described, e.g., in Enzymes are numbered as follows 1) Trp 2,3
dioxygenase (EC 1.13.11.11); 2) kynurenine formidase (EC 3.5.1.49); 3)
kynureninase
(EC 3.7.1.3); 4) tryptophanase (EC 4.1.99.1); 5) Trp aminotransferase (EC
2.6.1.27); 6)
indole lactate dehydrogenase (EC1.1.1.110); 7) Trp decarboxylase (EC
4.1.1.28); 8)
tryptamine oxidase (EC 1.4.3.4); 9) Trp side chain oxidase (EC 4.1.1.43); 10)
indole
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acetaldehyde dehydrogenase (EC 1.2.1.3); 11) indole acetic acid oxidase; 13)
Trp 2-
monooxygenase (EC 1.13.12.3); and 14) indole acetamide hydrolase (EC 3.5.1.0).
The
dotted lines ( __ ) indicate a spontaneous reaction. FIG. 37B Depicts a
schematic of
tryptophan derived pathways. Known AHR agonists are with asterisk.
Abbreviations are
as follows. Trp: Tryptophan; TrA: Tryptamine; IAAld: Indole-3-acetaldehyde;
IAA:
Indole-3-acetic acid; FICZ: 6-formylindolo(3,2-b)carbazole; IPyA: Indole-3-
pyruvic acid;
IAM: Indole-3-acetamine; IA0x: Indole-3-acetaldoxime; IAN: Indole-3-
acetonitrile; N-
formyl Kyn: N-formylkynurenine;; Kyn:Kynurenine; KynA: Kynurenic acid; I3C:
Indole-
3-carbinol; IAld: Indole-3-aldehyde; DIM: 3,3'-Diindolylmethane; ICZ:
Indolo(3,2-
b)carbazole. Enzymes are numbered as follows: 1. EC 1.13.11.11 (Tdo2, Bna2),
EC
1.13.11.11 (Idol); 2. EC 4.1.1.28 (Tdc); 3. EC 1.4.3.22, EC 1.4.3.4 (TynA); 4.
EC 1.2.1.3
(ladl), EC 1.2.3.7 (Aaol); 5. EC 3.5.1.9 (Afmid Bna3); 6. EC 2.6.1.7 (Cclbl,
Cc1b2,
Aadat, Got2); 7. EC 1.4.99.1 (TnaA); 8. EC 1.14.13.125 (CYP79B2, CYP79B3); 9.
EC
1.4.3.2 (Sta0), EC 2.6.1.27 (Aro9, aspC), EC 2.6.1.99 (Taal), EC 1.4.1.19
(TrpDH); 10.
EC 1.13.12.3 (laaM); 11. EC 4.1.1.74 (IpdC); 12. EC 1.14.13.168 (Yuc2); 13. EC
3.5.1.4
(IaaH); 14. EC 3.5.5.1. (Nitl); 15. EC 4.2.1.84 (Nitl); 16. EC 4.99.1.6
(CYP71A13); 17.
EC 3.2.1.147 (Pen2). In certain embodiments of the disclosure, the genetically
engineered
bacteria comprise gene cassettes comprising one or more of the bacterial
tryptophan
metabolism enzymes depicted in FIGs. 37A and 37B. In certain embodiments, the
genetically engineered bacteria comprise one or more gene cassettes which
produce one or
more of the metabolites depicted in FIGs. 37A and 37B. In certain embodiments,
the one
or more cassettes are on a plasmid; in other embodiments, the cassettes are
integrated into
the genome. In certain embodiments the one or more cassettes are under the
control of
inducible promoters which are induced under low-oxygen conditions, in the
presence of
certain molecules or metabolites, in the presence of molecules or metabolites
associated
with inflammation or an inflammatory response, or in the presence of some
other
metabolite that may or may not be present in the gut, such as arabinose.
[074] FIG. 38 depicts a schematic of the E. coli tryptophan synthesis pathway.
In
Escherichia coli, tryptophan is biosynthesized from chorismate, the principal
common
precursor of the aromatic amino acids tryptophan, tyrosine and phenylalanine,
as well as
the essential compounds tetrahydrofolate, ubiquinone-8, menaquinone-8 and
enterobactin
(enterochelin), as shown in the superpathway of chorismate metabolism. Five
genes
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encode five enzymes that catalyze tryptophan biosynthesis from chorismate. The
five
genes trpE trpD trpC trpB trpA form a single transcription unit, the trp
operon. A weak
internal promoter also exists within the trpD structural gene that provides
low, constitutive
levels of mRNA.
[075] FIG. 39 depicts one embodiment of the disclosure in which the E. coli
TRP
synthesis enzymes are expressed from a construct under the control of a
tetracycline
inducible system.
[076] FIG. 40A, FIG. 40B, FIG. 40C, and FIG. 40D depicts schematics of
exemplary embodiments of the disclosure, in which the genetically engineered
bacteria
comprise circuits for the production of tryptophan. Any of the gene(s), gene
sequence(s)
and/or gene circuit(s) or cassette(s) are optionally expressed from an
inducible promoter.
In certain embodiments the one or more cassettes are under the control of
constitutive
promoters. Exemplary inducible promoters which may control the expression of
the
gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen
level-
dependent promoters (e.g., FNR-inducible promoter), promoters induced by
inflammation
or an inflammatory response (RNS, ROS promoters), and promoters induced by a
metabolite that may or may not be naturally present (e.g., can be exogenously
added) in
the gut, e.g., arabinose and tetracycline. The bacteria may also include an
auxotrophy,
e.g., deletion of thyA (A thyA; thymidine dependence). FIG. 40A shows a
schematic
depicting an exemplary Tryptophan circuit. Tryptophan is produced from its
precursor,
chorismate, through expression of the trpE, trpG-D (also referred to as trpD),
trpC-F (also
referred to as trpC), trpB and trpA genes. Optional knockout of the tryptophan
repressor
trpR is also depicted. Optional production of chorismate through expression of
aroG/F/H
and aroB, aroD, aroE, aroK and aroC genes is also shown. The bacteria may
optionally
also include gene sequence(s) for the expression of YddG, which functions as a
tryptophan
exporter. The bacteria may optionally also comprise one or more gene
sequence(s)
depicted or described in FIG. 40B, and/or FIG. 40C, and/or FIG. 40D. FIG. 40B
depicts
a tryptophan producing strain, in which tryptophan is produced from the
chorismate
precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes.
AroG and
TrpE are replaced with feedback resistant versions to improve tryptophan
production.
Optionally, bacteria may comprise any of the transporters and/or additional
tryptophan
circuits depicted in FIG. 40A and/or described in the description of FIG. 40A.
The
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bacteria may optionally also comprise one or more gene sequence(s) depicted or
described
in FIG. 40C, and/or FIG. 40D. Optionally, trpR and/or the tnaA gene (encoding
a
tryptophanase converting tryptophan into indole) are deleted to further
increase levels of
tryptophan produced. FIG. 40C depicts a tryptophan producing strain, in which
tryptophan is produced from the chorismate precursor through expression of the
trpE,
trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback
resistant
versions to improve tryptophan production. The strain further comprises either
a wild type
or a feedback resistant SerA gene. Escherichia coli serA-encoded 3-
phosphoglycerate
(3PG) dehydrogenase catalyzes the first step of the major phosphorylated
pathway of L-
serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-
phosphohydroxypyruvate
(3PHP) with the concomitant reduction of NADI to NADH. E. coli uses one serine
for
each tryptophan produced. As a result, by expressing serA, tryptophan
production is
improved. Optionally, bacteria may comprise any of the transporters and/or
additional
tryptophan circuits depicted in FIG. 40A and/or described in the description
of FIG. 40A.
The bacteria may optionally also comprise one or more gene sequence(s)
depicted or
described in FIG. 40B, and/or FIG. 40D. Optionally, Trp Repressor and/or the
tnaA gene
are deleted to further increase levels of tryptophan produced. The bacteria
may optionally
also include gene sequence(s) for the expression of YddG, which functions as a
tryptophan
exporter. FIG. 40D depicts a non-limiting example of a tryptophan producing
strain, in
which tryptophan is produced from the chorismate precursor through expression
of the
trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with
feedback
resistant versions to improve tryptophan production. The strain further
optionally
comprises either a wild type or a feedback resistant SerA gene. Optionally,
bacteria may
comprise any of the transporters and/or additional tryptophan circuits
depicted in FIG.
40A and/or described in the description of FIG. 40A. The bacteria may
optionally also
comprise one or more gene sequence(s) depicted or described in FIG. 40B,
and/or FIG.
40C. Optionally, Trp Repressor and/or the tnaA gene are deleted to further
increase levels
of tryptophan produced. The bacteria may optionally also include gene
sequence(s) for
the expression of YddG, which functions as a tryptophan exporter. Optionally,
the bacteria
may also comprise a deletion in PheA, which prevents conversion of chorismate
into
phenylalanine and thereby promotes the production of anthranilate and
tryptophan.
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[077] FIG. 41A, FIG. 41B, FIG. 41D, FIG. 41D, FIG. 41E, FIG. 41F, FIG.
41G, and FIG. 41H depict schematics of non-limiting examples of embodiments of
the
disclosure. In all embodiments, optionally gene(s) which encode exporters may
also be
included. FIG. 41A depicts one embodiment of the disclosure, in which the
genetically
engineered bacteria produce tryptamine from tryptophan. In certain embodiments
the one
or more cassettes are under the control of inducible promoters. In certain
embodiments the
one or more cassettes are under the control of constitutive promoters. The
bacteria may
comprise any of the transporters and/or tryptophan circuits depicted and
described in FIG.
40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D for the
production of
tryptophan. Alternatively, optionally, tryptophan can be imported through a
transporter. In
addition, the genetically engineered bacteria comprise a circuit for
Tryptophan
decarboxylase, e.g., from Catharanthus roseus, which converts tryptophan to
tryptamine,
e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG.
41B depicts
one embodiment of the disclosure, in which the genetically engineered bacteria
produce
indole-3-acetaldehyde and FICZ from tryptophan. The bacteria may comprise any
of the
transporters and/or tryptophan circuits depicted and described in FIG. 40A
and/or FIG.
40B, and/or FIG. 40C, and/or FIG. 40D for the production of tryptophan.
Alternatively,
optionally, tryptophan can be imported through a transporter. In addition, the
genetically
engineered bacteria comprise a circuit for aro9 ( L-tryptophan
aminotransferase, e.g.,
from S. cerevisae) or aspC (aspartate aminotransferase, e.g., from E. coli, or
taal (L-
tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or sta0
(L-
tryptophan oxidase, e.g., from streptomyces sp. TP-A0274) or trpDH (Tryptophan
dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-
pyruvate
decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-
3-
acetaldehyde and FICZ from tryptophan, e.g., under the control of an inducible
promoter
e.g., an FNR promoter. FIG. 41C depicts one embodiment of the disclosure, in
which the
genetically engineered bacteria produce indole-3-acetaldehyde and FICZ from
tryptophan.
The bacteria may comprise any of the transporters and/or tryptophan circuits
depicted and
described in FIG. 40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D
for the
production of tryptophan. Alternatively, optionally, tryptophan can be
imported through a
transporter. In addition, the genetically engineered bacteria comprise a
circuit comprising
tdc (Tryptophan decarboxylase, e.g., from Catharanthus roseus and/or
Clostridium
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sporogenes), and tynA (Monoamine oxidase, e.g., from E. coli), which converts
tryptophan to indole-3-acetaldehyde and FICZ, e.g., under the control of an
inducible
promoter e.g., an FNR promoter. FIG. 41D depicts one embodiment of the
disclosure, in
which the genetically engineered bacteria produce indole-3-acetonitrile from
tryptophan.
The bacteria may comprise any of the transporters and/or tryptophan circuits
depicted and
described in FIG. 40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D
for the
production of tryptophan. Alternatively, optionally, tryptophan can be
imported through a
transporter. In addition, the genetically engineered bacteria comprise a
circuit for
cyp79B2, (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana) or
cyp79B3
(tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana), which together
convert
tryptophan to indole-3-acetonitrile, e.g., under the control of an inducible
promoter e.g., an
FNR promoter. FIG. 41E depicts one embodiment of the disclosure, in which the
genetically engineered bacteria produce kynurenine from tryptophan. The
bacteria may
comprise any of the transporters and/or tryptophan circuits depicted and
described in FIG.
40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D for the
production of
tryptophan. Alternatively, optionally, tryptophan can be imported through a
transporter. In
addition, the genetically engineered bacteria comprise a circuit comprising
ID01(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TD02 (tryptophan
2,3-
dioxygenase, e.g., from homo sapiens) or BNA2 (indoleamine 2,3-dioxygenase,
e.g.,
from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse) or
BNA3
(kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae) which
together convert
tryptophan to kynurenine, e.g., under the control of an inducible promoter
e.g., an FNR
promoter. FIG. 41F depicts one embodiment of the disclosure, in which the
genetically
engineered bacteria produce kynureninic acid from tryptophan. The bacteria may
comprise
any of the transporters and/or tryptophan circuits depicted and described in
FIG. 40A
and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D for the production of
tryptophan. Alternatively, optionally, tryptophan can be imported through a
transporter. In
addition, the genetically engineered bacteria comprise a circuit comprising
ID01(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TD02 (tryptophan
2,3-
dioxygenase, e.g., from homo sapiens) or BNA2 (indoleamine 2,3-dioxygenase,
e.g.,
from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse) or
BNA3
(kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae) and GOT2
(Aspartate
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aminotransferase, mitochondrial, e.g.,from homo sapiens or AADAT
(Kynurenine/alpha-
aminoadipate aminotransferase, mitochondrial, e.g., from homo sapiens), or
CCLB1
(Kynurenine--oxoglutarate transaminase 1, e.g., from homo sapiens) or CCLB2
(kynurenine--oxoglutarate transaminase 3, e.g., from homo sapiens, which
together
produce kynureninic acid from tryptophan, under the control of an inducible
promoter,
e.g., an FNR promoter. FIG. 41G depicts one embodiment of the disclosure, in
which the
genetically engineered bacteria produce indole from tryptophan. The bacteria
may
comprise any of the transporters and/or tryptophan circuits depicted and
described in FIG.
40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D for the
production of
tryptophan. Alternatively, optionally, tryptophan can be imported through a
transporter. In
addition, the genetically engineered bacteria comprise a circuit for tnaA
(tryptophanase,
e.g., from E. coli), which converts tryptophan to indole, e.g., under the
control of an
inducible promoter e.g., an FNR promoter. FIG. 41H depicts one embodiment of
the
disclosure, in which the genetically engineered bacteria produce indole-3-
carbinol, indole-
3-aldehyde, 3,3' diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from
indole
glucosinolate taken up through the diet. The genetically engineered bacteria
comprise a
circuit comprising pne2 (myrosinase, e.g., from Arabidopsis thaliana) under
the control of
an inducible promoter, e.g. an FNR promoter. The engineered bacterium shown in
any of
FIG. 41A, FIG. 41B, FIG. 41D, FIG. 41D, FIG. 41E, FIG. 41F, FIG. 41G and FIG.
41H may also have an auxotrophy, e.g., in one example, the thyA gene can be
been
mutated in the E. coli Nissle genome, so thymidine must be supplied in the
culture
medium to support growth.
[078] FIG. 42A, FIG. 42B, FIG. 42C, FIG. 42D, and FIG. 42E depict
schematics of exemplary embodiments of the disclosure, in which the
genetically
engineered bacteria convert tryptophan into indole-3-acetic acid. In certain
embodiments,
the one or more cassettes are under the control of inducible promoters. In
certain
embodiments, the one or more cassettes are under the control of constitutive
promoters. In
FIG. 42A, the optional circuits for tryptophan production are as depicted and
described in
FIG. 40A. The strain optionally comprises additional circuits as depicted
and/or described
in FIG. 40B and/or FIG. 40C and/or FIG. 40D. Alternatively, optionally,
tryptophan can
be imported through a transporter. In addition, the genetically engineered
bacteria
comprise a circuit comprising aro9 ( L-tryptophan aminotransferase, e.g., from
S.
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cerevisae) or aspC (aspartate aminotransferase, e.g., from E. coli, or taal (L-
tryptophan-
pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or sta0 (L-
tryptophan oxidase,
e.g., from streptomyces sp. TP-A0274) or trpDH (Tryptophan dehydrogenase,
e.g., from
Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g.,
from
Enterobacter cloacae) and iadl ( Indole-3-acetaldehyde dehydrogenase, e.g.,
from
Ustilago maydis) or AA01 (Indole-3-acetaldehyde oxidase, e.g., from
Arabidopsis
thaliana) which together produce indole-3-acetic acid from tryptophan, e.g.,
under the
control of an inducible promoter e.g., an FNR promoter. In FIG. 42B the
optional circuits
for tryptophan production are as depicted and described in FIG. 40A. The
strain optionally
comprises additional circuits as depicted and/or described in FIG. 40B and/or
FIG. 40C
and/or FIG. 40D. Alternatively, optionally, tryptophan can be imported through
a
transporter. In addition, the genetically engineered bacteria comprise a
circuit comprising
tdc (Tryptophan decarboxylase, e.g.,from Catharanthus roseus and/or
Clostridium
sporogenes) ot tynA (Monoamine oxidase, e.g., from E. coli) and or iadl
(Indole-3-
acetaldehyde dehydrogenase, e.g., from Ustilago maydis) or AA01 (Indole-3-
acetaldehyde oxidase, e.g., from Arabidopsis thaliana), e.g., under the
control of an
inducible promoter e.g., an FNR promoter. In FIG. 42C the optional circuits
for
tryptophan production are as depicted and described in FIG. 40A. The strain
optionally
comprises additional circuits as depicted and/or described in FIG. 40B and/or
FIG. 40C
and/or FIG. 40D. Alternatively, optionally, tryptophan can be imported through
a
transporter. In addition, the genetically engineered bacteria comprise a
circuit comprising
aro9 ( L-tryptophan aminotransferase, e.g., from S. cerevisae) or aspC
(aspartate
aminotransferase, e.g., from E. coli, or taal (L-tryptophan-pyruvate
aminotransferase,
e.g., from Arabidopsis thaliana) or sta0 (L-tryptophan oxidase, e.g., from
streptomyces
sp. TP-A0274) or trpDH (Tryptophan dehydrogenase, e.g., from Nostoc
punctiforme
NIES-2108) and yuc2 ( indole-3-pyruvate monoxygenase, e.g., from Arabidopsis
thaliana) e.g., under the control of an inducible promoter e.g., an FNR
promoter. In FIG.
42D the optional circuits for tryptophan production are as depicted and
described in FIG.
40A. The strain optionally comprises additional circuits as depicted and/or
described in
FIG. 40B and/or FIG. 40C and/or FIG. 40D. Alternatively, optionally,
tryptophan can be
imported through a transporter. In addition, the genetically engineered
bacteria comprise a
circuit comprising IaaM (Tryptophan 2-monooxygenase e.g., from Pseudomonas
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savastanoi) and iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas
savastanoi),
e.g., under the control of an inducible promoter e.g., an FNR promoter. In
FIG. 42E the
optional circuits for tryptophan production are as depicted and described in
FIG. 40A. The
strain optionally comprises additional circuits as depicted and/or described
in FIG. 40B
and/or FIG. 40C and/or FIG. 40D. Alternatively, optionally, tryptophan can be
imported
through a transporter. In addition, the genetically engineered bacteria
comprise a circuit
comprising cyp79B2 (tryptophan N-monooxygenase, e.g., from Arabidopsis
thaliana) or
cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana and
cyp71a13
(indoleacetaldoxime dehydratase, e.g., from Arabidopis thaliana) and nitl
(Nitrilase, e.g.,
from Arabidopsis thaliana) and iaaH (Indoleacetamide hydrolase, e.g., from
Pseudomonas
savastanoi), e.g., under the control of an inducible promoter e.g., an FNR
promoter. the
engineered bacterium shown in any of FIG. 42A, FIG. 42B, FIG. 42C, FIG. 42D,
and
FIG. 42E may also have an auxotrophy, e.g., in one example, the thyA gene can
be been
mutated in the E. coli Nissle genome, so thymidine must be supplied in the
culture
medium to support growth.
[079] In FIG. 42F the optional circuits for tryptophan production are as
depicted
and described in FIG. 40A. The strain optionally comprises additional circuits
as depicted
and/or described in FIG. 40B and/or FIG. 40C and/or FIG. 40D. Alternatively,
optionally, tryptophan can be imported through a transporter. Additionally,
the strain
comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-
2108)
and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae)
which
together produce indole-3-acetaldehyde and FICZ though an (indo1-3y1)pyruvate
intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from
Ustilago
maydis), which converts indole-3-acetaldehyde into indole-3-acetate.
[080] FIG. 43A, FIG. 43B, and FIG. 43C depict schematics of exemplary
embodiments of the disclosure, in which the genetically engineered bacteria
comprise
circuits for the production of tryptophan, tryptamine, indole acetic acid, and
indole
propionic acid. Any of the gene(s), gene sequence(s) and/or gene circuit(s) or
cassette(s)
are optionally expressed from an inducible promoter. In certain embodiments,
the one or
more cassettes are under the control of constitutive promoters. Exemplary
inducible
promoters which may control the expression of the gene(s), gene sequence(s)
and/or gene
circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR-
inducible
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promoter), promoters induced by inflammation or an inflammatory response (RNS,
ROS
promoters), and promoters induced by a metabolite that may or may not be
naturally
present (e.g., can be exogenously added) in the gut, e.g., arabinose and
tetracycline. The
bacteria may also include an auxotrophy, e.g., deletion of thyA (A thyA;
thymidine
dependence). FIG. 43A a depicts non-limiting example of a tryptamine producing
strain.
Tryptophan is optionally produced from chorismate precursor, and the strain
optionally
comprises circuits as depicted and/or described in FIG. 40A and/or FIG. 40B
and/or FIG.
40C and/or FIG. 40D. Additionally, the strain comprises tdc (tryptophan
decarboxylase,
e.g., from Catharanthus roseus and/or Clostridium sporogenes), which converts
tryptophan into tryptamine. FIG. 43B depicts a non-limiting example of an
indole-3-
acetate producing strain. Tryptophan is optionally produced from chorismate
precursor,
and the strain optionally comprises circuits as depicted and/or described in
FIG. 40A
and/or FIG. 40B and/or FIG. 40C and/or FIG. 40D. Additionally, the strain
comprises
trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and
ipdC
(Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which
together
produce indole-3-acetaldehyde and FICZ though an (indo1-3y1)pyruvate
intermediate, and
iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which
converts
indole-3-acetaldehyde into indole-3-acetate. FIG. 43C depicts a non-limiting
example of
an indole-3-propionate-producing strain. Tryptophan is optionally produced
from
chorismate precursor, and the strain optionally comprises circuits as depicted
and/or
described in FIG. 40A and/or FIG. 40B and/or FIG. 40C and/or FIG. 40D.
Additionally,
the strain comprises a circuit as described in FIG. 48, comprising trpDH
(Tryptophan
dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indo1-
3y1)pyruvate from tryptophan), fldA (indole-3-propionyl-CoA:indole-3-lactate
CoA
transferase, e.g., from Clostridium sporogenes, which converts converts indole-
3-lactate
and indo1-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-
CoA), fldB
and fldC (indole-3-lactate dehydratase e.g., from Clostridium sporogenes,
which converts
indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or AcuI: (indole-3-
acrylyl-CoA
reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase,
e.g., from
Rhodobacter sphaeroides, which convert indole-3-acrylyl-CoA to indole-3-
propionyl-
CoA). The circuits further comprisefldH/ and/or fldH2 (indole-3-lactate
dehydrogenase 1
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and/or 2, e.g., from Clostridium sporogenes), which converts (indo1-3-
yl)pyruvate into
indole-3-lactate).
[081] FIG. 44A and FIG. 44B depict schematics showing exemplary
engineering strategies which can be employed for tryptophan production. FIG.
44A depicts a schematic showing intermediates in tryptophan biosynthesis and
the gene
products catalyzing the production of these intermediates. Phosphoenolpyruvate
(PEP) and
D-erythrose 4-phosphate (E4P) are used to generate 3-deoxy-D-arabino-
heptulosonate 7-
phosphate (DAHP). DHAP is catabolized to chorismate and then anthranilate,
which is
converted to tryptophan (Trp) by the tryptophan operon. Alternatively,
chorismate can be
used in the synthesis of tyrosine (Tyr) and/or phenylalanine (Phe). In the
serine
biosynthesis pathway, D-3-phosphoglycerate is converted to serine, which can
also be a
source for tryptophan biosynthesis. AroG, AroF, AroH: DAHP synthase catalyzes
an aldol
reaction between phosphoenolpyruvate and D-erythrose 4-phosphate to generate 3-
deoxy-
D-arabino-heptulosonate 7-phosphate (DAHP). There are three isozymes of DAHP
synthase, each specifically feedback regulated by tyrosine (AroF),
phenylalanine (AroG)
or tryptophan(AroH). AroB: Dehydroquinate synthase (DHQ synthase) is involved
in the
second step of the chorismate pathway, which leads to the biosynthesis of
aromatic amino
acids. DHQ synthase catalyzes the cyclization of 3-deoxy-D-arabino-
heptulosonic acid 7-
phosphate (DAHP) to dehydroquinate (DHQ). AroD: 3-Dehydroquinate dehydratase
(DHQ dehydratase) is involved in the 3rd step of the chorismate pathway, which
leads to
the biosynthesis of aromatic amino acids. DHQ dehydratase catalyzes the
conversion of
DHQ to 3-dehydroshikimate and introduces the first double bond of the aromatic
ring.
AroE, YdiB: E. coli expresses two shikimate dehydrogenase paralogs, AroE and
YdiB.
Shikimate dehydrogenase is involved in the 4th step of the chorismate pathway,
which
leads to the biosynthesis of aromatic amino acids. This enzyme converts 3-
dehydroshikimate to shikimate by catalyzing the NADPH linked reduction of 3-
dehydro-
shikimate. AroL/AroK: Shikimate kinase is involved in the fifth step of the
chorismate
pathway, which leads to the biosynthesis of aromatic amino acids. Shikimate
kinase
catalyzes the formation of shikimate 3-phosphate from shikimate and ATP. There
are two
shikimate kinase enzymes, I (AroK) and II (AroL). AroA: 3-Phosphoshikimate-1-
carboxyvinyltransferase (EPSP synthase) is involved in the 6th step of the
chorismate
pathway, which leads to the biosynthesis of aromatic amino acids. EPSP
synthase
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catalyzes the transfer of the enolpyruvoyl moiety from phosphoenolpyruvate to
the
hydroxyl group of carbon 5 of shikimate 3-phosphate with the elimination of
phosphate to
produce 5-enolpyruvoyl shikimate 3-phosphate (EPSP). AroC: Chorismate synthase
(AroC) is involved in the 7th and last step of the chorismate pathway, which
leads to the
biosynthesis of aromatic amino acids. This enzyme catalyzes the conversion of
5-
enolpyruvylshikimate 3-phosphate into chorismate, which is the branch point
compound
that serves as the starting substrate for the three terminal pathways of
aromatic amino acid
biosynthesis. This reaction introduces a second double bond into the aromatic
ring system.
TrpEDCAB (E coli trp operon): TrpE (anthranilate synthase) converts chorismate
and L-
glutamine into anthranilate, pyruvate and L-glutamate. Anthranilate
phosphoribosyl
transferase (TrpD) catalyzes the second step in the pathway of tryptophan
biosynthesis.
TrpD catalyzes a phosphoribosyltransferase reaction that generates N-(5'-
phosphoribosyl)-
anthranilate. The phosphoribosyl transferase and anthranilate synthase
contributing
portions of TrpD are present in different portions of the protein.
Bifunctional
phosphoribosylanthranilate isomerase / indole-3-glycerol phosphate synthase
(TrpC)
carries out the third and fourth steps in the tryptophan biosynthesis pathway.
The
phosphoribosylanthranilate isomerase activity of TrpC catalyzes the Amadori
rearrangement of its substrate into carboxyphenylaminodeoxyribulo se
phosphate. The
indole-glycerol phosphate synthase activity of TrpC catalyzes the ring closure
of this
product to yield indole-3-glycerol phosphate. The TrpA polypeptide (TSase a)
functions
as the a subunit of the tetrameric (a2-02) tryptophan synthase complex. The
TrpB
polypeptide functions as the 0 subunit of the complex, which catalyzes the
synthesis of L-
tryptophan from indole and L-serine, also termed the 0 reaction. TnaA:
Tryptophanase or
tryptophan indole-lyase (TnaA) is a pyridoxal phosphate (PLP)-dependent enzyme
that
catalyzes the cleavage of L-tryptophan to indole, pyruvate and NH4+. PheA:
Bifunctional
chorismate mutase / prephenate dehydratase (PheA) carries out the shared first
step in the
parallel biosynthetic pathways for the aromatic amino acids tyrosine and
phenylalanine, as
well as the second step in phenylalanine biosynthesis. TyrA: Bifunctional
chorismate
mutase / prephenate dehydrogenase (TyrA) carries out the shared first step in
the parallel
biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine,
as well as
the second step in tyrosine biosynthesis. TyrB, ilvE, AspC: Tyrosine
aminotransferase
(TyrB), also known as aromatic-amino acid aminotransferase, is a broad-
specificity
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enzyme that catalyzes the final step in tyrosine, leucine, and phenylalanine
biosynthesis.
TyrB catalyzes the transamination of 2-ketoisocaproate, p-
hydroxyphenylpyruvate, and
phenylpyruvate to yield leucine, tyrosine, and phenylalanine, respectively.
TyrB overlaps
with the catalytic activities of branched-chain amino-acid aminotransferase
(IlvE), which
also produces leucine, and aspartate aminotransferase, PLP-dependent (AspC),
which also
produces phenylalanine. SerA: D-3-phosphoglycerate dehydrogenase catalyzes the
first
committed step in the biosynthesis of L-serine. SerC: The serC-encoded enzyme,
phosphoserine/phosphohydroxythreonine aminotransferase, functions in the
biosythesis of
both serine and pyridoxine, by using different substrates. Pyridoxal 5'-
phosphate is a
cofactor for both enzyme activities. SerB: Phosphoserine phosphatase catalyzes
the last
step in serine biosynthesis. Steps which are negatively regulated by the Trp
Repressor (2),
Tyr Repressor (1), or tyrosine (3), phenylalanine (4), or tryptophan (4) or
positively
regulated by trptophan (6) are indicated. FIG. 44B depicts a schematic showing
exemplary engineering strategies which can improve tryptophan production. Each
of these
exemplary strategies can be used alone or two or more strategies can be
combined to
increase tryptophan production. Intervention points are in bold, italics and
underlined. In
one embodiment of the disclosure, bacteria are engineered to express a
feedback resistant
from of AroG (AroGfbr). In one embodiment, bacteria are engineered to express
AroL. In
one embodiment, bacteria are engineered to comprise one or more copies of a
feedback
resistant form of TrpE (TrpEfbr). In one embodiment, bacteria are engineered
to comprise
one or more additional copies of the Trp operon, e.g., TrpE, e.g. TrpEtbr,
and/or TrpD,
and/or TrpC, and/or TrpA, and/or TrpB. In one embodiment, endogenous TnaA is
knocked out through mutation(s) and/or deletion(s). In one embodiment,
bacteria are
engineered to comprise one or more additional copies of SerA. In one
embodiment,
bacteria are engineered to comprise one or more additional copies of YddG, a
tryptophan
exporter. In one embodiment, endogenous PheA is knocked out through
mutation(s)
and/or deletion(s). In one embodiment, two or more of the strategies depicted
in the
schematic of FIG. 44B are engineered into a bacterial strain. Alternatively,
other gene
products in this pathway may be mutated or overexpressed.
[082] FIG.45A and FIG. 45B and FIG. 45C depict bar graphs showing
tryptophan production by various engineered bacterial strains. FIG.45A depicts
a bar
graph showing tryptophan production by various tryptophan producing strains.
The data
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show expressing a feedback resistant form of AroG (Arodbl.) is necessary to
get
tryptophan production. Additionally, using a feedback resistant trpE (trpE
tbr) has a
positive effect on tryptophan production. FIG. 45B shows tryptophan production
from a
strain comprising a tet-trpEtbrDCBA, tet-aroGthr construct, comparing glucose
and
glucuronate as carbon sources in the presence and absence of oxygen. It takes
E. coli two
molecules of phosphoenolpyruvate (PEP) to produce one molecule of tryptophan.
When
glucose is used as the carbon source, 50% of all available PEP is used to
import glucose
into the cell through the PTS system (Phosphotransferase system). Tryptophan
production
is improved by using a non-PTS sugar (glucuronate) aerobically. The data also
show the
positive effect of deleting tnaA (only at early time point aerobically). FIG.
45C depicts a
bar graph showing improved tryptophan production by engineered strain
comprising
AtrpRAtnaA, tet-trperDCBA, tet-arodbr through the addition of serine.
[083] FIG. 46 depicts a bar graph showing a comparison in tryptophan
production in strains SYN2126, SYN2323, SYN2339, SYN2473, and SYN2476.
SYN2126 AtrpRAtnaA. AtrpRAtnaA, tet-aroGfbr. SYN2339 comprises AtrpRAtnaA, tet-
aroGfbr, tet-trpEfbrDCBA. SYN2473 comprises AtrpRAtnaA, tet-aroGthr-serA, tet-
trpEfbrDCBA. SYN2476 comprises AtrpRAtnaA, tet-trpEtbrDCBA. Results indicate
that
expressing aroG is not sufficient nor necessary under these conditions to get
Trp
production and that expressing serA is beneficial for tryptophan production.
[084] FIG. 47 depicts a schematic of an indole-3-propionic acid (IPA)
synthesis
circuit. IPA produced by the gut microbiota has a significant positive effect
on barrier
integrity. IPA does not signal through AhR, but rather through a different
receptor (PXR)
(Venkatesh et al., Symbiotic Bacterial Metabolites Regulate Gastrointestinal
Bardrier
Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41,
296-310,
August 21, 2014). In some embodiments, IPA can be produced in a synthetic
circuit by
expressing two enzymes, a tryptophan ammonia lyase and an indole-3-acrylate
reductase
(e.g., Tryptophan ammonia lyase (WAL) (e.g., from Rubrivivax benzoatilyticus)
and
indole-3-acrylate reductase (e.g., from Clostridum botulinum). Tryptophan
ammonia lyase
converts tryptophan to indole-3-acrylic acid, and indole-3-acrylate reductase
converts
indole-3-acrylic acid into IPA. Without wishing to be bound by theory, no
oxygen is
needed for this reaction, allowing it to proceed under low or no oxygen
conditions, e.g., as
those found in the mammalian gut. In some embodiments, the genetically
engineered
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bacteria further comprise one or more circuits for the production of
tryptophan, e.g., as
shown in FIG. 40 (A-D) and FIG. 44 and as described elsewhere herein. In some
embodiments, AroG and/or TrpE are replaced with feedback resistant versions to
improve
tryptophan production in the genetically engineered bacteria. In some
embodiments, trpR
and/or the tnaA gene (encoding a tryptophanase converting tryptophan into
indole) are
deleted to further increase levels of tryptophan produced.
[085] FIG. 48 depicts a schematic of indole-3-propionic acid (IPA), indole
acetic
acid (IAA), and tryptamine synthesis(TrA) circuits. Enzymes are as follows :
1. TrpDH:
tryptophan dehydrogenase, e.g., from from Nostoc punctiforme NIES-2108;
FldHl/F1dH2: indole-3-lactate dehydrogenase, e.g., from Clostridium
sporogenes; FldA:
indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from
Clostridium
sporogenes; FldBC: indole-3-lactate dehydratase, e.g., from Clostridium
sporogenes;
FldD: indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes; AcuI:
acrylyl-
CoA reductase, e.g., from Rhodobacter sphaeroides. 1pdC: Indole-3-pyruvate
decarboxylase, e.g., from Enterobacter cloacae; lad 1: Indole-3-acetaldehyde
dehydrogenase, e.g., from Ustilago maydis; Tdc: Tryptophan decarboxylase,
e.g., from
Catharanthus roseus or from Clostridium sporogenes.
[086] Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the
reversible chemical reaction converting L-tryptophan, NAD(P) and water to
(indo1-3-
yl)pyruvate (IPyA), NH3, NAD(P)H and H. Indole-3-lactate dehydrogenase ((EC
1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) converts
(indo1-
3y1)pyruvate (IpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+. Indole-
3-
propionyl-CoA:indole-3-lactate CoA transferase (F1dA ) converts indole-3-
lactate (ILA)
and indo1-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-
lactate-CoA.
Indole-3-acrylyl-CoA reductase (F1dD ) and acrylyl-CoA reductase (AcuI)
convert indole-
3-acrylyl-CoA to indole-3-propionyl-CoA. Indole-3-lactate dehydratase (FldBC )
converts
indole-3-lactate-CoA to indole-3 -acrylyl-CoA. Indole-3-pyruvate decarboxylase
(1pdC:)
converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAA1d) ladl:
Indole-3-
acetaldehyde dehydrogenase coverts Indole-3-acetaldehyde (IAA1d) into Indole-3-
acetic
acid (IAA) Tdc: Tryptophan decarboxylase converts tryptophan (Trp) into
tryptamine
(TrA). In some embodiments, the genetically engineered bacteria further
comprise one or
more circuits for the production of tryptophan, e.g., as shown in FIG. 40 (A-
D) and FIG.
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44 and as described elsewhere herein. In some embodiments, AroG and/or TrpE
are
replaced with feedback resistant versions to improve tryptophan production in
the
genetically engineered bacteria. In some embodiments, trpR and/or the tnaA
gene
(encoding a tryptophanase converting tryptophan into indole) are deleted to
further
increase levels of tryptophan produced.
[087] FIG. 49 depicts a bar graph showing tryptophan and indole acetic acid
production for strains SYN2126, SYN2339 and SYN2342. SYN2126: comprises AtrpR
and AtnaA (AtrpRAtnaA). SYN2339 comprises circuitry for the production of
tryptophan
(AtrpRAtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr (p15A)).
SYN2342
comprises the same tryptophan production circuitry as the parental strain
SYN2339, and
additionally comprises ipdC-iadl incorporated at the end of the second
construct
(AtrpRAtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr-trpDH-ipdC-iadl
(p15A)). SYN2126 produced no tryptophan, SYN2339 produces increasing
tryptophan
over the time points measured, and SYN2342 converts all trypophan it produces
into IAA.
[088] FIG. 50 depicts a bar graph showing tryptophan and tryptamine production
for strains SYN2339, SYN2340, and SYN2794. SYN2339 is used as a control which
can
produce tryptophan but cannot convert it to tryptamine and comprises
AtrpRAtnaA, tetR-
Ptet-trpEtbrDCBA (pSC101), tetR-PteraroGthr (p15A). SYN2340 comprises
AtrpRAtnaA,
tetR-Ptet-trpEtbrDCBA (pSC101), tetR-PteraroGtk-tdcc, (p15A). SYN2794
comprises
AtrpRAtnaA, tetR-Ptet-trpEtbrDCBA (pSC101), tetR-PteraroGthr-tdccs (p15A).
Results
indicate that Tdccs from Clostridium sporo genes is more efficient the Tdcc,
from
Catharanthus roseus in tryptamine production and converts all the tryptophan
produced
into tryptamine.
[089] FIG. 51A, FIG. 51B, FIG. 51C, FIG. 51D, FIG. 51E depict schematics of
non-limiting examples of genetically engineered bacteria of the disclosure
which
comprises one or more gene sequence(s) and/or gene cassette(s) as described
herein.
[090] FIG. 52 depicts a map of integration sites within the E. coil Nissle
chromosome. These sites indicate regions where circuit components may be
inserted into
the chromosome without interfering with essential gene expression. Backslashes
(/) are
used to show that the insertion will occur between divergently or convergently
expressed
genes. Insertions within biosynthetic genes, such as thyA, can be useful for
creating
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nutrient auxotrophies. In some embodiments, an individual circuit component is
inserted
into more than one of the indicated sites.
[091] FIG. 53 depicts an exemplary schematic of the E. coli 1917 Nissle
chromosome comprising multiple mechanisms of action (MoAs).
[092] FIG. 54A and FIG. 54B depict schematics of bacterial chromosomes, for
example the E. coli Nissle 1917 Chromosome. For example, FIG. 54A depicts a
schematic of an engineered bacterium comprising, a circuit for butyrate
production, a
circuit for propionate production, and a circuit for production of one or more
interleukins
relevant to IBD. Fig. 54B depicts a schematic of an engineered bacterium
comprising
three circuits, a circuit for butyrate production, a circuit for GLP-2
expression and and a
circuit for production of one or more interleukins relevant to IBD.
[093] FIG. 55 depicts a schematic of a secretion system based on the flagellar
type III secretion in which an incomplete flagellum is used to secrete a
therapeutic peptide
of interest (star) by recombinantly fusing the peptide to an N-terminal
flagellar secretion
signal of a native flagellar component so that the intracellularly expressed
chimeric
peptide can be mobilized across the inner and outer membranes into the
surrounding host
environment.
[094] FIG. 56 depicts a schematic of a type V secretion system for the
extracellular production of recombinant proteins in which a therapeutic
peptide (star) can
be fused to an N-terminal secretion signal, a linker and the beta-domain of an
autotransporter. In this system, the N-terminal signal sequence directs the
protein to the
SecA-YEG machinery which moves the protein across the inner membrane into the
periplasm, followed by subsequent cleavage of the signal sequence. The beta-
domain is
recruited to the Bam complex where the beta-domain is folded and inserted into
the outer
membrane as a beta-barrel structure. The therapeutic peptide is then thread
through the
hollow pore of the beta-barrel structure ahead of the linker sequence. The
therapeutic
peptide is freed from the linker system by an autocatalytic cleavage or by
targeting of a
membrane-associated peptidase (scissors) to a complementary protease cut site
in the
linker.
[095] FIG. 57 depicts a schematic of a type I secretion system, which
translocates a passenger peptide directly from the cytoplasm to the
extracellular space
using HlyB (an ATP-binding cassette transporter); HlyD (a membrane fusion
protein); and
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To1C (an outer membrane protein) which form a channel through both the inner
and outer
membranes. The secretion signal-containing C-terminal portion of HlyA is fused
to the C-
terminal portion of a therapeutic peptide (star) to mediate secretion of this
peptide.
[096] FIG. 58 depicts a schematic of the outer and inner membranes of a gram-
negative bacterium, and several deletion targets for generating a leaky or
destabilized
outer membrane, thereby facilitating the translocation of a therapeutic
polypeptides to the
extracellular space, e.g., therapeutic polypeptides of eukaryotic origin
containing
disulphide bonds. Deactivating mutations of one or more genes encoding a
protein that
tethers the outer membrane to the peptidoglycan skeleton, e.g., 1pp, ompC,
ompA, ompF,
tolA, to1B, pal, and/or one or more genes encoding a periplasmic protease,
e.g., degS,
degP, nlpl, generates a leaky phenotype. Combinations of mutations may
synergistically
enhance the leaky phenotype.
[097] FIG. 59 depicts a modified type 3 secretion system (T3SS) to allow the
bacteria to inject secreted therapeutic proteins into the gut lumen. An
inducible promoter
(small arrow, top), e.g. a FNR-inducible promoter, drives expression of the T3
secretion
system gene cassette (3 large arrows, top) that produces the apparatus that
secretes tagged
peptides out of the cell. An inducible promoter (small arrow, bottom), e.g. a
FNR-
inducible promoter, drives expression of a regulatory factor, e.g. T7
polymerase, that then
activates the expression of the tagged therapeutic peptide (hexagons).
[098] FIGs. 60A- 60C depict other non-limiting embodiments of the disclosure,
wherein the expression of a heterologous gene is activated by an exogenous
environmental
signal. In the absence of arabinose, the AraC transcription factor adopts a
conformation
that represses transcription. In the presence of arabinose, the AraC
transcription factor
undergoes a conformational change that allows it to bind to and activate the
ParaBAD
promoter (P
\ - araBAD), which induces expression of the Tet repressor (TetR) and an anti-
toxin.
The anti-toxin builds up in the recombinant bacterial cell, while TetR
prevents expression
of a toxin (which is under the control of a promoter having a TetR binding
site). However,
when arabinose is not present, both the anti-toxin and TetR are not expressed.
Since TetR
is not present to repress expression of the toxin, the toxin is expressed and
kills the cell.
FIG. 60A also depicts another non-limiting embodiment of the disclosure,
wherein the
expression of an essential gene not found in the recombinant bacteria is
activated by an
exogenous environmental signal. In the absence of arabinose, the AraC
transcription
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factor adopts a conformation that represses transcription of the essential
gene under the
control of the araBAD promoter and the bacterial cell cannot survive. In the
presence of
arabinose, the AraC transcription factor undergoes a conformational change
that allows it
to bind to and activate the araBAD promoter, which induces expression of the
essential
gene and maintains viability of the bacterial cell. FIG. 60B depicts a non-
limiting
embodiment of the disclosure, where an anti-toxin is expressed from a
constitutive
promoter, and expression of a heterologous gene is activated by an exogenous
environmental signal. In the absence of arabinose, the AraC transcription
factor adopts a
conformation that represses transcription. In the presence of arabinose, the
AraC
transcription factor undergoes a conformational change that allows it to bind
to and
activate the araBAD promoter, which induces expression of TetR, thus
preventing
expression of a toxin. However, when arabinose is not present, TetR is not
expressed, and
the toxin is expressed, eventually overcoming the anti-toxin and killing the
cell. The
constitutive promoter regulating expression of the anti-toxin should be a
weaker promoter
than the promoter driving expression of the toxin. The araC gene is under the
control of a
constitutive promoter in this circuit. FIG. 60C depicts another non-limiting
embodiment
of the disclosure, wherein the expression of a heterologous gene is activated
by an
exogenous environmental signal. In the absence of arabinose, the AraC
transcription
factor adopts a conformation that represses transcription. In the presence of
arabinose, the
AraC transcription factor undergoes a conformational change that allows it to
bind to and
activate the araBAD promoter, which induces expression of the Tet repressor
(TetR) and
an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell,
while TetR
prevents expression of a toxin (which is under the control of a promoter
having a TetR
binding site). However, when arabinose is not present, both the anti-toxin and
TetR are
not expressed. Since TetR is not present to repress expression of the toxin,
the toxin is
expressed and kills the cell. The araC gene is either under the control of a
constitutive
promoter or an inducible promoter (e.g., AraC promoter) in this circuit.
[099] FIG. 61 depicts one non-limiting embodiment of the disclosure, where an
exogenous environmental condition or one or more environmental signals
activates
expression of a heterologous gene and at least one recombinase from an
inducible
promoter or inducible promoters. The recombinase then flips a toxin gene into
an
activated conformation, and the natural kinetics of the recombinase create a
time delay in
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expression of the toxin, allowing the heterologous gene to be fully expressed.
Once the
toxin is expressed, it kills the cell.
[0100] FIG. 62 depicts another non-limiting embodiment of the disclosure,
where
an exogenous environmental condition or one or more environmental signals
activates
expression of a heterologous gene, an anti-toxin, and at least one recombinase
from an
inducible promoter or inducible promoters. The recombinase then flips a toxin
gene into
an activated conformation, but the presence of the accumulated anti-toxin
suppresses the
activity of the toxin. Once the exogenous environmental condition or cue(s) is
no longer
present, expression of the anti-toxin is turned off. The toxin is
constitutively expressed,
continues to accumulate, and kills the bacterial cell.
[0101] FIG. 63 depicts another non-limiting embodiment of the disclosure,
where
an exogenous environmental condition or one or more environmental signals
activates
expression of a heterologous gene and at least one recombinase from an
inducible
promoter or inducible promoters. The recombinase then flips at least one
excision enzyme
into an activated conformation. The at least one excision enzyme then excises
one or
more essential genes, leading to senescence, and eventual cell death. The
natural kinetics
of the recombinase and excision genes cause a time delay, the kinetics of
which can be
altered and optimized depending on the number and choice of essential genes to
be
excised, allowing cell death to occur within a matter of hours or days. The
presence of
multiple nested recombinases can be used to further control the timing of cell
death.
[0102] FIG. 64 depicts one non-limiting embodiment of the disclosure, where an
exogenous environmental condition or one or more environmental signals
activates
expression of a heterologous gene and a first recombinase from an inducible
promoter or
inducible promoters. The recombinase then flips a second recombinase from an
inverted
orientation to an active conformation. The activated second recombinase flips
the toxin
gene into an activated conformation, and the natural kinetics of the
recombinase create a
time delay in expression of the toxin, allowing the heterologous gene to be
fully
expressed. Once the toxin is expressed, it kills the cell.
[0103] FIG. 65 depicts the use of GeneGuards as an engineered safety
component.
All engineered DNA is present on a plasmid which can be conditionally
destroyed. See,
e.g., Wright et al., "GeneGuard: A Modular Plasmid System Designed for
Biosafety,"
ACS Synthetic Biology (2015) 4: 307-316.
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[0104] FIG. 66 depicts P-galactosidase levels in samples comprising bacteria
harboring a low-copy plasmid expressing lacZ from an FNR-responsive promoter
selected
from the exemplary FNR promoters shown in the tables (Pfnr1-5). Different FNR-
responsive promoters were used to create a library of anaerobic-inducible
reporters with a
variety of expression levels and dynamic ranges. These promoters included
strong
ribosome binding sites. Bacterial cultures were grown in either aerobic (+02)
or anaerobic
conditions (-02). Samples were removed at 4 hrs and the promoter activity
based on f3-
galactosidase levels was analyzed by performing standard P-galactosidase
colorimetric
assays.
[0105] FIGs. 67A-67C depict a schematic representation of the lacZ gene under
the control of an exemplary FNR promoter (Pfnrs) and corresponding graphical
data.
FIGs. 67A depicts a schematic representation of the lacZ gene under the
control of an
exemplary FNR promoter (Pfnrs). LacZ encodes the P-galactosidase enzyme and is
a
common reporter gene in bacteria. FIG. 67B depicts FNR promoter activity as a
function
of P-galactosidase activity in 5YN340. 5YN340, an engineered bacterial strain
harboring
a low-copy fnrS-lacZ fusion gene, was grown in the presence or absence of
oxygen.
Values for standard P-galactosidase colorimetric assays are expressed in
Miller units
(Miller, 1972). These data suggest that the fnrS promoter begins to drive high-
level gene
expression within 1 hr under anaerobic conditions. FIG. 67C depicts the growth
of
bacterial cell cultures expressing lacZ over time, both in the presence and
absence of
oxygen.
[0106] FIGs. 68A-68D depict bar graphs, schematic, and dot blot, respectively,
showing the structure or activity of reporter constructs. FIG. 68A and FIG.
68B depict
bar graphs of reporter constructs activity. FIG. 68A depicts a graph of an ATC-
inducible
reporter construct expression and FIG. 68B depicts a graph of a nitric oxide-
inducible
reporter construct expression. These constructs, when induced by their cognate
inducer,
lead to expression of GFP. Nissle cells harboring plasmids with either the
control, ATC-
inducible Ptet-GFP reporter construct or the nitric oxide inducible P
- nsr12.-GFP reporter
construct induced across a range of concentrations. Promoter activity is
expressed as
relative florescence units. FIG. 68C depicts a schematic of the constructs.
FIG. 68D
depicts a dot blot of bacteria harboring a plasmid expressing NsrR under
control of a
constitutive promoter and the reporter gene gfp (green fluorescent protein)
under control
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of an NsrR-inducible promoter. DSS-treated mice serve as exemplary models for
HE. As
in HE subjects, the guts of mice are damaged by supplementing drinking water
with 2-3%
dextran sodium sulfate (DSS). Chemiluminescent is shown for NsrR-regulated
promoters
induced in DSS-treated mice.
[0107] FIG. 69 depicts a graph of Nissle residence in vivo. Streptomycin-
resistant
Nissle was administered to mice via oral gavage without antibiotic pre-
treatment. Fecal
pellets from 6 total mice were monitored post-administration to determine the
amount of
administered Nissle still residing within the mouse gastrointestinal tract.
The bars
represent the number of bacteria administered to the mice. The line represents
the number
of Nissle recovered from the fecal samples each day for 10 consecutive days.
[0108] FIG. 70 depicts a bar graph of residence over time for streptomycin
resistant Nissle in various compartments of the intestinal tract at 1, 4, 8,
12, 24, and 30
hours post gavage. Mice were treated with approximately 109 CFU, and at each
timepoint,
animals (n=4) were euthanized, and intestine, cecum, and colon were removed.
The small
intestine was cut into three sections, and the large intestine and colon each
into two
sections. Intestinal effluents gathered and CFUs in each compartment were
determined by
serial dilution plating.
[0109] FIG. 71A and FIG. 71B depict a schematic diagrams of a wild-type clbA
construct (FIG. 71A) and a schematic diagram of a clbA knockout construct
(FIG. 71B).
[0110] FIG. 72 depicts a schematic of a design-build-test cycle. Steps are as
follows: 1: Define the disease pathway; 2. Identify target metabolites; 3.
Design genetic
circuits; 4. Build synthetic biotic; 5. Activate circuit in vivo; 6.
Characterize circuit
activation kinetics; 7. Optimize in vitro productivity to disease threshold;
8. Test optimize
circuit in animla disease model; 9. Assimilate into the microbiome; 10.
Develop
understanding of in vivo PK and dosing regimen.
[0111] FIG. 73 depicts a schematic of non-limiting manufacturing processes for
upstream and downstream production of the genetically engineered bacteria of
the present
disclosure. Step 1 depicts the parameters for starter culture 1 (SC1): loop
full ¨ glycerol
stock, duration overnight, temperature 37 C, shaking at 250 rpm. Step 2
depicts the
parameters for starter culture 2 (5C2): 1/100 dilution from SC1, duration 1.5
hours,
temperature 37 C, shaking at 250 rpm. Step 3 depicts the parameters for the
production
bioreactor: inoculum ¨ 5C2, temperature 37 C, pH set point 7.00, pH dead band
0.05,
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dissolved oxygen set point 50%, dissolved oxygen cascade agitation/gas FLO,
agitation
limits 300-1200 rpm, gas FLO limits 0.5-20 standard liters per minute,
duration 24 hours.
Step 4 depicts the parameters for harvest: centrifugation at speed 4000 rpm
and duration
30 minutes, wash 1X 10% glycerol/PBS, centrifugation, re-suspension 10%
glycerol/PBS.
Step 5 depicts the parameters for vial fill/storage: 1-2 mL aliquots, -80 C.
[0112] Fig. 74 depicts three bacterial strains which constitutively express
red
fluorescent protein (RFP). In strains 1-3, the rfp gene has been inserted into
different sites
within the bacterial chromosome, and results in varying degrees of brightness
under
fluorescent light. Unmodified E. coli Nissle (strain 4) is non-fluorescent.
[0113] Fig. 75A depicts a graph showing bacterial cell growth of a Nissle thyA
auxotroph strain (thyA knock-out) in various concentrations of thymidine. A
chloramphenicol-resistant Nissle thyA auxotroph strain was grown overnight in
LB +
10mM thymidine at 37C. The next day, cells were diluted 1:100 in 1 mL LB +
10mM
thymidine, and incubated at 37C for 4 hours. The cells were then diluted 1:100
in 1 mL
LB + varying concentrations of thymidine in triplicate in a 96-well plate. The
plate is
incubated at 37C with shaking, and the 0D600 is measured every 5 minutes for
720
minutes. This data shows that Nissle thyA auxotroph does not grow in
environments
lacking thymidine.
[0114] Fig. 75B depicts a bar graph of Nissle residence in vivo of wildtype
Nissle
versus Nissle thyA auxotroph (thyA knock-out). Streptomycin- resistant Nissle
(wildtype
or thyA auxotroph) was administered to mice via oral gavage without antibiotic
pre-
treatment. Fecal pellets from 6 total mice were monitored post-administration
to
determine the amount of administered Nissle still residing within the mouse
gastrointestinal tract. Each bar represents the number of Nissle recovered
from the fecal
samples each day for 7 consecutive days. There were no bacteria recovered in
fecal
samples from mice gavaged with Nissle thyA auxotroph bacteria after day 3.
This data
shows that the Nissle thyA auxotroph does not persist in vivo in mice.
[0115] Fig. 76 depicts a one non-limiting embodiment of the disclosure, which
comprises a plasmid stability system with a plasmid that produces both a short-
lived anti-
toxin and a long-lived toxin. When the cell loses the plasmid, the anti-toxin
is no longer
produced, and the toxin kills the cell. In one embodiment, the genetically
engineered
bacteria produce an equal amount of a Hok toxin and a short-lived Sok
antitoxin. In the
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upper panel, the cell produces equal amounts of toxin and anti-toxin and is
stable. In the
center panel, the cell loses the plasmid and anti-toxin begins to decay. In
the lower panel,
the anti-toxin decays completely, and the cell dies.
[0116] Figs. 77A-77D depict schematics of non-limiting examples of the gene
organization of plasmids, which function as a component of a bio safety system
(Fig. 77A
and Fig. 77B), which also contains a chromosomal component (shown in Fig. 77C
and
Fig. 77D). The bosafety plasmid system vector comprises Kid Toxin and R6K
minimal
ori, dapA (Fig. 77A) and thyA (Fig. 77B) and promoter elements driving
expression of
these components. In some embodiments, bla is knocked out and replaced with
one or
more constructs described herein, in which a first protein of interest (POI1)
and/or a
second protein of interest, e.g., a transporter (P0I2), and/or a third protein
of interest
(P0I3) are expressed from an inducible or constitutive promoter. Fig. 77C and
Fig. 77D
depict schematics of the gene organization of the chromosomal component of a
biosafety
system. Fig. 77C depicts a construct comprising low copy Rep (Pi) and Kis
antitoxin, in
which transcription of Pi (Rep), which is required for the replication of the
plasmid
component of the system, is driven by a low copy RBS containing promoter. Fig.
77D
depicts a construct comprising a medium-copy Rep (Pi) and Kis antitoxin, in
which
transcription of Pi (Rep), which is required for the replication of the
plasmid component of
the system, is driven by a medium copy RBS containing promoter. If the plasmid
containing the functional DapA is used (as shown in Fig. 77A), then the
chromosomal
constructs shown in Fig. 77C and Fig. 77D are knocked into the DapA locus. If
the
plasmid containing the functional ThyA is used (as shown in Fig. 77B), then
the
chromosomal constructs shown in Fig. 77C and Fig. 77D are knocked into the
ThyA
locus. In this system, the bacteria comprising the chromosomal construct and a
knocked
out dapA or thyA gene can grow in the absence of dap or thymidine only in the
presence
of the plasmid.
[0117] Fig. 78 depicts a schematic of a polypeptide of interest displayed on
the
surface of the bacterium. A non-limiting example of such a therapeutic protein
is a scFv.
The polypeptide is expressed as a fusion protein, which comprises a outer
membrane
anchor from another protein, which was developed as part of a display system.
Non-
limiting examples of such anchors are described herein and include LppOmpA,
NGIgAsig-NGIgAP, InaQ, Intimin, Invasin, pe1B-PAL, and blcA/BAN. In a
nonlimiting
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example a bacterial strain which has one or more diffusible outer membrane
phenotype
("leaky membrane") mutation, e.g., as described herein.
[0118] Fig. 79 depicts the gene organization of exemplary construct comprising
FNRS24Y driven by the arabinose inducible promoter and araC in reverse
direction.
[0119] Fig. 80A depicts a "Oxygen bypass switch" useful for aerobic pre-
induction of a strain comprising one or proteins of interest (POI), e.g., one
or more anti-
cancer molecules or immune modulatory effectors (POII) and a second set of one
or more
proteins of interest (P0I2), e.g., one or more transporter(s)/importer(s)
and/or exporter(s),
under the control of a low oxygen FNR promoter in vitro in a culture vessel
(e.g., flask,
fermenter or other vessel, e.g., used during with cell growth, cell expansion,
fermentation,
recovery, purification, formulation, and/or manufacture). In some embodiments,
it is
desirable to pre-load a strain with active effector molecules prior to
administration. This
can be done by pre-inducing the expression of these effectors as the strains
are propagated,
(e.g., in flasks, fermenters or other appropriate vesicles) and are prepared
for in vivo
administration. In some embodiments, strains are induced under anaerobic
and/or low
oxygen conditions, e.g. to induce FNR promoter activity and drive expression
of one or
more effectors or proteins of interest. In some embodiments, it is desirable
to prepare, pre-
load and pre-induce the strains under aerobic or microaerobic conditions with
one or more
effectors or proteins of interest. This allows more efficient growth and, in
some cases,
reduces the build-up of toxic metabolites.
[0120] FNRS24Y is a mutated form of FNR which is more resistant to
inactivation
by oxygen, and therefore can activate FNR promoters under aerobic conditions
(see e.g.,
Jervis AJ, The 02 sensitivity of the transcription factor FNR is controlled by
Ser24
modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci
U S A.
2009 Mar 24;106(12):4659-64, the contents of which is herein incorporated by
reference
in its entirety). In this oxygen bypass system, FNRS24Y is induced by addition
of
arabinose and then drives the expression of one or more POIs by binding and
activating
the FNR promoter under aerobic conditions. Thus, strains can be grown,
produced or
manufactured efficiently under aerobic conditions, while being effectively pre-
induced
and pre-loaded, as the system takes advantage of the strong FNR promoter
resulting in of
high levels of expression of one or more POIs. This system does not interfere
with or
compromise in vivo activation, since the mutated FNRS24Y is no longer
expressed in the
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absence of arabinose, and wild type FNR then binds to the FNR promoter and
drives
expression of the POIs in vivo. In some embodiments, a Lad I promoter and IPTG
induction are used in this system (in lieu of Para and arabinose induction).
In some
embodiments, a rhamnose inducible promoter is used in this system. In some
embodiments, a temperature sensitive promoter is used to drive expression of
FNRS24Y.
[0121] Fig. 80B depicts a strategy to allow the expression of one or more
POI(s)
under aerobic conditions through the arabinose inducible expression of
FNRS24Y. By
using a ribosome binding site optimization strategy, the levels of Fnrs24Y
expression can
be fine-tuned, e.g., under optimal inducing conditions (adequate amounts of
arabinose for
full induction). Fine-tuning is accomplished by selection of an appropriate
RBS with the
appropriate translation initiation rate. Bioinformatics tools for optimization
of RBS are
known in the art.
[0122] Fig. 80C depicts a strategy to fine-tune the expression of a Para-POI
construct by using a ribosome binding site optimization strategy.
Bioinformatics tools for
optimization of RBS are known in the art. In one strategy, arabinose
controlled POI genes
can be integrated into the chromosome to provide for efficient aerobic growth
and pre-
induction of the strain (e.g., in flasks, fermenters or other appropriate
vesicles), while
integrated versions of Pthrs-POI constructs are maintained to allow for strong
in vivo
induction.
[0123] Fig. 81 depicts the gene organization of an exemplary construct, e.g.,
comprised in SYN-PKU401, comprising a cloned POI gene under the control of a
Tet
promoter sequence and a Tet repressor gene.
[0124] Fig. 82 depicts the gene organization of an exemplary construct
comprising
Lad I in reverse orientation, and a IPTG inducible promoter driving the
expression of one
or more POIs. In some embodiments, this construct is useful for pre-induction
and pre-
loading of a therapeutic strain prior to in vivo administration under aerobic
conditions and
in the presence of inducer, e.g., IPTG. In some embodiments, this construct is
used alone.
In some embodiments, the construct is used in combination with other
constitutive or
inducible POI constructs, e.g., low oxygen, arabinose or IPTG inducible
constructs. In
some embodiments, the construct is used in combination with a low-oxygen
inducible
construct which is active in an in vivo setting.
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[0125] In some embodiments, the construct is located on a plasmid, e.g., a low
copy or a high copy plasmid. In some embodiments, the construct is located on
a plasmid
component of a biosafety system. In some embodiments, the construct is
integrated into
the bacterial chromosome at one or more locations. In some embodiments, the
construct is
used in combination with construct expressing a second POI, e.g., a
transporter, which can
either be provided on a plasmid or is integrated into the bacterial chromosome
at one or
more locations. P012 expression may be constitutive or driven by an inducible
promoter,
e.g., low-oxygen, arabinose, or IPTG. In some embodiments, the construct is
located on a
plasmid, e.g., a low or high copy plasmid. In some embodiments, the construct
is
employed in a biosafety system, such as the system shown in Fig. 77A, Fig.
77B, Fig.
77C, and Fig. 77D. In some embodiments, the construct is integrated into the
genome at
one or more locations described herein.
[0126] Fig. 83A, Fig. 83B, and Fig. 83C depict schematics of non-limiting
examples of constructs for the expression of proteins of interest POI(s). Fig
83A depicts a
schematic of a non-limiting example of the organization of a construct for POI
expression
under the control a lambda CI inducible promoter. The construct also provides
the coding
sequence of a mutant of CI, CI857, which is a temperature sensitive mutant of
CI. The
temperature sensitive CI repressor mutant, CI857, binds tightly at 30 degrees
C but is
unable to bind (repress) at temperatures of 37 C and above. In some
embodiments, this
construct is used alone. In some embodiments, the temperature sensitive
construct is used
in combination with other constitutive or inducible POI constructs, e.g., low
oxygen,
arabinose, rhamnose, or IPTG inducible constructs. In some embodiments, the
construct
allows pre-induction and pre-loading of a POI1 and/or a P012 prior to in vivo
administration. In some embodiments, the construct provides in vivo activity.
In some
embodiments, the construct is located on a plasmid, e.g., a low copy or a high
copy
plasmid. In some embodiments, the construct is located on a plasmid component
of a
biosafety system. In some embodiments, the construct is integrated into the
bacterial
chromosome at one or more locations. In some embodiments, the construct is
used in
combination with a P012 construct, which can either be provided on a plasmid
or is
integrated into the bacterial chromosome at one or more locations. P012
expression may
be constitutive or driven by an inducible promoter, e.g., low-oxygen,
arabinose, rhamnose,
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or temperature sensitive. In some embodiments, the construct is used in
combination with
a P013 expression construct.
[0127] In some embodiments, a temperature sensitive system can be used to set
up
a conditional auxotrophy. In a a strain comprising deltaThyA or deltaDapA, a
dapA or
thyA gene can be introduced into the strain under the control of a
thermoregulated
promoter system. The strain can grow in the absence of Thy and Dap only at the
permissive temperature, e.g., 37 C (and not lower).
[0128] Fig. 84A depicts a schematic of the gene organization of a PssB
promoter.
The ssB gene product protects ssDNA from degradation; SSB interacts directly
with
numerous enzymes of DNA metabolism and is believed to have a central role in
organizing the nucleoprotein complexes and processes involved in DNA
replication (and
replication restart), recombination and repair. The PssB promoter was cloned
in front of a
LacZ reporter and beta-galactosidase activity was measured.
[0129] Fig. 84B depicts a bar graph showing the reporter gene activity for the
PssB promoter under aerobic and anaerobic conditions. Briefly, cells were
grown
aerobically overnight, then diluted 1:100 and split into two different tubes.
One tube was
placed in the anaerobic chamber, and the other was kept in aerobic conditions
for the
length of the experiment. At specific times, the cells were analyzed for
promoter
induction. The Pssb promoter is active under aerobic conditions, and shuts off
under
anaerobic conditions. This promoter can be used to express a gene of interest
under
aerobic conditions. This promoter can also be used to tightly control the
expression of a
gene product such that it is only expressed under anaerobic and/or low oxygen
conditions.
In this case, the oxygen induced PssB promoter induces the expression of a
repressor,
which represses the expression of a gene of interest. Thus, the gene of
interest is only
expressed in the absence of the repressor, i.e., under anaerobic and/or low
oxygen
conditions. This strategy has the advantage of an additional level of control
for improved
fine-tuning and tighter control. In one non-limiting example, this strategy
can be used to
control expression of thyA and/or dapA, e.g., to make a conditional auxotroph.
The
chromosomal copy of dapA or ThyA is knocked out. Under anaerobic and/or low
oxygen
conditions, dapA or thyA -as the case may be- are expressed, and the strain
can grow in
the absence of dap or thymidine. Under aerobic conditions, dapA or thyA
expression is
shut off, and the strain cannot grow in the absence of dap or thymidine. Such
a strategy
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can, for example be employed to allow survival of bacteria under anaerobic
and/or low
oxygen conditions, e.g., the gut, but prevent survival under aerobic
conditions (biosafety
switch).
[0130] Fig. 85A depicts a schematic diagram of a wild-type clbA construct.
[0131] Fig. 85B depicts a schematic diagram of a clbA knockout construct.
Description of Embodiments
[0132] The present disclosure includes genetically engineered bacteria,
pharmaceutical compositions thereof, and methods of reducing gut inflammation,
enhancing gut barrier function, and/or treating or preventing autoimmune
disorders. In
some embodiments, the genetically engineered bacteria comprise at least one
non-native
gene and/or gene cassette for producing a non-native anti-inflammation and/or
gut barrier
function enhancer molecule(s). In some embodiments, the at least one gene
and/or gene
cassette is further operably linked to a regulatory region that is controlled
by a
transcription factor that is capable of sensing an inducing condition, e.g., a
low-oxygen
environment, the presence of ROS, or the presence of RNS. The genetically
engineered
bacteria are capable of producing the anti-inflammation and/or gut barrier
function
enhancer molecule(s) in inducing environments, e.g., in the gut. Thus, the
genetically
engineered bacteria and pharmaceutical compositions comprising those bacteria
may be
used to treat or prevent autoimmune disorders and/or diseases or conditions
associated
with gut inflammation and/or compromised gut barrier function, including IBD.
[0133] In order that the disclosure may be more readily understood, certain
terms
are first defined. These definitions should be read in light of the remainder
of the
disclosure and as understood by a person of ordinary skill in the art. Unless
defined
otherwise, all technical and scientific terms used herein have the same
meaning as
commonly understood by a person of ordinary skill in the art. Additional
definitions are
set forth throughout the detailed description.
[0134] As used herein, "diseases and conditions associated with gut
inflammation
and/or compromised gut barrier function" include, but are not limited to,
inflammatory
bowel diseases, diarrheal diseases, and related diseases. "Inflammatory bowel
diseases"
and "IBD" are used interchangeably herein to refer to a group of diseases
associated with
gut inflammation, which include, but are not limited to, Crohn's disease,
ulcerative colitis,
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collagenous colitis, lymphocytic colitis, diversion colitis, Behcet's disease,
and
indeterminate colitis. As used herein, "diarrheal diseases" include, but are
not limited to,
acute watery diarrhea, e.g., cholera; acute bloody diarrhea, e.g., dysentery;
and persistent
diarrhea. As used herein, related diseases include, but are not limited to,
short bowel
syndrome, ulcerative proctitis, proctosigmoiditis, left-sided colitis,
pancolitis, and
fulminant colitis.
[0135] Symptoms associated with the aforementioned diseases and conditions
include, but are not limited to, one or more of diarrhea, bloody stool, mouth
sores, perianal
disease, abdominal pain, abdominal cramping, fever, fatigue, weight loss, iron
deficiency,
anemia, appetite loss, weight loss, anorexia, delayed growth, delayed pubertal
development, inflammation of the skin, inflammation of the eyes, inflammation
of the
joints, inflammation of the liver, and inflammation of the bile ducts.
[0136] A disease or condition associated with gut inflammation and/or
compromised gut barrier function may be an autoimmune disorder. A disease or
condition
associated with gut inflammation and/or compromised gut barrier function may
be co-
morbid with an autoimmune disorder. As used herein, "autoimmune disorders"
include,
but are not limited to, acute disseminated encephalomyelitis (ADEM), acute
necrotizing
hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia
areata,
amyloidosis, ankylo sing spondylitis, anti-GBM/anti-TBM nephritis,
antiphospholipid
syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune
dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune
hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease
(AIED),
autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis,
autoimmune
retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid
disease,
autoimmune urticarial, axonal & neuronal neuropathies, Balo disease, Behcet's
disease,
bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas
disease,
chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent
multifocal
ostomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign
muco sal
pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease,
congenital heart
block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia,
demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's
disease
(neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis,
eosinophilic
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esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic
encephalomyelitis, Evans syndrome, fibrosing alveolitis, giant cell arteritis
(temporal
arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's
syndrome,
granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barre
syndrome,
Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-
Schonlein
purpura, herpes gestationis, hypogammaglobulinemia, idiopathic
thrombocytopenic
purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease,
immunoregulatory
lipoproteins, inclusion body myositis, interstitial cystitis, juvenile
arthritis, juvenile
idiopathic arthritis, juvenile myositis, Kawasaki syndrome, Lambert-Eaton
syndrome,
leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous
conjunctivitis, linear
IgA disease (LAD), lupus (systemic lupus erythematosus), chronic Lyme disease,
Meniere's disease, microscopic polyangiitis, mixed connective tissue disease
(MCTD),
Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia
gravis,
myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular
cicatricial
pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric
Autoimmune
Neuropsychiatric Disorders Associated with Streptococcus), paraneoplastic
cerebellar
degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg
syndrome,
Parsonnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus,
peripheral
neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome,
polyarteritis nodosa, type I, II, & III autoimmune polyglandular syndromes,
polymyalgia
rheumatic, polymyositis, postmyocardial infarction syndrome,
postpericardiotomy
syndrome, progesterone dermatitis, primary biliary cirrhosis, primary
sclerosing
cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis,
pyoderma
gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis,
reflex
sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless
legs
syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis,
sarcoidosis,
Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm &
testicular
autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE),
Susac's
syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal
arteritis/giant cell
arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse
myelitis,
type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective
tissue disease
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(UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and
Wegener's
granulomatosis.
[0137] As used herein, "anti-inflammation molecules" and/or "gut barrier
function
enhancer molecules" include, but are not limited to, short-chain fatty acids,
butyrate,
propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and
analogs, GLP-1,
IL-10, IL-27, TGF-(31, TGF-(32, N-acylphosphatidylethanolamines (NAPEs),
elafin (also
called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin,
tryptophan, PGD2, and
kynurenic acid, indole metabolites, and other tryptophan metabolites, as well
as other
molecules disclosed herein. Such molecules may also include compounds that
inhibit pro-
inflammatory molecules, e.g., a single-chain variable fragment (scFv),
antisense RNA,
siRNA, or shRNA that neutralizes TNF-a, IFN-y, IL-113, IL-6, IL-8, IL-17,
and/or
chemokines, e.g., CXCL-8 and CCL2. Such molecules also include AHR agonists
(e.g.,
which result in IL-22 production, e.g., indole acetic acid, indole-3-aldehyde,
and indole)
and and PXR agonists (e.g., IPA), as described herein. Such molecules also
include
HDAC inhibitors (e.g., butyrate), activators of GPR41 and/or GPR43 (e.g.,
butyrate and/or
propionate and/or acetate), activtators of GPR109A (e.g., butyrate),
inhibitors of NF-
kappaB signaling (e.g., butyrate), and modulators of PPARgamma (e.g.,
butyrate),
activators of AMPK signaling (e.g., acetate), and modulators of GLP-1
secretion. Such
molecules also include hydroxyl radical scavengers and antioxidants (e.g.,
IPA). A
molecule may be primarily anti-inflammatory, e.g., IL-10, or primarily gut
barrier function
enhancing, e.g., GLP-2. A molecule may be both anti-inflammatory and gut
barrier
function enhancing. An anti-inflammation and/or gut barrier function enhancer
molecule
may be encoded by a single gene, e.g., elafin is encoded by the P13 gene.
Alternatively,
an anti-inflammation and/or gut barrier function enhancer molecule may be
synthesized by
a biosynthetic pathway requiring multiple genes, e.g., butyrate. These
molecules may also
be referred to as therapeutic molecules. In some instances, the "anti-
inflammation
molecules" and/or "gut barrier function enhancer molecules" are referred to
herein as
"effector molecules" or "therapeutic molecules" or "therapeutic polypeptides".
[0138] As used herein, the term "recombinant microorganism" refers to a
microorganism, e.g., bacterial, yeast, or viral cell, or bacteria, yeast, or
virus, that has been
genetically modified from its native state. Thus, a "recombinant bacterial
cell" or
"recombinant bacteria" refers to a bacterial cell or bacteria that have been
genetically
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modified from their native state. For instance, a recombinant bacterial cell
may have
nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and
nucleotide
modifications introduced into their DNA. These genetic modifications may be
present in
the chromosome of the bacteria or bacterial cell, or on a plasmid in the
bacteria or
bacterial cell. Recombinant bacterial cells disclosed herein may comprise
exogenous
nucleotide sequences on plasmids. Alternatively, recombinant bacterial cells
may
comprise exogenous nucleotide sequences stably incorporated into their
chromosome.
[0139] A "programmed or engineered microorganism" refers to a microorganism,
e.g., bacterial or viral cell, or bacteria or virus, that has been genetically
modified from its
native state to perform a specific function. Thus, a "programmed or engineered
bacterial
cell" or "programmed or engineered bacteria" refers to a bacterial cell or
bacteria that has
been genetically modified from its native state to perform a specific
function. In certain
embodiments, the programmed or engineered bacterial cell has been modified to
express
one or more proteins, for example, one or more proteins that have a
therapeutic activity or
serve a therapeutic purpose. The programmed or engineered bacterial cell may
additionally have the ability to stop growing or to destroy itself once the
protein(s) of
interest have been expressed.
[0140] As used herein, the term "gene" refers to a nucleic acid fragment that
encodes a protein or fragment thereof, optionally including regulatory
sequences
preceding (5' non-coding sequences) and following (3' non-coding sequences)
the coding
sequence. In one embodiment, a "gene" does not include regulatory sequences
preceding
and following the coding sequence. A "native gene" refers to a gene as found
in nature,
optionally with its own regulatory sequences preceding and following the
coding
sequence. A "chimeric gene" refers to any gene that is not a native gene,
optionally
comprising regulatory sequences preceding and following the coding sequence,
wherein
the coding sequences and/or the regulatory sequences, in whole or in part, are
not found
together in nature. Thus, a chimeric gene may comprise regulatory sequences
and coding
sequences that are derived from different sources, or regulatory and coding
sequences that
are derived from the same source, but arranged differently than is found in
nature.
[0141] As used herein, the term "gene sequence" is meant to refer to a genetic
sequence, e.g., a nucleic acid sequence. The gene sequence or genetic sequence
is meant
to include a complete gene sequence or a partial gene sequence. The gene
sequence or
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genetic sequence is meant to include sequence that encodes a protein or
polypeptide and is
also menat to include genetic sequence that does not encode a protein or
polypeptide, e.g.,
a regulatory sequence, leader sequence, signal sequence, or other non-protein
coding
sequence.
[0142] In some embodiments, the term "gene" or "gene sequence" is meant to
refer to a nucleic acid sequence encoding any of the anti-inflammatory and gut
barrier
function enhancing molecules described herein, e.g., IL-2, IL-22, superoxide
dismutase
(SOD), kynurenine, GLP-2, GLP-1, IL-10, IL-27, TGF-01, TGF-02, N-
acylphosphatidylethanolamines (NAPEs), elafin, and trefoil factor, as well as
others. The
nucleic acid sequence may comprise the entire gene sequence or a partial gene
sequence
encoding a functional molecule. The nucleic acid sequence may be a natural
sequence or
a synthetic sequence. The nucleic acid sequence may comprise a native or wild-
type
sequence or may comprise a modified sequence having one or more insertions,
deletions,
substitutions, or other modifications, for example, the nucleic acid sequence
may be
codon-optimized.
[0143] As used herein, a "heterologous" gene or "heterologous sequence" refers
to
a nucleotide sequence that is not normally found in a given cell in nature. As
used herein,
a heterologous sequence encompasses a nucleic acid sequence that is
exogenously
introduced into a given cell and can be a native sequence (naturally found or
expressed in
the cell) or non-native sequence (not naturally found or expressed in the
cell) and can be a
natural or wild-type sequence or a variant, non-natural, or synthetic
sequence.
"Heterologous gene" includes a native gene, or fragment thereof, that has been
introduced
into the host cell in a form that is different from the corresponding native
gene. For
example, a heterologous gene may include a native coding sequence that is a
portion of a
chimeric gene to include non-native regulatory regions that is reintroduced
into the host
cell. A heterologous gene may also include a native gene, or fragment thereof,
introduced
into a non-native host cell. Thus, a heterologous gene may be foreign or
native to the
recipient cell; a nucleic acid sequence that is naturally found in a given
cell but expresses
an unnatural amount of the nucleic acid and/or the polypeptide which it
encodes; and/or
two or more nucleic acid sequences that are not found in the same relationship
to each
other in nature. As used herein, the term "endogenous gene" refers to a native
gene in its
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natural location in the genome of an organism. As used herein, the term
"transgene" refers
to a gene that has been introduced into the host organism, e.g., host
bacterial cell, genome.
[0144] As used herein, a "non-native" nucleic acid sequence refers to a
nucleic
acid sequence not normally present in a microorganism, e.g., an extra copy of
an
endogenous sequence, or a heterologous sequence such as a sequence from a
different
species, strain, or substrain of bacteria or virus, or a sequence that is
modified and/or
mutated as compared to the unmodified sequence from bacteria or virus of the
same
subtype. In some embodiments, the non-native nucleic acid sequence is a
synthetic, non-
naturally occurring sequence (see, e.g., Purcell et al., 2013). The non-native
nucleic acid
sequence may be a regulatory region, a promoter, a gene, and/or one or more
genes in
gene cassette. In some embodiments, "non-native" refers to two or more nucleic
acid
sequences that are not found in the same relationship to each other in nature.
The non-
native nucleic acid sequence may be present on a plasmid or chromosome. In
some
embodiments, the genetically engineered microorganism of the disclosure
comprises a
gene that is operably linked to a promoter that is not associated with said
gene in nature.
For example, in some embodiments, the genetically engineered bacteria
disclosed herein
comprise a gene that is operably linked to a directly or indirectly inducible
promoter that is
not associated with said gene in nature, e.g., an FNR responsive promoter (or
other
promoter disclosed herein) operably linked to an anti-inflammatory or gut
barrier enhancer
molecule. In some embodiments, the genetically engineered virus of the
disclosure
comprises a gene that is operably linked to a directly or indirectly inducible
promoter that
is not associated with said gene in nature, e.g., a promoter operably linked
to a gene
encoding an anti-inflammatory or gut barrier enhancer molecule.
[0145] As used herein, the term "coding region" refers to a nucleotide
sequence
that codes for a specific amino acid sequence. The term "regulatory sequence"
refers to a
nucleotide sequence located upstream (5' non-coding sequences), within, or
downstream
(3' non-coding sequences) of a coding sequence, and which influences the
transcription,
RNA processing, RNA stability, or translation of the associated coding
sequence.
Examples of regulatory sequences include, but are not limited to, promoters,
translation
leader sequences, effector binding sites, signal sequences, and stem-loop
structures. In
one embodiment, the regulatory sequence comprises a promoter, e.g., an FNR
responsive
promoter or other promoter disclosed herein.
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[0146] As used herein, a "gene cassette" or "operon" encoding a biosynthetic
pathway refers to the two or more genes that are required to produce an anti-
inflammatory
or gut barrier enhancer molecule. In addition to encoding a set of genes
capable of
producing said molecule, the gene cassette or operon may also comprise
additional
transcription and translation elements, e.g., a ribosome binding site.
[0147] A "butyrogenic gene cassette," "butyrate biosynthesis gene cassette,"
and
"butyrate operon" are used interchangeably to refer to a set of genes capable
of producing
butyrate in a biosynthetic pathway. Unmodified bacteria that are capable of
producing
butyrate via an endogenous butyrate biosynthesis pathway include, but are not
limited to,
Clostridium, Peptoclostridium, Fusobacterium, Butyrivibrio, Eubacterium, and
Treponema. The genetically engineered bacteria of the invention may comprise
butyrate
biosynthesis genes from a different species, strain, or substrain of bacteria,
or a
combination of butyrate biosynthesis genes from different species, strains,
and/or
substrains of bacteria. A butyrogenic gene cassette may comprise, for example,
the eight
genes of the butyrate production pathway from Peptoclostridium difficile (also
called
Clostridium difficile): bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk,
which encode
butyryl-CoA dehydrogenase subunit, electron transfer flavoprotein subunit
beta, electron
transfer flavoprotein subunit alpha, acetyl-CoA C-acetyltransferase, 3-
hydroxybutyryl-
CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate
kinase,
respectively (Aboulnaga et al., 2013). One or more of the butyrate
biosynthesis genes may
be functionally replaced or modified, e.g., codon optimized. Peptoclostridium
difficile
strain 630 and strain 1296 are both capable of producing butyrate, but
comprise different
nucleic acid sequences for etfA3, thiAl, hbd, crt2, pbt, and buk. A
butyrogenic gene
cassette may comprise bcd2, etfB3, etfA3, and thiAl from Peptoclostridium
difficile strain
630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296.
Alternatively,
a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA
reductase) is
capable of functionally replacing all three of the bcd2, etfB3, and etfA3
genes from
Peptoclostridium difficile. Thus, a butyrogenic gene cassette may comprise
thiAl, hbd,
crt2, pbt, and buk from Peptoclostridium difficile and ter from Treponema
denticola. The
butyrogenic gene cassette may comprise genes for the aerobic biosynthesis of
butyrate
and/or genes for the anaerobic or microaerobic biosynthesis of butyrate. In
another
example of a butyrate gene cassette, the pbt and buk genes are replaced with
tesB (e.g.,
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from E coli). Thus a butyrogenic gene cassette may comprise ter, thiAl, hbd,
crt2, and
tesB.
[0148] Likewise, a "propionate gene cassette" or "propionate operon" refers to
a
set of genes capable of producing propionate in a biosynthetic pathway.
Unmodified
bacteria that are capable of producing propionate via an endogenous propionate
biosynthesis pathway include, but are not limited to, Clostridium propionicum,
Megasphaera elsdenii, and Prevotella ruminicola. The genetically engineered
bacteria of
the invention may comprise propionate biosynthesis genes from a different
species, strain,
or substrain of bacteria, or a combination of propionate biosynthesis genes
from different
species, strains, and/or substrains of bacteria. In some embodiments, the
propionate gene
cassette comprises acrylate pathway propionate biosynthesis genes, e.g., pct,
lcdA, lcdB,
lcdC, e0, acrB, and acrC, which encode propionate CoA-transferase, lactoyl-CoA
dehydratase A, lactoyl-CoA dehydratase B, lactoyl-CoA dehydratase C, electron
transfer
flavoprotein subunit A, acryloyl-CoA reductase B, and acryloyl-CoA reductase
C,
respectively (Hetzel et al., 2003, Selmer et al., 2002, and Kandasamy 2012
Engineering
Escherichia coli with acrylate pathway genes for propionic acid synthesis and
its impact
on mixed-acid fermentation). This operon catalyses the reduction of lactate to
propionate.
Dehydration of (R)-lactoyl-CoA leads to the production of the intermediate
acryloyl-CoA
by lactoyl-CoA dehydratase (LcdABC). Acrolyl-CoA is converted to propionyl-CoA
by
acrolyl-CoA reductase (EtfA, AcrBC). In some embodiments, the rate limiting
step
catalyzed by the enzymes encoded by etfA, acrB and acrC, are replaced by the
actd gene
from R. sphaeroides. This gene product catalyzes the NADPH-dependent acrylyl-
CoA
reduction to produce propionyl-CoA (Acrylyl-Coenzyme A Reductase, an Enzyme
Involved in the Assimilation of 3-Hydroxypropionate by Rhodobacter
sphaeroides; Asao
2013). Thus the propionate cassette comprises pct, lcdA, lcdB, lcdC, and actd.
In another
embodiment, the homolog of AcuI in E coli, YhdH is used (see.e.g., Structure
of
Escherichia coli YhdH, a putative quinone oxidoreductase. Sulzenbacher 2004).
This the
propionate cassette comprises pct, lcdA, lcdB, lcdC, and yhdH. In alternate
embodiments,
the propionate gene cassette comprises pyruvate pathway propionate
biosynthesis genes
(see, e.g., Tseng et al., 2012), e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE,
aceF, and 1pd, which
encode homoserine dehydrogenase 1, homoserine kinase, L-threonine synthase, L-
threonine dehydratase, pyruvate dehydrogenase, dihydrolipoamide
acetyltrasferase, and
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dihydrolipoyl dehydrogenase, respectively. In some embodiments, the propionate
gene
cassette further comprises tesB, which encodes acyl-CoA thioesterase.
[0149] In another example of a propionate gene cassette comprises the genes of
the
Sleeping Beauty Mutase operon, e.g., from E. coli (sbm, ygfD, ygfG, ygfH).
Recently, this
pathway has been considered and utilized for the high yield industrial
production of
propionate from glycerol (Akawi et al., Engineering Escherichia coli for high-
level
production of propionate; J Ind Microbiol Biotechnol (2015) 42:1057-1072, the
contents
of which is herein incorporated by reference in its entirety). In addition, as
described
herein, it has been found that this pathway is also suitable for production of
proprionate
from glucose, e.g. by the genetically engineered bacteria of the disclosure.
The SBM
pathway is cyclical and composed of a series of biochemical conversions
forming
propionate as a fermentative product while regenerating the starting molecule
of succinyl-
CoA. Sbm (methylmalonyl-CoA mutase) converts succinyl CoA to L-
methylmalonylCoA,
YgfD is a Sbm-interacting protein kinase with GTPase activity, ygfG
(methylmalonylCoA
decarboxylase) converts L-methylmalonylCoA into PropionylCoA, and ygfH
(propionyl-
CoA/succinylCoA transferase) converts propionylCoA into propionate and
succinate into
succinylCoA (Sleeping beauty mutase (sbm) is expressed and interacts with ygfd
in
Escherichia coli; Froese 2009). This pathway is very similar to the oxidative
propionate
pathway of Propionibacteria, which also converts succinate to propionate.
Succinyl-CoA
is converted to R-methylmalonyl-CoA by methymalonyl-CoA mutase (mutAB). This
is in
turn converted to S-methylmalonyl-CoA via methymalonyl-CoA epimerase
(GI:18042134). There are three genes which encode methylmalonyl-CoA
carboxytransferase (mmdA, PFREUD 18870, bccp) which converts methylmalonyl-CoA
to propionyl-CoA.
[01501 The propionate gene cassette may comprise genes for the aerobic
biosynthesis of propionate and/or genes for the anaerobic or microaerobic
biosynthesis of
propionate. One or more of the propionate biosynthesis genes may be
functionally
replaced or modified, e.g., codon optimized.
[0151] An "acetate gene cassette" or "acetate operon" refers to a set of genes
capable of producing acetate in a biosynthetic pathway. Bacteria "synthesize
acetate from
a number of carbon and energy sources," including a variety of substrates such
as
cellulose, lignin, and inorganic gases, and utilize different biosynthetic
mechanisms and
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genes, which are known in the art (Ragsdale et al., 2008). The genetically
engineered
bacteria of the invention may comprise acetate biosynthesis genes from a
different species,
strain, or substrain of bacteria, or a combination of acetate biosynthesis
genes from
different species, strains, and/or substrains of bacteria. Escherichia coli
are capable of
consuming glucose and oxygen to produce acetate and carbon dioxide during
aerobic
growth (Kleman et al., 1994). Several bacteria, such as Acetitomaculum,
Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium,
Clostridium,
Moorella, Oxobacter, Sporomusa, and Thermoacetogenium, are acetogenic
anaerobes that
are capable of converting CO or CO2 + H2 into acetate, e.g., using the Wood-
Ljungdahl
pathway (Schiel-Bengelsdorf et al, 2012). Genes in the Wood-Ljungdahl pathway
for
various bacterial species are known in the art. The acetate gene cassette may
comprise
genes for the aerobic biosynthesis of acetate and/or genes for the anaerobic
or
microaerobic biosynthesis of acetate. One or more of the acetate biosynthesis
genes may
be functionally replaced or modified, e.g., codon optimized.
[0152] Each gene or gene cassette may be present on a plasmid or bacterial
chromosome. In addition, multiple copies of any gene, gene cassette, or
regulatory region
may be present in the bacterium, wherein one or more copies of the gene, gene
cassette, or
regulatory region may be mutated or otherwise altered as described herein. In
some
embodiments, the genetically engineered bacteria are engineered to comprise
multiple
copies of the same gene, gene cassette, or regulatory region in order to
enhance copy
number or to comprise multiple different components of a gene cassette
performing
multiple different functions.
[0153] Each gene or gene cassette may be operably linked to a promoter that is
induced under low-oxygen conditions. "Operably linked" refers a nucleic acid
sequence,
e.g., a gene or gene cassette for producing an anti-inflammatory or gut
barrier enhancer
molecule, that is joined to a regulatory region sequence in a manner which
allows
expression of the nucleic acid sequence, e.g., acts in cis. A regulatory
region "Operably
linked" refers to the association of nucleic acid sequences on a single
nucleic acid
fragment so that the function of one is affected by the other. A regulatory
element is
operably linked with a coding sequence when it is capable of affecting the
expression of
the gene coding sequence, regardless of the distance between the regulatory
element and
the coding sequence. More specifically, operably linked refers to a nucleic
acid sequence,
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e.g., a gene encoding an anti-inflammatory or gut barrier enhancer molecule,
that is joined
to a regulatory sequence in a manner which allows expression of the nucleic
acid
sequence, e.g., the gene encoding the anti-inflammatory or gut barrier
enhancer molecule.
In other words, the regulatory sequence acts in cis. In one embodiment, a gene
may be
"directly linked" to a regulatory sequence in a manner which allows expression
of the
gene. In another embodiment, a gene may be "indirectly linked" to a regulatory
sequence
in a manner which allows expression of the gene. In one embodiment, two or
more genes
may be directly or indirectly linked to a regulatory sequence in a manner
which allows
expression of the two or more genes. A regulatory region or sequence is a
nucleic acid that
can direct transcription of a gene of interest and may comprise promoter
sequences,
enhancer sequences, response elements, protein recognition sites, inducible
elements,
promoter control elements, protein binding sequences, 5' and 3' untranslated
regions,
transcriptional start sites, termination sequences, polyadenylation sequences,
and introns.
[0154] A "promoter" as used herein, refers to a nucleotide sequence that is
capable
of controlling the expression of a coding sequence or gene. Promoters are
generally
located 5' of the sequence that they regulate. Promoters may be derived in
their entirety
from a native gene, or be composed of different elements derived from
promoters found in
nature, and/or comprise synthetic nucleotide segments. Those skilled in the
art will
readily ascertain that different promoters may regulate expression of a coding
sequence or
gene in response to a particular stimulus, e.g., in a cell- or tissue-specific
manner, in
response to different environmental or physiological conditions, or in
response to specific
compounds. Prokaryotic promoters are typically classified into two classes:
inducible and
constitutive. A "constitutive promoter" refers to a promoter that allows for
continual
transcription of the coding sequence or gene under its control.
[0155] "Constitutive promoter" refers to a promoter that is capable of
facilitating
continuous transcription of a coding sequence or gene under its control and/or
to which it
is operably linked. Constitutive promoters and variants are well known in the
art and
include, but are not limited to, Ptac promoter, BBa J23100, a constitutive
Escherichia coli
GS promoter (e.g., an osmY promoter (International Genetically Engineered
Machine
(iGEM) Registry of Standard Biological Parts Name BBa J45992; BBa J45993)), a
constitutive Escherichia coli a32 promoter (e.g., htpG heat shock promoter
(BBa J45504)), a constitutive Escherichia coli a70 promoter (e.g., lacq
promoter
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(BBa J54200; BBa J56015), E. coli CreABCD phosphate sensing operon promoter
(BBa J64951), GlnRS promoter (BBa K088007), lacZ promoter (BBa K119000;
BBa K119001); M13K07 gene I promoter (BBa M13101); M13K07 gene II promoter
(BBa M13102), M13K07 gene III promoter (BBa M13103), M13K07 gene IV promoter
(BBa M13104), M13K07 gene V promoter (BBa M13105), M13K07 gene VI promoter
(BBa M13106), M13K07 gene VIII promoter (BBa M13108), M13110 (BBa M13110)),
a constitutive Bacillus subtilis GA promoter (e.g., promoter veg (BBa
K143013),
promoter 43 (BBa K143013), PliaG (BBa K823000), PlepA (BBa K823002), Pveg
(BBa K823003)), a constitutive Bacillus subtilis GB promoter (e.g., promoter
ctc
(BBa K143010), promoter gsiB (BBa K143011)), a Salmonella promoter (e.g.,
Pspv2
from Salmonella (BBa K112706), Pspv from Salmonella (BBa K112707)), a
bacteriophage T7 promoter (e.g., T7 promoter (BBa I712074; BBa I719005;
BBa J34814; BBa J64997; BBa K113010; BBa K113011; BBa K113012; BBa R0085;
BBa R0180; BBa R0181; BBa R0182; BBa R0183; BBa Z0251; BBa Z0252;
BBa Z0253)), and a bacteriophage 5P6 promoter (e.g., 5P6 promoter (BBa
J64998)).
[0156] An "inducible promoter" refers to a regulatory region that is operably
linked to one or more genes, wherein expression of the gene(s) is increased in
the presence
of an inducer of said regulatory region. An "inducible promoter" refers to a
promoter that
initiates increased levels of transcription of the coding sequence or gene
under its control
in response to a stimulus or an exogenous environmental condition. A "directly
inducible
promoter" refers to a regulatory region, wherein the regulatory region is
operably linked to
a gene encoding a protein or polypeptide, where, in the presence of an inducer
of said
regulatory region, the protein or polypeptide is expressed. An "indirectly
inducible
promoter" refers to a regulatory system comprising two or more regulatory
regions, for
example, a first regulatory region that is operably linked to a first gene
encoding a first
protein, polypeptide, or factor, e.g., a transcriptional regulator, which is
capable of
regulating a second regulatory region that is operably linked to a second
gene, the second
regulatory region may be activated or repressed, thereby activating or
repressing
expression of the second gene. Both a directly inducible promoter and an
indirectly
inducible promoter are encompassed by "inducible promoter." Exemplary
inducible
promoters described herein include oxygen level-dependent promoters (e.g., FNR-
inducible promoter), promoters induced by inflammation or an inflammatory
response
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(RNS, ROS promoters), and promoters induced by a metabolite that may or may
not be
naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose
and
tetracycline. Examples of inducible promoters include, but are not limited to,
an FNR
responsive promoter, a ParaC promoter, a ParaBAD promoter, and a PTetR
promoter,
each of which are described in more detail herein. Examples of other inducible
promoters
are provided herein below.
[0157] As used herein, "stably maintained" or "stable" bacterium is used to
refer to
a bacterial host cell carrying non-native genetic material, e.g., a gene
encoding one or
more anti-inflammation and/or gut barrier enhancer molecule(s), which is
incorporated
into the host genome or propagated on a self-replicating extra-chromosomal
plasmid, such
that the non-native genetic material is retained, expressed, and propagated.
The stable
bacterium is capable of survival and/or growth in vitro, e.g., in medium,
and/or in vivo,
e.g., in the gut. For example, the stable bacterium may be a genetically
engineered
bacterium comprising a gene encoding a encoding a payload, e.g., one or more
anti-
inflammation and/or gut barrier enhancer molecule(s), in which the plasmid or
chromosome carrying the gene is stably maintained in the bacterium, such that
the payload
can be expressed in the bacterium, and the bacterium is capable of survival
and/or growth
in vitro and/or in vivo. In some embodiments, copy number affects the
stability of
expression of the non-native genetic material. In some embodiments, copy
number affects
the level of expression of the non-native genetic material.
[0158] As used herein, the term "expression" refers to the transcription and
stable
accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid,
and/or to
translation of an mRNA into a polypeptide.
[0159] As used herein, the term "plasmid" or "vector" refers to an
extrachromosomal nucleic acid, e.g., DNA, construct that is not integrated
into a bacterial
cell's genome. Plasmids are usually circular and capable of autonomous
replication.
Plasmids may be low-copy, medium-copy, or high-copy, as is well known in the
art.
Plasmids may optionally comprise a selectable marker, such as an antibiotic
resistance
gene, which helps select for bacterial cells containing the plasmid and which
ensures that
the plasmid is retained in the bacterial cell. A plasmid disclosed herein may
comprise a
nucleic acid sequence encoding a heterologous gene, e.g., a gene encoding an
anti-
inflammatory or gut barrier enhancer molecule.
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[0160] As used herein, the term "transform" or "transformation" refers to the
transfer of a nucleic acid fragment into a host bacterial cell, resulting in
genetically-stable
inheritance. Host bacterial cells comprising the transformed nucleic acid
fragment are
referred to as "recombinant" or "transgenic" or "transformed" organisms.
[0161] The term "genetic modification," as used herein, refers to any genetic
change. Exemplary genetic modifications include those that increase, decrease,
or abolish
the expression of a gene, including, for example, modifications of native
chromosomal or
extrachromosomal genetic material. Exemplary genetic modifications also
include the
introduction of at least one plasmid, modification, mutation, base deletion,
base addition,
base substitution, and/or codon modification of chromosomal or
extrachromosomal
genetic sequence(s), gene over-expression, gene amplification, gene
suppression, promoter
modification or substitution, gene addition (either single or multi-copy),
antisense
expression or suppression, or any other change to the genetic elements of a
host cell,
whether the change produces a change in phenotype or not. Genetic modification
can
include the introduction of a plasmid, e.g., a plasmid comprising an anti-
inflammatory or
gut barrier enhancer molecule operably linked to a promoter, into a bacterial
cell. Genetic
modification can also involve a targeted replacement in the chromosome, e.g.,
to replace a
native gene promoter with an inducible promoter, regulated promoter, strong
promoter, or
constitutive promoter. Genetic modification can also involve gene
amplification, e.g.,
introduction of at least one additional copy of a native gene into the
chromosome of the
cell. Alternatively, chromosomal genetic modification can involve a genetic
mutation.
[0162] As used herein, the term "genetic mutation" refers to a change or
changes
in a nucleotide sequence of a gene or related regulatory region that alters
the nucleotide
sequence as compared to its native or wild-type sequence. Mutations include,
for
example, substitutions, additions, and deletions, in whole or in part, within
the wild-type
sequence. Such substitutions, additions, or deletions can be single nucleotide
changes
(e.g., one or more point mutations), or can be two or more nucleotide changes,
which may
result in substantial changes to the sequence. Mutations can occur within the
coding
region of the gene as well as within the non-coding and regulatory sequence of
the gene.
The term "genetic mutation" is intended to include silent and conservative
mutations
within a coding region as well as changes which alter the amino acid sequence
of the
polypeptide encoded by the gene. A genetic mutation in a gene coding sequence
may, for
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example, increase, decrease, or otherwise alter the activity (e.g., enzymatic
activity) of the
gene's polypeptide product. A genetic mutation in a regulatory sequence may
increase,
decrease, or otherwise alter the expression of sequences operably linked to
the altered
regulatory sequence.
[0163] As used herein, the term "transporter" is meant to refer to a
mechanism,
e.g., protein, proteins, or protein complex, for importing a molecule, e.g.,
amino acid,
peptide (di-peptide, tri-peptide, polypeptide, etc), toxin, metabolite,
substrate, as well as
other biomolecules into the microorganism from the extracellular milieu.
[0164] As used herein, the phrase "exogenous environmental condition" or
"exogenous environment signal" refers to settings, circumstances, stimuli, or
biological
molecules under which a promoter described herein is directly or indirectly
induced. The
phrase "exogenous environmental conditions" is meant to refer to the
environmental
conditions external to the engineered micororganism, but endogenous or native
to the host
subject environment. Thus, "exogenous" and "endogenous" may be used
interchangeably
to refer to environmental conditions in which the environmental conditions are
endogenous to a mammalian body, but external or exogenous to an intact
microorganism
cell. In some embodiments, the exogenous environmental conditions are specific
to the
gut of a mammal. In some embodiments, the exogenous environmental conditions
are
specific to the upper gastrointestinal tract of a mammal. In some embodiments,
the
exogenous environmental conditions are specific to the lower gastrointestinal
tract of a
mammal. In some embodiments, the exogenous environmental conditions are
specific to
the small intestine of a mammal. In some embodiments, the exogenous
environmental
conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the
environment of the mammalian gut. In some embodiments, exogenous environmental
conditions are molecules or metabolites that are specific to the mammalian
gut, e.g.,
propionate. In some embodiments, the exogenous environmental condition is a
tissue-
specific or disease-specific metabolite or molecule(s). In some embodiments,
the
exogenous environmental condition is specific to an inflammatory disease. In
some
embodiments, the exogenous environmental condition is a low-pH environment. In
some
embodiments, the genetically engineered microorganism of the disclosure
comprises a pH-
dependent promoter. In some embodiments, the genetically engineered
microorganism of
the diclosure comprise an oxygen level-dependent promoter. In some aspects,
bacteria
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have evolved transcription factors that are capable of sensing oxygen levels.
Different
signaling pathways may be triggered by different oxygen levels and occur with
different
kinetics. An "oxygen level-dependent promoter" or "oxygen level-dependent
regulatory
region" refers to a nucleic acid sequence to which one or more oxygen level-
sensing
transcription factors is capable of binding, wherein the binding and/or
activation of the
corresponding transcription factor activates downstream gene expression.
[0165] Examples of oxygen level-dependent transcription factors include, but
are
not limited to, FNR (fumarate and nitrate reductase), ANR, and DNR.
Corresponding
FNR-responsive promoters, ANR (anaerobic nitrate respiration)-responsive
promoters,
and DNR (dissimilatory nitrate respiration regulator)-responsive promoters are
known in
the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand
et al., 1991;
Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003), and non-
limiting
examples are shown in Table 1A.
[0166] In a non-limiting example, a promoter (PfnrS) was derived from the E.
coli
Nissle fumarate and nitrate reductase gene S (fnrS) that is known to be highly
expressed
under conditions of low or no environmental oxygen (Durand and Storz, 2010;
Boysen et
al, 2010). The PfnrS promoter is activated under anaerobic conditions by the
global
transcriptional regulator FNR that is naturally found in Nissle. Under
anaerobic
conditions, FNR forms a dimer and binds to specific sequences in the promoters
of
specific genes under its control, thereby activating their expression.
However, under
aerobic conditions, oxygen reacts with iron-sulfur clusters in FNR dimers and
converts
them to an inactive form. In this way, the PfnrS inducible promoter is adopted
to
modulate the expression of proteins or RNA. PfnrS is used interchangeably in
this
application as FNRS, fnrs, FNR, P-FNRS promoter and other such related
designations to
indicate the promoter PfnrS.
Table 1A. Examples of transcription factors and responsive genes and
regulatory
regions
Transcription Examples of responsive genes,
Factor promoters, and/or regulatory regions:
FNR nirB, ydfZ, pdhR, focA, ndH, hlyE, narK,
narX, narG, yfiD, tdcD
ANR arcDABC
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DNR norb, norC
[0167] As used herein, a "tunable regulatory region" refers to a nucleic acid
sequence under direct or indirect control of a transcription factor and which
is capable of
activating, repressing, derepressing, or otherwise controlling gene expression
relative to
levels of an inducer. In some embodiments, the tunable regulatory region
comprises a
promoter sequence. The inducer may be RNS, or other inducer described herein,
and the
tunable regulatory region may be a RNS-responsive regulatory region or other
responsive
regulatory region described herein. The tunable regulatory region may be
operatively
linked to a gene sequence(s) or gene cassette for the production of one or
more payloads,
e.g., a butyrogenic or other gene cassette or gene sequence(s). For example,
in one
specific embodiment, the tunable regulatory region is a RNS-derepressible
regulatory
region, and when RNS is present, a RNS-sensing transcription factor no longer
binds to
and/or represses the regulatory region, thereby permitting expression of the
operatively
linked gene or gene cassette. In this instance, the tunable regulatory region
derepresses
gene or gene cassette expression relative to RNS levels. Each gene or gene
cassette may
be operatively linked to a tunable regulatory region that is directly or
indirectly controlled
by a transcription factor that is capable of sensing at least one RNS.
[0168] In some embodiments, the exogenous environmental conditions are the
presence or absence of reactive oxygen species (ROS). In other embodiments,
the
exogenous environmental conditions are the presence or absence of reactive
nitrogen
species (RNS). In some embodiments, exogenous environmental conditions are
biological
molecules that are involved in the inflammatory response, for example,
molecules present
in an inflammatory disorder of the gut. In some embodiments, the exogenous
environmental conditions or signals exist naturally or are naturally absent in
the
environment in which the recombinant bacterial cell resides. In some
embodiments, the
exogenous environmental conditions or signals are artificially created, for
example, by the
creation or removal of biological conditions and/or the administration or
removal of
biological molecules.
[0169] In some embodiments, the exogenous environmental condition(s) and/or
signal(s) stimulates the activity of an inducible promoter. In some
embodiments, the
exogenous environmental condition(s) and/or signal(s) that serves to activate
the inducible
promoter is not naturally present within the gut of a mammal. In some
embodiments, the
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inducible promoter is stimulated by a molecule or metabolite that is
administered in
combination with the pharmaceutical composition of the disclosure, for
example,
tetracycline, arabinose, or any biological molecule that serves to activate an
inducible
promoter. In some embodiments, the exogenous environmental condition(s) and/or
signal(s) is added to culture media comprising a recombinant bacterial cell of
the
disclosure. In some embodiments, the exogenous environmental condition that
serves to
activate the inducible promoter is naturally present within the gut of a
mammal (for
example, low oxygen or anaerobic conditions, or biological molecules involved
in an
inflammatory response). In some embodiments, the loss of exposure to an
exogenous
environmental condition (for example, in vivo) inhibits the activity of an
inducible
promoter, as the exogenous environmental condition is not present to induce
the promoter
(for example, an aerobic environment outside the gut). "Gut" refers to the
organs, glands,
tracts, and systems that are responsible for the transfer and digestion of
food, absorption of
nutrients, and excretion of waste. In humans, the gut comprises the
gastrointestinal (GI)
tract, which starts at the mouth and ends at the anus, and additionally
comprises the
esophagus, stomach, small intestine, and large intestine. The gut also
comprises accessory
organs and glands, such as the spleen, liver, gallbladder, and pancreas. The
upper
gastrointestinal tract comprises the esophagus, stomach, and duodenum of the
small
intestine. The lower gastrointestinal tract comprises the remainder of the
small intestine,
i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum,
colon, rectum,
and anal canal. Bacteria can be found throughout the gut, e.g., in the
gastrointestinal tract,
and particularly in the intestines.
[0170] As used herein, the term "low oxygen" is meant to refer to a level,
amount,
or concentration of oxygen (02) that is lower than the level, amount, or
concentration of
oxygen that is present in the atmosphere (e.g., <21% 02, <160 ton 02)). Thus,
the term
"low oxygen condition or conditions" or "low oxygen environment" refers to
conditions or
environments containing lower levels of oxygen than are present in the
atmosphere. In
some embodiments, the term "low oxygen" is meant to refer to the level,
amount, or
concentration of oxygen (02) found in a mammalian gut, e.g., lumen, stomach,
small
intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal
sigmoid colon,
rectum, and anal canal. In some embodiments, the term "low oxygen" is meant to
refer to
a level, amount, or concentration of 02 that is 0-60 mmHg 02 (0-60 ton 02)
(e.g., 0, 1, 2,
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3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48,
49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, and 60 mmHg 02), including any and all incremental
fraction(s)
thereof (e.g., 0.2 mmHg, 0.5 mmHg 02, 0.75 mmHg 02, 1.25 mmHg 02, 2.175 mmHg
02,
3.45 mmHg 02, 3.75 mmHg 02, 4.5 mmHg 02, 6.8 mmHg 02, 11.35 mmHg 02, 46.3
mmHg 02, 58.75 mmHg, etc., which exemplary fractions are listed here for
illustrative
purposes and not meant to be limiting in any way). In some embodiments, "low
oxygen"
refers to about 60 mmHg 02 or less (e.g., 0 to about 60 mmHg 02). The term
"low
oxygen" may also refer to a range of 02 levels, amounts, or concentrations
between 0-60
mmHg 02 (inclusive), e.g., 0-5 mmHg 02, < 1.5 mmHg 02, 6-10 mmHg, < 8 mmHg, 47-
60 mmHg, etc. which listed exemplary ranges are listed here for illustrative
purposes and
not meant to be limiting in any way. See, for example, Albenberg et al.,
Gastroenterology, 147(5): 1055-1063 (2014); Bergofsky et al., J Clin. Invest.,
41(11):
1971- 1980 (1962); Crompton et al., J Exp. Biol., 43: 473-478 (1965); He et
al., PNAS
(USA), 96: 4586-4591 (1999); McKeown, Br. J. Radiol., 87:20130676 (2014) (doi:
10.1259/brj.20130676), each of which discusses the oxygen levels found in the
mammalian gut of various species and each of which are incorportated by
reference
herewith in their entireties. In some embodiments, the term "low oxygen" is
meant to
refer to the level, amount, or concentration of oxygen (02) found in a
mammalian organ or
tissue other than the gut, e.g., urogenital tract, tumor tissue, etc. in which
oxygen is present
at a reduced level, e.g., at a hypoxic or anoxic level. In some embodiments,
"low oxygen"
is meant to refer to the level, amount, or concentration of oxygen (02)
present in partially
aerobic, semi aerobic, microaerobic, nanoaerobic, microoxic, hypoxic, anoxic,
and/or
anaerobic conditions. For example, Table 1B summarizes the amount of oxygen
present in
various organs and tissues. In some embodiments, the level, amount, or
concentration of
oxygen (02) is expressed as the amount of dissolved oxygen ("DO") which refers
to the
level of free, non-compound oxygen (02) present in liquids and is typically
reported in
milligrams per liter (mg/L), parts per million (ppm; lmg/L = 1 ppm), or in
micromoles
(umole) (1 umole 02 = 0.022391 mg/L 02). Fondriest Environmental, Inc.,
"Dissolved
Oxygen", Fundamentals of Environmental Measurements, 19 Nov 2013,
www.fondriest.com/environmental-measurements/parameters/water-
quality/dissolved-
oxygen/>. In some embodiments, the term "low oxygen" is meant to refer to a
level,
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amount, or concentration of oxygen (02) that is about 6.0 mg/L DO or less,
e.g., 6.0 mg/L,
5.0 mg/L, 4.0 mg/L, 3.0 mg/L, 2.0 mg/L, 1.0 mg/L, or 0 mg/L, and any fraction
therein,
e.g., 3.25 mg/L, 2.5 mg/L, 1.75 mg/L, 1.5 mg/L, 1.25 mg/L, 0.9 mg/L, 0.8 mg/L,
0.7
mg/L, 0.6 mg/L, 0.5 mg/L, 0.4 mg/L, 0.3 mg/L, 0.2 mg/L and 0.1 mg/L DO, which
exemplary fractions are listed here for illustrative purposes and not meant to
be limiting in
any way. The level of oxygen in a liquid or solution may also be reported as a
percentage
of air saturation or as a percentage of oxygen saturation (the ratio of the
concentration of
dissolved oxygen (02) in the solution to the maximum amount of oxygen that
will dissolve
in the solution at a certain temperature, pressure, and salinity under stable
equilibrium).
Well-aerated solutions (e.g., solutions subjected to mixing and/or stirring)
without oxygen
producers or consumers are 100% air saturated. In some embodiments, the term
"low
oxygen" is meant to refer to 40% air saturation or less, e.g., 40%, 39%, 38%,
37%, 36%,
35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%,
20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%,
3%, 2%, 1%, and 0% air saturation, including any and all incremental
fraction(s) thereof
(e.g., 30.25%, 22.70%, 15.5%, 7.7%, 5.0%, 2.8%, 2.0%, 1.65%, 1.0%, 0.9%, 0.8%,
0.75%, 0.68%, 0.5%. 0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%,
0.04%.
0.032%, 0.025%, 0.01%, etc.) and any range of air saturation levels between 0-
40%,
inclusive (e.g., 0-5%, 0.05- 0.1%, 0.1-0.2%, 0.1-0.5%, 0.5 - 2.0%, 0-10%, 5-
10%, 10-
15%, 15-20%, 20-25%, 25-30%, etc.). The exemplary fractions and ranges listed
here are
for illustrative purposes and not meant to be limiting in any way. In some
embodiments,
the term "low oxygen" is meant to refer to 9% 02 saturation or less, e.g., 9%,
8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0%, 02 saturation, including any and all incremental
fraction(s)
thereof (e.g., 6.5%, 5.0%, 2.2%, 1.7%, 1.4%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%.
0.44%,
0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%. 0.032%, 0.025%, 0.01%,
etc.)
and any range of 02 saturation levels between 0-9%, inclusive (e.g., 0-5%,
0.05 - 0.1%,
0.1-0.2%, 0.1-0.5%, 0.5 - 2.0%, 0-8%, 5-7%, 0.3-4.2% 02, etc.). The exemplary
fractions
and ranges listed here are for illustrative purposes and not meant to be
limiting in any way.
Table 1B.
Compartment Oxygen Tension
stomach -60 torr (e.g., 58 +/- 15 ton)
duodenum and first part of -30 ton (e.g., 32 +/- 8 ton); -20% oxygen in
jejunum ambient air
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Ileum (mid- small intestine) ¨10 torr; ¨6% oxygen in ambient air (e.g., 11 +/-
3
ton)
Distal sigmoid colon ¨ 3 ton (e.g., 3 +/- 1 torr)
colon <2torr
Lumen of cecum <1 ton
tumor <32 ton (most tumors are <15 ton)
[0171] "Microorganism" refers to an organism or microbe of microscopic,
submicroscopic, or ultramicroscopic size that typically consists of a single
cell. Examples
of microrganisms include bacteria, viruses, parasites, fungi, certain algae,
yeast, e.g.,
Saccharomyces, and protozoa. In some aspects, the microorganism is engineered
("engineered microorganism") to produce one or more therpauetic molecules,
e.g., an
antinflammatory or barrier enhancer molecule. In certain embodiments, the
engineered
microorganism is an engineered bacterium. In certain embodiments, the
engineered
microorganism is an engineered virus.
[0172] "Non-pathogenic bacteria" refer to bacteria that are not capable of
causing
disease or harmful responses in a host. In some embodiments, non-pathogenic
bacteria are
Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-
positive bacteria. In some embodiments, non-pathogenic bacteria do not contain
lipopolysaccharides (LPS). In some embodiments, non-pathogenic bacteria are
commensal bacteria. Examples of non-pathogenic bacteria include, but are not
limited to
certain strains belonging to the genus Bacillus, Bacteroides, Bifidobacterium,
Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus,
Lactococcus,
Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus
subtilis,
Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron,
Bifidobacterium
bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium lon
gum,
Clostridium butyricum, Enterococcus faecium, Escherichia coli, Escherichia
coli Nissle,
Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei,
Lactobacillus
johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus
reuteri,
Lactobacillus rhamnosus,Lactococcus lactis and Saccharomyces boulardii
(Sonnenborn et
al., 2009; Dinleyici et al., 2014; U.S. Patent No. 6,835,376; U.S. Patent No.
6,203,797;
U.S. Patent No. 5,589,168; U.S. Patent No. 7,731,976). Non-pathogenic bacteria
also
include commensal bacteria, which are present in the indigenous microbiota of
the gut. In
one embodiment, the disclosure further includes non-pathogenic Saccharomyces,
such as
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Saccharomyces boulardii. Naturally pathogenic bacteria may be genetically
engineered to
reduce or eliminate pathogenicity.
[0173] "Probiotic" is used to refer to live, non-pathogenic microorganisms,
e.g.,
bacteria, which can confer health benefits to a host organism that contains an
appropriate
amount of the microorganism. In some embodiments, the host organism is a
mammal. In
some embodiments, the host organism is a human. In some embodiments, the
probiotic
bacteria are Gram-negative bacteria. In some embodiments, the probiotic
bacteria are
Gram-positive bacteria. Some species, strains, and/or subtypes of non-
pathogenic bacteria
are currently recognized as probiotic bacteria. Examples of probiotic bacteria
include, but
are not limited to, certain strains belonging to the genus Bifidobacteria,
Escherichia Coli,
Lactobacillus, and Saccharomyces e.g., Bifidobacterium bifidum, Enterococcus
faecium,
Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus
bulgaricus,
Lactobacillus paracasei, and Lactobacillus plantarum, and Saccharomyces
boulardii
(Dinleyici et al., 2014; U.S. Patent No. 5,589,168; U.S. Patent No. 6,203,797;
U.S. Patent
6,835,376). The probiotic may be a variant or a mutant strain of bacterium
(Arthur et al.,
2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006).
Non-
pathogenic bacteria may be genetically engineered to enhance or improve
desired
biological properties, e.g., survivability. Non-pathogenic bacteria may be
genetically
engineered to provide probiotic properties. Probiotic bacteria may be
genetically
engineered to enhance or improve probiotic properties.
[0174] As used herein, the term "modulate" and its cognates means to alter,
regulate, or adjust positively or negatively a molecular or physiological
readout, outcome,
or process, to effect a change in said readout, outcome, or process as
compared to a
normal, average, wild-type, or baseline measurement. Thus, for example,
"modulate" or
"modulation" includes up-regulation and down-regulation. A non-limiting
example of
modulating a readout, outcome, or process is effecting a change or alteration
in the normal
or baseline functioning, activity, expression, or secretion of a biomolecule
(e.g. a protein,
enzyme, cytokine, growth factor, hormone, metabolite, short chain fatty acid,
or other
compound). Another non-limiting example of modulating a readout, outcome, or
process
is effecting a change in the amount or level of a biomolecule of interest,
e.g. in the serum
and/or the gut lumen. In another non-limiting example, modulating a readout,
outcome, or
process relates to a phenotypic change or alteration in one or more disease
symptoms.
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Thus, "modulate" is used to refer to an increase, decrease, masking, altering,
overriding or
restoring the normal functioning, activity, or levels of a readout, outcome or
process (e.g,
biomolecule of interest, and/or molecular or physiological process, and/or a
phenotypic
change in one or more disease symptoms).
[0175] As used herein, the term "auxotroph" or "auxotrophic" refers to an
organism that requires a specific factor, e.g., an amino acid, a sugar, or
other nutrient) to
support its growth. An "auxotrophic modification" is a genetic modification
that causes
the organism to die in the absence of an exogenously added nutrient essential
for survival
or growth because it is unable to produce said nutrient. As used herein, the
term "essential
gene" refers to a gene which is necessary to for cell growth and/or survival.
Essential
genes are described in more detail infra and include, but are not limited to,
DNA synthesis
genes (such as thyA), cell wall synthesis genes (such as dapA), and amino acid
genes (such
as serA and metA).
[0176] As used herein, the terms "modulate" and "treat" a disease and their
cognates refer to an amelioration of a disease, disorder, and/or condition, or
at least one
discernible symptom thereof. In another embodiment, "modulate" and "treat"
refer to an
amelioration of at least one measurable physical parameter, not necessarily
discernible by
the patient. In another embodiment, "modulate" and "treat" refer to inhibiting
the
progression of a disease, disorder, and/or condition, either physically (e.g.,
stabilization of
a discernible symptom), physiologically (e.g., stabilization of a physical
parameter), or
both. In another embodiment, "modulate" and "treat" refer to slowing the
progression or
reversing the progression of a disease, disorder, and/or condition. As used
herein,
"prevent" and its cognates refer to delaying the onset or reducing the risk of
acquiring a
given disease, disorder and/or condition or a symptom associated with such
disease,
disorder, and/or condition.
[0177] Those in need of treatment may include individuals already having a
particular medical disorder, as well as those at risk of having, or who may
ultimately
acquire the disorder. The need for treatment is assessed, for example, by the
presence of
one or more risk factors associated with the development of a disorder, the
presence or
progression of a disorder, or likely receptiveness to treatment of a subject
having the
disorder. Treating autoimmune disorders and/or diseases and conditions
associated with
gut inflammation and/or compromised gut barrier function may encompass
reducing or
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eliminating excess inflammation and/or associated symptoms, and does not
necessarily
encompass the elimination of the underlying disease.
[0178] Treating the diseases described herein may encompass increasing levels
of
butyrate, increasing levels of acetate, increasing levels of butyrate and
increasing GLP-2,
IL-22, and/o rIL-10, and/or modulating levels of tryptophan and/or its
metabolites (e.g.,
kynurenine), and/or providing any other anti-inflammation and/or gut barrier
enhancer
molecule and does not necessarily encompass the elimination of the underlying
disease.
[0179] As used herein a "pharmaceutical composition" refers to a preparation
of
genetically engineered microorganism of the disclosure, e.g., genetically
engineered
bacteria or virus, with other components such as a physiologically suitable
carrier and/or
excipient.
[0180] The phrases "physiologically acceptable carrier" and "pharmaceutically
acceptable carrier" which may be used interchangeably refer to a carrier or a
diluent that
does not cause significant irritation to an organism and does not abrogate the
biological
activity and properties of the administered bacterial or viral compound. An
adjuvant is
included under these phrases.
[0181] The term "excipient" refers to an inert substance added to a
pharmaceutical
composition to further facilitate administration of an active ingredient.
Examples include,
but are not limited to, calcium bicarbonate, sodium bicarbonate calcium
phosphate,
various sugars and types of starch, cellulose derivatives, gelatin, vegetable
oils,
polyethylene glycols, and surfactants, including, for example, polysorbate 20.
[0182] The terms "therapeutically effective dose" and "therapeutically
effective
amount" are used to refer to an amount of a compound that results in
prevention, delay of
onset of symptoms, or amelioration of symptoms of a condition, e.g.,
inflammation,
diarrhea.an autoimmune disorder. A therapeutically effective amount may, for
example,
be sufficient to treat, prevent, reduce the severity, delay the onset, and/or
reduce the risk of
occurrence of one or more symptoms of an autoimmune a disorder and/or a
disease or
condition associated with gut inflammation and/or compromised gut barrier
function. A
therapeutically effective amount, as well as a therapeutically effective
frequency of
administration, can be determined by methods known in the art and discussed
below.
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[0183] As used herein, the term "bacteriostatic" or "cytostatic" refers to a
molecule
or protein which is capable of arresting, retarding, or inhibiting the growth,
division,
multiplication or replication of recombinant bacterial cell of the disclosure.
[0184] As used herein, the term "bactericidal" refers to a molecule or protein
which is capable of killing the recombinant bacterial cell of the disclosure.
[0185] As used herein, the term "toxin" refers to a protein, enzyme, or
polypeptide
fragment thereof, or other molecule which is capable of arresting, retarding,
or inhibiting
the growth, division, multiplication or replication of the recombinant
bacterial cell of the
disclosure, or which is capable of killing the recombinant bacterial cell of
the disclosure.
The term "toxin" is intended to include bacteriostatic proteins and
bactericidal proteins.
The term "toxin" is intended to include, but not limited to, lytic proteins,
bacteriocins
(e.g., microcins and colicins), gyrase inhibitors, polymerase inhibitors,
transcription
inhibitors, translation inhibitors, DNases, and RNases. The term "anti-toxin"
or
"antitoxin," as used herein, refers to a protein or enzyme which is capable of
inhibiting the
activity of a toxin. The term anti-toxin is intended to include, but not
limited to, immunity
modulators, and inhibitors of toxin expression. Examples of toxins and
antitoxins are
known in the art and described in more detail infra.
[0186] As used herein, "payload" refers to one or more molecules of interest
to be
produced by a genetically engineered microorganism, such as a bacteria or a
virus. In
some embodiments, the payload is a therapeutic payload, e.g. and
antiinflammatory or gut
barrier enhancer molecule, e.g. butyrate, acetate, propionate, GLP-2, IL-10,
IL-22, IL-2,
other interleukins, and/or tryptophan and/or one or more of its metabolites.
In some
embodiments, the payload is a regulatory molecule, e.g., a transcriptional
regulator such as
FNR. In some embodiments, the payload comprises a regulatory element, such as
a
promoter or a repressor. In some embodiments, the payload comprises an
inducible
promoter, such as from FNRS. In some embodiments the payload comprises a
repressor
element, such as a kill switch. In some embodiments the payload comprises an
antibiotic
resistance gene or genes. In some embodiments, the payload is encoded by a
gene,
multiple genes, gene cassette, or an operon. In alternate embodiments, the
payload is
produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or
biochemical pathway may optionally be endogenous to the microorganism. In
alternate
embodiments, the payload is produced by a biosynthetic or biochemical pathway,
wherein
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the biosynthetic or biochemical pathway is not endogenous to the
microorganism. In
some embodiments, the genetically engineered microorganism comprises two or
more
payloads.
[0187] As used herein, the term "conventional treatment" or "conventional
therapy" refers to treatment or therapy that is currently accepted, considered
current
standard of care, and/or used by most healthcare professionals for treating a
disease or
disorder associated with BCAA. It is different from alternative or
complementary
therapies, which are not as widely used.
[0188] As used herein, the term "polypeptide" includes "polypeptide" as well
as
"polypeptides," and refers to a molecule composed of amino acid monomers
linearly
linked by amide bonds (i.e., peptide bonds). The term "polypeptide" refers to
any chain or
chains of two or more amino acids, and does not refer to a specific length of
the product.
Thus, "peptides," "dipeptides," "tripeptides, "oligopeptides," "protein,"
"amino acid
chain," or any other term used to refer to a chain or chains of two or more
amino acids, are
included within the definition of "polypeptide," and the term "polypeptide"
may be used
instead of, or interchangeably with any of these terms. The term "polypeptide"
is also
intended to refer to the products of post-expression modifications of the
polypeptide,
including but not limited to glycosylation, acetylation, phosphorylation,
amidation,
derivatization, proteolytic cleavage, or modification by non-naturally
occurring amino
acids. A polypeptide may be derived from a natural biological source or
produced by
recombinant technology. In other embodiments, the polypeptide is produced by
the
genetically engineered bacteria or virus of the current invention. A
polypeptide of the
invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or
more, 25 or
more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or
more, or
2,000 or more amino acids. Polypeptides may have a defined three-dimensional
structure,
although they do not necessarily have such structure. Polypeptides with a
defined three-
dimensional structure are referred to as folded, and polypeptides, which do
not possess a
defined three-dimensional structure, but rather can adopt a large number of
different
conformations, are referred to as unfolded. The term "peptide" or
"polypeptide" may refer
to an amino acid sequence that corresponds to a protein or a portion of a
protein or may
refer to an amino acid sequence that corresponds with non-protein sequence,
e.g., a
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sequence selected from a regulatory peptide sequence, leader peptide sequence,
signal
peptide sequence, linker peptide sequence, and other peptide sequence.
[0189] An "isolated" polypeptide or a fragment, variant, or derivative thereof
refers to a polypeptide that is not in its natural milieu. No particular level
of purification is
required. Recombinantly produced polypeptides and proteins expressed in host
cells,
including but not limited to bacterial or mammalian cells, are considered
isolated for
purposed of the invention, as are native or recombinant polypeptides which
have been
separated, fractionated, or partially or substantially purified by any
suitable technique.
Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides
or proteins
produced by recombinant DNA techniques, i.e. produced from cells, microbial or
mammalian, transformed by an exogenous recombinant DNA expression construct
encoding the polypeptide. Proteins or peptides expressed in most bacterial
cultures will
typically be free of glycan. Fragments, derivatives, analogs or variants of
the foregoing
polypeptides, and any combination thereof are also included as polypeptides.
The terms
"fragment," "variant," "derivative" and "analog" include polypeptides having
an amino
acid sequence sufficiently similar to the amino acid sequence of the original
peptide and
include any polypeptides, which retain at least one or more properties of the
corresponding
original polypeptide. Fragments of polypeptides of the present invention
include
proteolytic fragments, as well as deletion fragments. Fragments also include
specific
antibody or bioactive fragments or immunologically active fragments derived
from any
polypeptides described herein. Variants may occur naturally or be non-
naturally occurring.
Non-naturally occurring variants may be produced using mutagenesis methods
known in
the art. Variant polypeptides may comprise conservative or non-conservative
amino acid
substitutions, deletions or additions.
[0190] Polypeptides also include fusion proteins. As used herein, the term
"variant" includes a fusion protein, which comprises a sequence of the
original peptide or
sufficiently similar to the original peptide. As used herein, the term "fusion
protein" refers
to a chimeric protein comprising amino acid sequences of two or more different
proteins.
Typically, fusion proteins result from well known in vitro recombination
techniques.
Fusion proteins may have a similar structural function (but not necessarily to
the same
extent), and/or similar regulatory function (but not necessarily to the same
extent), and/or
similar biochemical function (but not necessarily to the same extent) and/or
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immunological activity (but not necessarily to the same extent) as the
individual original
proteins which are the components of the fusion proteins."Derivatives" include
but are not
limited to peptides, which contain one or more naturally occurring amino acid
derivatives
of the twenty standard amino acids. "Similarity" between two peptides is
determined by
comparing the amino acid sequence of one peptide to the sequence of a second
peptide.
An amino acid of one peptide is similar to the corresponding amino acid of a
second
peptide if it is identical or a conservative amino acid substitution.
Conservative
substitutions include those described in Dayhoff, M. 0., ed., The Atlas of
Protein
Sequence and Structure 5, National Biomedical Research Foundation, Washington,
D.C.
(1978), and in Argos, EMBO J. 8 (1989), 779-785. For example, amino acids
belonging to
one of the following groups represent conservative changes or substitutions: -
Ala, Pro,
Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met, Ala, Phe; -
Lys, Arg, His;
-Phe, Tyr, Trp, His; and -Asp, Glu.
[0191] An antibody generally refers to a polypeptide of the immunoglobulin
family or a polypeptide comprising fragments of an immunoglobulin that is
capable of
noncovalently, reversibly, and in a specific manner binding a corresponding
antigen. An
exemplary antibody structural unit comprises a tetramer. Each tetramer is
composed of
two identical pairs of polypeptide chains, each pair having one "light" (about
25 kD) and
one "heavy" chain (about 50-70 kD), connected through a disulfide bond. The
recognized
immunoglobulin genes include the ic, k, a, y, 6, , and 11 constant region
genes, as well as
the myriad immunoglobulin variable region genes. Light chains are classified
as either lc
or k. Heavy chains are classified as y, 1,t, a, 6, or , which in turn define
the
immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. The N-
terminus of
each chain defines a variable region of about 100 to 110 or more amino acids
primarily
responsible for antigen recognition. The terms variable light chain (VL) and
variable
heavy chain (VH) refer to these regions of light and heavy chains
respectively.
[0192] As used herein, the term "antibody" or "antibodies"is meant to
encompasses all variations of antibody and fragments thereof that possess one
or more
particular binding specificities. Thus, the term "antibody" or "antibodies" is
meant to
include full length antibodies, chimeric antibodies, humanized antibodies,
single chain
antibodies (ScFv, camelids), Fab, Fab', multimeric versions of these fragments
(e.g.,
F(ab')2), single domain antibodies (sdAB, VHH framents), heavy chain
antibodies
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(HCAb), nanobodies, diabodies, and minibodies. Antibodies can have more than
one
binding specificity, e.g., be bispecific. The term "antibody" is also meant to
include so-
called antibody mimetics. Antibody mimetics refers to small molecules, e.g., 3-
30 kDa,
which can be single amino acid chain molecules, which can specifically bind
antigens but
do not have an antibody-related structure. Antibody mimetics, include, but are
not limited
to, Affibody molecules (Z domain of Protein A), Affilins (Gamma-B
crystalline),
Ubiquitin, Affimers (Cystatin), Affitins (Sac7d (from Sulfolobus
acidocaldarius),
Alphabodies (Triple helix coiled coil), Anticalins (Lipocalins), Avimers
(domains of
various membrane receptors), DARPins (Ankyrin repeat motif), Fynomers (SH3
domain
of Fyn), Kunitz domain peptides Kunitz domains of various protease
inhibitors),
Ecallantide (Kalbitor), and Monobodies. In certain aspects, the term
"antibody" or
"antibodies" is meant to refer to a single chain antibody(ies), single domain
antibody(ies),
and camelid antibody(ies). Utility of antibodies in the treatment of cancer
and additional
anti cancer antibodies can for example be found in Scott et al., Antibody
Therapy for
Cancer, Nature Reviews Cancer April 2012 Volume 12, incorporated by reference
in its
entirety.
[0193] A "single-chain antibody" or "single-chain antibodies" typically refers
to a
peptide comprising a heavy chain of an immunoglobulin, a light chain of an
immunoglobulin, and optionally a linker or bond, such as a disulfide bond. The
single-
chain antibody lacks the constant Fc region found in traditional antibodies.
In some
embodiments, the single-chain antibody is a naturally occurring single-chain
antibody,
e.g., a camelid antibody. In some embodiments, the single-chain antibody is a
synthetic,
engineered, or modified single-chain antibody. In some embodiments, the single-
chain
antibody is capable of retaining substantially the same antigen specificity as
compared to
the original immunoglobulin despite the addition of a linker and the removal
of the
constant regions. In some aspects, the single chain antibody can be a "scFv
antibody",
which refers to a fusion protein of the variable regions of the heavy (VH) and
light chains
(VL) of immunoglobulins (without any constant regions), optionally connected
with a
short linker peptide of ten to about 25 amino acids, as described, for
example, in U.S.
Patent No. 4,946,778, the contents of which is herein incorporated by
reference in its
entirety. The Fv fragment is the smallest fragment that holds a binding site
of an antibody,
which binding site may, in some aspects, maintain the specificity of the
original antibody.
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Techniques for the production of single chain antibodies are described in U.S.
Patent No.
4,946,778. The Vh and VL sequences of the scFv can be connected via the N-
terminus of
the VH connecting to the C-terminus of the VL or via the C-terminus of the VH
connecting to the N-terminus of the VL. ScFv fragments are independent folding
entities
that can be fused indistinctively on either end to other epitope tags or
protein domains.
Linkers of varying length can be used to link the Vh and VL sequences, which
the linkers
can be glycine rich (provides flexibility) and serine or threonine rich
(increases solubility).
Short linkers may prevent association of the two domains and can result in
multimers
(diabodies, tribodies, etc.). Long linkers may result in proteolysis or weak
domain
association (described in Voelkel et al el., 2011). Linkers of length between
15 and 20
amino acids or 18 and 20 amino acids are most often used. Additional non-
limiting
examples of linkers, including other flexible linkers are described in Chen et
al., 2013
(Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Fusion Protein Linkers:
Property,
Design and Functionality), the contents of which is herein incorporated by
reference in its
entirety. Flexible linkers are also rich in small or polar amino acids such as
Glycine and
Serine, but can contain additional amino acids such as Threonine and Alanine
to maintain
flexibility, as well as polar amino acids such as Lysine and Glutamate to
improve
solubility. Exemplary linkers include, but are not limited to, (Gly-Gly-Gly-
Gly-Ser)n,
KESGSVSSEQLAQFRSLD and EGKSSGSGSESKST, (Gly)8, and Gly and Ser rich
flexible linker, GSAGSAAGSGEF. "Single chain antibodies" as used herein also
include
single-domain antibodies, which include camelid antibodies and other heavy
chain
antibodies, light chain antibodies, including nanobodies and single domains VH
or VL
domains derived from human, mouse or other species. Single domain antibodies
may be
derived from any species including, but not limited to mouse, human, camel,
llama, fish,
shark, goat, rabbit, and bovine. Single domain antibodies include domain
antigen-binding
units which have a camelid scaffold, derived from camels, llamas, or alpacas.
Camelids
produce functional antibodies devoid of light chains. The heavy chain variable
(VH)
domain folds autonomously and functions independently as an antigen-binding
unit. Its
binding surface involves only three CDRs as compared to the six CDRs in
classical
antigen-binding molecules (Fabs) or single chain variable fragments (scFvs).
Camelid
antibodies are capable of attaining binding affinities comparable to those of
conventional
antibodies. Camelid scaffold-based antibodies can be produced using methods
well known
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in the art. Cartilaginous fishes also have heavy-chain antibodies (IgNAR,
'immunoglobulin
new antigen receptor'), from which single-domain antibodies called VNAR
fragments can
be obtained. Alternatively, the dimeric variable domains from IgG from humans
or mice
can be split into monomers. Nanobodies are single chain antibodies derived
from light
chains. The term "single chain antibody" also refers to antibody mimetics.
[0194] In some embodiments, the antibodies expressed by the engineered
microorganisms are bispecfic. In certain embodiments, a bispecific antibody
molecule
comprises a scFv, or fragment thereof, have binding specificity for a first
epitope and a
scFv, or fragment thereof, have binding specificity for a second epitope.
Antigen-binding
fragments or antibody portions include bivalent scFv (diabody), bispecific
scFv antibodies
where the antibody molecule recognizes two different epitopes, single binding
domains
(dAbs), and minibodies. Monomeric single-chain diabodies (scDb) are readily
assembled
in bacterial and mammalian cells and show improved stability under
physiological
conditions (Voelkel et al., 2001 and references therein; Protein Eng. (2001)
14 (10): 815-
823 (describes optimized linker sequences for the expression of monomeric and
dimeric
bispecific single-chain diabodies).
[0195] As used herein, the term "sufficiently similar" means a first amino
acid
sequence that contains a sufficient or minimum number of identical or
equivalent amino
acid residues relative to a second amino acid sequence such that the first and
second amino
acid sequences have a common structural domain and/or common functional
activity. For
example, amino acid sequences that comprise a common structural domain that is
at least
about 45%, at least about 50%, at least about 55%, at least about 60%, at
least about 65%,
at least about 70%, at least about 75%, at least about 80%, at least about
85%, at least
about 90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%,
at least about 95%, at least about 96%, at least about 97%, at least about
98%, at least
about 99%, or at least about 100%, identical are defined herein as
sufficiently similar.
Preferably, variants will be sufficiently similar to the amino acid sequence
of the peptides
of the invention. Such variants generally retain the functional activity of
the peptides of
the present invention. Variants include peptides that differ in amino acid
sequence from
the native and wt peptide, respectively, by way of one or more amino acid
deletion(s),
addition(s), and/or substitution(s). These may be naturally occurring variants
as well as
artificially designed ones.
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[0196] As used herein the term "linker", "linker peptide" or "peptide linkers"
or
"linker" refers to synthetic or non-native or non-naturally-occurring amino
acid sequences
that connect or link two polypeptide sequences, e.g., that link two
polypeptide domains.
As used herein the term "synthetic" refers to amino acid sequences that are
not naturally
occurring. Exemplary linkers are described herein. Additional exemplary
linkers are
provided in US 20140079701, the contents of which are herein incorporated by
reference
in its entirety.
[0197] As used herein the term "codon-optimized" refers to the modification of
codons in the gene or coding regions of a nucleic acid molecule to reflect the
typical
codon usage of the host organism without altering the polypeptide encoded by
the nucleic
acid molecule. Such optimization includes replacing at least one, or more than
one, or a
significant number, of codons with one or more codons that are more frequently
used in
the genes of the host organism. A "codon-optimized sequence" refers to a
sequence, which
was modified from an existing coding sequence, or designed, for example, to
improve
translation in an expression host cell or organism of a transcript RNA
molecule transcribed
from the coding sequence, or to improve transcription of a coding sequence.
Codon
optimization includes, but is not limited to, processes including selecting
codons for the
coding sequence to suit the codon preference of the expression host organism.
Many
organisms display a bias or preference for use of particular codons to code
for insertion of
a particular amino acid in a growing polypeptide chain. Codon preference or
codon bias,
differences in codon usage between organisms, is allowed by the degeneracy of
the
genetic code, and is well documented among many organisms. Codon bias often
correlates
with the efficiency of translation of messenger RNA (mRNA), which is in turn
believed to
be dependent on, inter alia, the properties of the codons being translated and
the
availability of particular transfer RNA (tRNA) molecules. The predominance of
selected
tRNAs in a cell is generally a reflection of the codons used most frequently
in peptide
synthesis. Accordingly, genes can be tailored for optimal gene expression in a
given
organism based on codon optimization.
[0198] As used herein, the terms "secretion system" or "secretion protein"
refers to
a native or non-native secretion mechanism capable of secreting or exporting a
biomolecule, e.g., polypeptide from the microbial, e.g., bacterial cytoplasm.
The secretion
system may comprise a single protein or may comprise two or more proteins
assembled in
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a complex e.g.,HlyBD. Non-limiting examples of secretion systems for gram
negative
bacteria include the modified type III flagellar, type I (e.g., hemolysin
secretion system),
type II, type IV, type V, type VI, and type VII secretion systems, resistance-
nodulation-
division (RND) multi-drug efflux pumps, various single membrane secretion
systems.
Non-liming examples of secretion systems for gram positive bacteria include
Sec and TAT
secretion systems. In some embodiments, the polypeptide to be secreted include
a
"secretion tag" of either RNA or peptide origin to direct the polypeptide to
specific
secretion systems. In some embodiments, the secretion system is able to remove
this tag
before secreting the polyppetide from the engineered bacteria. For example, in
Type
V auto-secretion-mediated secretion the N-terminal peptide secretion tag is
removed upon
translocation of the "passenger" peptide from the cytoplasm into the
periplasmic
compartment by the native Sec system. Further, once the auto-secretor is
translocated
across the outer membrane the C-terminal secretion tag can be removed by
either an
autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the
antinflammatory or barrier enhancer molecule(s) into the extracellular milieu.
In some
embodiments, the secretion system involves the generation of a "leaky" or de-
stabilized
outer membrane, which may be accomplished by deleting or mutagenizing genes
responsible for tethering the outer membrane to the rigid peptidoglycan
skeleton,
including for example, 1pp, ompC, ompA, ompF, tolA, to1B, pal, degS, degP, and
nlpl.
Lpp functions as the primary 'staple' of the bacterial cell wall to the
peptidoglycan. To1A-
PAL and OmpA complexes function similarly to Lpp and are other deletion
targets to
generate a leaky phenotype. Additionally, leaky phenotypes have been observed
when
periplasmic proteases, such as degS, degP or nlpl, are deactivated. Thus, in
some
embodiments, the engineered bacteria have one or more deleted or mutated
membrane
genes, e.g., selected from 1pp, ompA, ompA, ompF, tolA, to1B, and pal genes.
In some
embodiments, the engineered bacteria have one or more deleted or mutated
periplasmic
protease genes, e.g., selected from degS, degP, and nlpl. In some embodiments,
the
engineered bacteria have one or more deleted or mutated gene(s), selected from
1pp,
ompA, ompA, ompF, tolA, to1B, pal, degS, degP, and nlpl genes.
[0199] The articles "a" and "an," as used herein, should be understood to mean
"at
least one," unless clearly indicated to the contrary.
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[0200] The phrase "and/or," when used between elements in a list, is intended
to
mean either (1) that only a single listed element is present, or (2) that more
than one
element of the list is present. For example, "A, B, and/or C" indicates that
the selection
may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C.
The
phrase "and/or" may be used interchangeably with "at least one of' or "one or
more of'
the elements in a list.
[0201] Ranges provided herein are understood to be shorthand for all of the
values
within the range. For example, a range of 1 to 50 is understood to include any
number,
combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5,
6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
Bacteria
[0202] The genetically engineered microorganisms, or programmed
microorganisms, such as genetically engineered bacteria of the disclosure are
capable of
producing one or more non-native anti-inflammation and/or gut barrier function
enhancer
molecules. In certain embodiments, the genetically engineered bacteria are
obligate
anaerobic bacteria. In certain embodiments, the genetically engineered
bacteria are
facultative anaerobic bacteria. In certain embodiments, the genetically
engineered bacteria
are aerobic bacteria. In some embodiments, the genetically engineered bacteria
are Gram-
positive bacteria. In some embodiments, the genetically engineered bacteria
are Gram-
positive bacteria and lack LPS. In some embodiments, the genetically
engineered bacteria
are Gram-negative bacteria. In some embodiments, the genetically engineered
bacteria are
Gram-positive and obligate anaerobic bacteria. In some embodiments, the
genetically
engineered bacteria are Gram-positive and facultative anaerobic bacteria. In
some
embodiments, the genetically engineered bacteria are non-pathogenic bacteria.
In some
embodiments, the genetically engineered bacteria are commensal bacteria. In
some
embodiments, the genetically engineered bacteria are probiotic bacteria. In
some
embodiments, the genetically engineered bacteria are naturally pathogenic
bacteria that are
modified or mutated to reduce or eliminate pathogenicity. Exemplary bacteria
include, but
are not limited to, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria,
Caulobacter,
Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus,
Listeria,
Mycobacterium, Saccharomyces, Salmonella, Staphylococcus, Streptococcus,
Vibrio,
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Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides
subtilis, Bacteroides
thetaiotaomicron, Bifidobacterium adolescentis, Bifidobacterium bifidum,
Bifidobacterium breve UCC2003, Bifidobacterium infantis, Bifidobacterium
lactis,
Bifidobacterium longum, Clostridium acetobutylicum, Clostridium butyricum,
Clostridium butyricum M-55, Clostridium cochlearum, Clostridium felsineum,
Clostridium histolyticum, Clostridium multifermentans, Clostridium novyi-NT,
Clostridium paraputrificum, Clostridium pasteureanum, Clostridium
pectinovorum,
Clostridium perfringens, Clostridium roseum, Clostridium sporogenes,
Clostridium
tertium, Clostridium tetani, Clostridium tyrobutyricum, Corynebacterium
parvum,
Escherichia coli MG1655, Escherichia coli Nissle 1917, Listeria monocytogenes,
Mycobacterium bovis, Salmonella choleraesuis, Salmonella typhimurium, and
Vibrio
cholera. In certain embodiments, the genetically engineered bacteria are
selected from the
group consisting of Enterococcus faecium, Lactobacillus acidophilus,
Lactobacillus
bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus
paracasei,
Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus,
Lactococcus
lactis, and Saccharomyces boulardii, Clostridium clusters IV and XIVa of
Firmicutes
(including species of Eubacterium), Roseburia, Faecalibacterium, Enterobacter,
Faecalibacterium prausnitzii, Clostridium difficile, Subdoligranulum,
Clostridium
sporogenes, Campylobacter jejuni, Clostridium saccharolyticum, Klebsiella,
Citrobacter,
Pseudobutyrivibrio, and Ruminococcus. In certain embodiments, the the
genetically
engineered bacteria are selected from Bacteroides fragilis, Bacteroides
thetaiotaomicron,
Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis,
Bifidobacterium
lactis, Clostridium butyricum, Escherichia coli, Escherichia coli Nissle,
Lactobacillus
acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus
lactis
[0203] In some embodiments, the genetically engineered bacterium is a Gram-
positive bacterium, e.g., Clostridium, that is naturally capable of producing
high levels of
butyrate. In some embodiments, the genetically engineered bacterium is
selected from the
group consisting of C. butyricum ZJUCB, C. butyricum S21, C. thermobutyricum
ATCC
49875, C. beijerinckii, C. populeti ATCC 35295, C. tyrobutyricum JM1, C.
tyrobutyricum
CIP 1-776, C. tyrobutyricum ATCC 25755, C. tyrobutyricum CNRZ 596, and C.
tyrobutyricum ZJU 8235. In some embodiments, the genetically engineered
bacterium is
C. butyricum CBM588, a probiotic bacterium that is highly amenable to protein
secretion
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and has demonstrated efficacy in treating IBD (Kanai et al., 2015). In some
embodiments, the genetically engineered bacterium is Bacillus, a probiotic
bacterium that
is highly genetically tractable and has been a popular chassis for industrial
protein
production; in some embodiments, the bacterium has highly active secretion
and/or no
toxic byproducts (Cutting, 2011).
[0204] In one embodiment, the bacterial cell is a Bacteroides fragilis
bacterial cell.
In one embodiment, the bacterial cell is a Bacteroides thetaiotaomicron
bacterial cell. In
one embodiment, the bacterial cell is a Bacteroides subtilis bacterial cell.
In one
embodiment, the bacterial cell is a Bifidobacterium bifidum bacterial cell. In
one
embodiment, the bacterial cell is a Bifidobacterium infantis bacterial cell.
In one
embodiment, the bacterial cell is a Bifidobacterium lactis bacterial cell. In
one
embodiment, the bacterial cell is a Clostridium butyricum bacterial cell. In
one
embodiment, the bacterial cell is an Escherichia coli bacterial cell. In one
embodiment,
the bacterial cell is a Lactobacillus acidophilus bacterial cell. In one
embodiment, the
bacterial cell is a Lactobacillus plantarum bacterial cell. In one embodiment,
the bacterial
cell is a Lactobacillus reuteri bacterial cell. In one embodiment, the
bacterial cell is a
Lactococcus lactis bacterial cell.
[0205] In some embodiments, the genetically engineered bacteria are
Escherichia
coli strain Nissle 1917 (E. coli Nissle), a Gram-negative bacterium of the
Enterobacteriaceae family that has evolved into one of the best characterized
probiotics
(Ukena et al., 2007). The strain is characterized by its complete harmlessness
(Schultz,
2008), and has GRAS (generally recognized as safe) status (Reister et al.,
2014, emphasis
added). Genomic sequencing confirmed that E. coli Nissle lacks prominent
virulence
factors (e.g., E. coli a-hemolysin, P-fimbrial adhesins) (Schultz, 2008). In
addition, it has
been shown that E. coli Nissle does not carry pathogenic adhesion factors,
does not
produce any enterotoxins or cytotoxins, is not invasive, and not uropathogenic
(Sonnenborn et al., 2009). As early as in 1917, E. coli Nissle was packaged
into medicinal
capsules, called Mutaflor, for therapeutic use. E. coli Nissle has since been
used to treat
ulcerative colitis in humans in vivo (Rembacken et al., 1999), to treat
inflammatory bowel
disease, Crohn's disease, and pouchitis in humans in vivo (Schultz, 2008), and
to inhibit
enteroinvasive Salmonella, Legionella, Yersinia, and Shigella in vitro
(Altenhoefer et al.,
2004). It is commonly accepted that E. coli Nissle's therapeutic efficacy and
safety have
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convincingly been proven (Ukena et al., 2007). In some embodiments, the
genetically
engineered bacteria are E. coli Nissle and are naturally capable of promoting
tight
junctions and gut barrier function. In some embodiments, the genetically
engineered
bacteria are E. coli and are highly amenable to recombinant protein
technologies.
[0206] One of ordinary skill in the art would appreciate that the genetic
modifications disclosed herein may be adapted for other species, strains, and
subtypes of
bacteria. It is known, for example, that the clostridial butyrogenic pathway
genes are
widespread in the genome-sequenced clostridia and related species (Aboulnaga
et al.,
2013). Furthermore, genes from one or more different species of bacteria can
be
introduced into one another, e.g., the butyrogenic genes fromPeptoclostridium
difficile
have been expressed in Escherichia coli (Aboulnaga et al., 2013).
[0207] . In one embodiment, the recombinant bacterial cell does not colonize
the
subject having the disorder. Unmodified E. coli Nissle and the genetically
engineered
bacteria of the invention may be destroyed, e.g., by defense factors in the
gut or blood
serum (Sonnenborn et al., 2009) or by activation of a kill switch, several
hours or days
after administration. Thus, the genetically engineered bacteria may require
continued
administration. Residence time in vivo may be calculated for the genetically
engineered
bacteria. In some embodiments, the residence time is calculated for a human
subject. In
some embodiments, residence time in vivo is calculated for the genetically
engineered
bacteria of the invention, e.g. as described herein.
[0208] In some embodiments, the bacterial cell is a genetically engineered
bacterial cell. In another embodiment, the bacterial cell is a recombinant
bacterial cell. In
some embodiments, the disclosure comprises a colony of bacterial cells
disclosed herein.
[0209] In another aspect, the disclosure provides a recombinant bacterial
culture
which comprises bacterial cells disclosed herein.
[0210] In some embodiments, the genetically engineered bacteria comprising an
anti-inflammatory or gut barrier enhancer molecule further comprise a kill-
switch circuit,
such as any of the kill-switch circuits provided herein. For example, in some
embodiments, the genetically engineered bacteria further comprise one or more
genes
encoding one or more recombinase(s) under the control of an inducible
promoter, and an
inverted toxin sequence. In some embodiments, the genetically engineered
bacteria
further comprise one or more genes encoding an antitoxin. In some embodiments,
the
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engineered bacteria further comprise one or more genes encoding one or more
recombinase(s) under the control of an inducible promoter and one or more
inverted
excision genes, wherein the excision gene(s) encode an enzyme that deletes an
essential
gene. In some embodiments, the genetically engineered bacteria further
comprise one or
more genes encoding an antitoxin. In some embodiments, the engineered bacteria
further
comprise one or more genes encoding a toxin under the control of a promoter
having a
TetR repressor binding site and a gene encoding the TetR under the control of
an inducible
promoter that is induced by arabinose, such as ParaBAD. In some embodiments,
the
genetically engineered bacteria further comprise one or more genes encoding an
antitoxin.
[0211] In some embodiments, the genetically engineered bacteria is an
auxotroph
comprising gene sequence encoding an anti-inflammatory or gut barrier enhancer
molecule and further comprises a kill-switch circuit, such as any of the kill-
switch circuits
described herein.
[0212] In some embodiments of the above described genetically engineered
bacteria, the gene encoding an anti-inflammatory or gut barrier enhancer
molecule is
present on a plasmid in the bacterium. In some embodiments, the gene
sequence(s)
encoding an anti-inflammatory or gut barrier enhancer molecule is present in
the bacterial
chromosome. In some embodiments, a gene sequence encoding a secretion protein
or
protein complex, such as any of the secretion systems disclosed herein, for
secreting a
biomolecule (e.g. an anti-inflammatory or gut barrier enhancer molecule), is
present on a
plasmid in the bacterium. In some embodiments, the gene sequence encoding a
secretion
protein or protein complex for secreting a biomolecule, such as any of the
secretion
systems disclosed herein, is present in the bacterial chromosome. In some
embodiments,
the gene sequence(s) encoding an antibiotic resistance gene is present on a
plasmid in the
bacterium. In some embodiments, the gene sequence(s) encoding an antibiotic
resistance
gene is present in the bacterial chromosome.
Anti-inflammation and/or gut barrier function enhancer molecules
[0213] The genetically engineered bacteria comprise one or more gene
sequence(s)
and/or gene cassette(s) for producing a non-native anti-inflammation and/or
gut barrier
function enhancer molecule. In some embodiments, the genetically engineered
bacteria
comprise one or more gene sequence(s) for producing a non-native anti-
inflammation
and/or gut barrier function enhancer molecule. For example, the genetically
engineered
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bacteria may comprise two or more gene sequence(s) for producing a non-native
anti-
inflammation and/or gut barrier function enhancer molecule. In some
embodiments, the
two or more gene sequences are multiple copies of the same gene. In some
emodiments,
the two or more gene sequences are sequences encoding different genes. In some
emodiments, the two or more gene sequences are sequences encoding multiple
copies of
one or more different genes. In some embodiments, the genetically engineered
bacteria
comprise one or more gene cassette(s) for producing a non-native anti-
inflammation
and/or gut barrier function enhancer molecule. For example, the genetically
engineered
bacteria may comprise two or more gene cassette(s) for producing a non-native
anti-
inflammation and/or gut barrier function enhancer molecule. In some
embodiments, the
two or more gene cassettes are multiple copies of the same gene cassette. In
some
emodiments, the two or more gene cassettes are different gene cassettes for
producing
either the same or different anti-inflammation and/or gut barrier function
enhancer
molecule(s). In some emodiments, the two or more gene cassettes are gene
cassettes for
producing multiple copies of one or more different anti-inflammation and/or
gut barrier
function enhancer molecule(s). In some embodiments, the anti-inflammation
and/or gut
barrier function enhancer molecule is selected from the group consisting of a
short-chain
fatty acid, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase
(SOD), GLP-2,
GLP-1, IL-10 (human or viral), IL-27, TGF-(31, TGF-(32, N-
acylphosphatidylethanolamines (NAPEs), elafin (also known as peptidase
inhibitor 3 or
SKALP), trefoil factor, melatonin, PGD2, kynurenic acid, kynurenine, typtophan
metabolite, indole, indole metabolite, a single-chain variable fragment
(scFv), antisense
RNA, siRNA, or shRNA that neutralizes TNF-a, IFN-y, IL-113, IL-6, IL-8, IL-17,
and/or
chemokines, e.g., CXCL-8 and CCL2, AHR agonist (e.g., indole acetic acid,
indole-3-
aldehyde, and indole), PXR agonist (e.g., IPA), HDAC inhibitor (e.g.,
butyrate), GPR41
and/or GPR43 activator (e.g., butyrate and/or propionate and/or acetate),
GPR109A
activator (e.g., butyrate), inhibitor of NF-kappaB signaling (e.g., butyrate),
modulator of
PPARgamma (e.g., butyrate), activator of AMPK signaling (e.g., acetate),
modulator of
GLP-1 secretion, and hydroxyl radical scavengers and antioxidants (e.g., IPA).
A
molecule may be primarily anti-inflammatory, e.g., IL-10, or primarily gut
barrier function
enhancing, e.g., GLP-2. Alternatively, a molecule may be both anti-
inflammatory and gut
barrier function enhancing.
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[0214] In some embodiments, the genetically engineered bacteria of the
invention
express one or more anti-inflammation and/or gut barrier function enhancer
molecule(s)
that is encoded by a single gene, e.g., the molecule is elafin and encoded by
the PI3 gene,
or the molecule is interleukin-10 and encoded by the IL10 gene. In alternate
embodiments, the genetically engineered bacteria of the invention encode one
or more an
anti-inflammation and/or gut barrier function enhancer molecule(s), e.g.,
butyrate, that is
synthesized by a biosynthetic pathway requiring multiple genes.
[0215] The one or more gene sequence(s) and/or gene cassette(s) may be
expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some
embodiments, expression from the plasmid may be useful for increasing
expression of the
anti-inflammation and/or gut barrier function enhancer molecule(s). In some
embodiments, expression from the chromosome may be useful for increasing
stability of
expression of the anti-inflammation and/or gut barrier function enhancer
molecule(s). In
some embodiments, the gene sequence(s)or gene cassette(s) for producing the
anti-
inflammation and/or gut barrier function enhancer molecule(s) is integrated
into the
bacterial chromosome at one or more integration sites in the genetically
engineered
bacteria. For example, one or more copies of the butyrate biosynthesis gene
cassette may
be integrated into the bacterial chromosome. In some embodiments, the gene
sequence(s)
or gene cassette(s) for producing the anti-inflammation and/or gut barrier
function
enhancer molecule(s) is expressed from a plasmid in the genetically engineered
bacteria.
In some embodiments, the gene sequence(s) or gene cassette(s) for producing
the anti-
inflammation and/or gut barrier function enhancer molecule(s) is inserted into
the bacterial
genome at one or more of the following insertion sites in E. coli Nissle:
malE/K,
araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see,
e.g., Fig. 52
for exemplary insertion sites). The insertion site may be anywhere in the
genome, e.g., in
a gene required for survival and/or growth, such as thyA (to create an
auxotroph); in an
active area of the genome, such as near the site of genome replication; and/or
in between
divergent promoters in order to reduce the risk of unintended transcription,
such as
between AraB and AraC of the arabinose operon.
Short chain Fatty Acids and Tryptophan Metabolites
[0216] One strategy in the treatment, prevention, and/or management of
inflammatory bowel disorders may include approaches to help maintain and/or
reestablish
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gut barrier function, e.g. through the prevention, treatment and/or management
of
inflammatory events at the root of increased permeability, e.g. through the
administration
of anti-inflammatory effectors.
[0217] For example, leading metabolites that play gut-protective roles are
short
chain fatty acids, e.g. acetate, butyrate and propionate, and those derived
from tryptophan
metabolism. These metabolites have been shown to play a major role in the
prevention of
inflammatory disease. As such one approach in the treatment, prevention,
and/or
management of gut barrier health may be to provide a treatment which contains
one or
more of such metabolites.
[0218] For example, butyrate and other SCFA, e.g., derived from the
microbiota,
are known to promote maintaining intestinal integrity (e.g., as reviewed in
Thorburn et al.,
Diet, Metabolites, and "Western-Lifestyle" Inflammatory Diseases; Immunity
Volume 40,
Issue 6, 19 June 2014, Pages 833-842). (A) SCFA-induced promotion of mucus by
gut
epithelial cells, possibly through signaling through metabolite sensing GPCRs;
(B) SCFA-
induced secretion of IgA by B cells; (C) SCFA-induced promotion of tissue
repair and
wound healing; (D) SCFA-induced promotion of Treg cell development in the gut
in a
process that presumably facilitates immunological tolerance; (E) SCFA-
mediated
enhancement of epithelial integrity in a process dependent on inflammasome
activation
(e.g., via NALP3) and IL-18 production; and (F) anti-inflammatory effects,
inhibition of
inflammatory cytokine production (e.g., TNF, 11-6, and IFN-gamma), and
inhibition of
NF-KB. Many of these actions of SCFAs in gut homeostatis can be ascribed to
GPR43 and
GPR109A, which are expressed by the colonic epithelium, by inflammatory
leukocytes
(e.g. neutrophils and marcophages) and by Treg cells. These receptors signal
through G
proteins, coupled to MAPK, PI3K and mTOR, as well as a separate arrestin-
pathway,
leading to NFkappa B inhibition. Other effects can be ascribed to SCFA-
mediated HDAC
inhibition, e.g. butyrate, which may regulate macrophage function and promote
TReg
cells.
[0219] In addition, a number of tryptophan metabolites, including kynurenine
and
kynurenic acid, as well as several indoles, such as indole-3 aldehyde, indole-
3 propionic
acid, and several other indole metabolites (which can be derived from
microbiota or the
diet) described infra, have been shown to be essential for gut homeostais and
promote gut-
barrier health. These metabolites bind to aryl hydrocarbon receptor (Ahr).
After agonist
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binding, AhR translocates to the nucleus, where it forms a heterodimer with
AhR nuclear
translocator (ARNT). AhR-dependent gene expression includes genes involved in
the
production of mediators important for gut homeostasis; these mediators include
IL-22,
antimicrobicidal factors, increased Th17 cell activity, and the maintenance of
intraepithelial lymphocytes and RORyt+ innate lymphoid cells.
[0220] Tryptophan can also be transported across the epithelium by transport
machinery comprising angiotensin I converting enzyme 2 (Ace2). Tryptophan is
degraded
to kynurenine, another AhR agonist, by the immune-regulatory enzyme
indoleamine 2,3-
dioxygenase (IDO), which is linked to suppression of T cell responses,
promotion of Treg
cells, and immune tolerance. Moreover, a number of tryptophan metabolites,
including
kynurenic acid and niacin, agonize metabolite-sensing GPCRs, such as GPR35 and
GPR109A and thus multiple elements of tryptophan catabolism facilitate gut
homeostasis.
[0221] In addition, some indole metabolites, e.g., indole 3-propionic acid
(IPA),
may exert their effect an acitvating ligand of Pregnane X receptor (PXR),
which is thought
to play a key role as an essential regulator of intestinal barrier function,
through
downregulation of TLR4 signaling (Venkatesh et al., 2014 Symbiotic Bacterial
Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic
Sensor PXR
and Toll-like Receptor 4; Immunity 41, 296-310, August 21, 2014). As a result,
indole
levels may through the activation of PXR regulate and balance the levels of
TLR4
expression to promote homeostasis and gut barrier health.
[0222] Thus, in some embodiments, the genetically engineered bacteria of the
disclosure produce one or more short chain fatty acids and/or one or more
tryprophan
metabolites.
Acetate
[0223] In some embodiments, the genetically engineered bacteria of the
invention
comprise an acetate gene cassette and are capable of producing acetate. The
genetically
engineered bacteria may include any suitable set of acetate biosynthesis
genes. In other
embodiments, the bacteria eomprise an endogenous acetate biosynthetic gene or
gene
cassette and naturally produce acetate. Unmodified bacteria comprising acetate
biosynthesis genes are known in the art and are capable of consuming various
substrates to
produce acetate under aerobic and/or anaerobic conditions (see, e.g.,
Ragsdale, 2008), and
these endogenous acetate biosynthesis pathways may be a source of genes for
the
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genetically engineered bacteria of the invention. In some embodiments, the
genetically
engineered bacteria of the invention comprise acetate biosynthesis genes from
a different
species, strain, or substrain of bacteria. In some embodiments, the native
acetate
biosynthesis genes in the genetically engineered bacteria are enhanced. In
some
embodiments, the genetically engineered bacteria comprise aerobic acetate
biosynthesis
genes, e.g., from Escherichia coli. In some embodiments, the genetically
engineered
bacteria comprise anaerobic acetate biosynthesis genes, e.g., from
Acetitomaculum,
Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium,
Clostridium,
Moorella, Oxobacter, Sporomusa, and/or The rmoacetogenium. The genetically
engineered bacteria may comprise genes for aerobic acetate biosynthesis or
genes for
anaerobic or microaerobic acetate biosynthesis. In some embodiments, the
genetically
engineered bacteria comprise both aerobic and anaerobic or microaerobic
acetate
biosynthesis genes. In some embodiments, the genetically engineered bacteria
comprise a
combination of acetate biosynthesis genes from different species, strains,
and/or substrains
of bacteria, and are capable of producing acetate. In some embodiments, one or
more of
the acetate biosynthesis genes is functionally replaced, modified, and/or
mutated in order
to enhance stability and/or acetate production. In some embodiments, the
genetically
engineered bacteria are capable of expressing the acetate biosynthesis
cassette and
producing acetate under inducing conditions. In some embodiments, the
genetically
engineered bacteria are capable of producing an alternate short-chain fatty
acid.
[0224] In E. coli Nissle, acetate is generated as an end product of
fermentation. In
E coli, glucose fermentation occurs in two steps, (1) the glycolysis reactions
and (2) the
NADH recycling reactions, i.e. these reactions re-oxidize the NAD+ generated
during the
fermentation process. E. coli employs the "mixed acid" fermentation pathway
(see, e.g.,
FIG 25). Through the "mixed acid" pathway, E coli generates several
alternative end
products and in variable amounts (e.g., lactate, acetate, formate, succinate,
ethanol, carbon
dioxide, and hydrogen) though various arms of the fermentation pathway, e.g.,
as shown
in FIG. 25. Without wishing to be bound by theory, prevention or reduction of
flux
through one or more metabolic arm(s) generating metabolites other than
acetate, e.g.
through mutation, deletion and/or inhibition of one or more gene(s) encoding
key enzymes
in these metabolic arms, results in an increase in production of acetate for
NAD recycling.
As disclosed herein, e.g., in Example 20, deletions in gene(s) encoding such
enzymes
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increase acetate production. Such enzymes include fumarate reductase (encoded
by the
frd genes), lactate dehydrogenase (encoded by the ldh gene), and aldehyde-
alcohol
dehydrogenase (encoded by the adhE gene).
[0225] LdhA is a soluble NAD-linked lactate dehydrogenase (LDH) that is
specific for the production of D-lactate and is a homotetramer and shows
positive
homotropic cooperativity under higher pH conditions. E. coli carrying ldhA
mutations
show no observable growth defect and can still ferment sugars to a variety of
products
other than lactate.
[0226] In some embodiments, the genetically engineered bacteria producing
acetate comprise a mutation and/or deletion in the endogenous ldhA gene.
[0227] AdhE is a homopolymeric protein with three catalytic functions: alcohol
dehydrogenase, coenzyme A-dependent acetaldehyde dehydrogenase, and pyruvate
formate-lyase deactivase. During fermentation, AdhE has catalyzes two steps
towards the
generation of ethanol: (1) the reduction of acetyl-CoA to acetaldehyde and (2)
the
reduction of acetaldehyde to to ethanol. Deletion of adhE has been employed to
enhance
production of certain metabolites inducstrially, including succinate, D-
lactate, and
polyhydroxyalkanoates (Singh et al, Manipulating redox and ATP balancing for
improved
production of succinate in E. coli.; Metab Eng. 2011 Jan;13(1):76-81; Zhou et
al.,
Evaluation of genetic manipulation strategies on D-lactate production by
Escherichia coli,
Curr Microbiol. 2011 Mar;62(3):981-9; Jian et al., Production of
polyhydroxyalkanoates
by Escherichia coli mutants with defected mixed acid fermentation pathways,
Appl
Microbiol Biotechnol. 2010 Aug;87(6):2247-56).
[0228] In some embodiments, the genetically engineered bacteria producing
acetate comprise a mutation and/or deletion in the endogenous adhE gene.
[0229] The fumarate reductase enzyme complex, encoded by the frdABCD
operon, allows Escherichia coli to utilize fumarate as a terminal electron
acceptor for
anaerobic oxidative phosphorylation. FrdA is one of two catalytic subunits in
the four
subunit fumarate reductase complex. FrdB is the second catalytic subinut of
the complex.
FrdC and FrdD are two integral membrane protein components of the fumarate
reductase
complex. In some embodiments, the genetically engineered bacteria comprise a
mutation
and/or deletion in the endogenous frdA gene.
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[0230] In some embodiments, the genetically engineered bacteria producing
acetate comprise a mutation and/or deletion in one or more endogenous genes
selected
from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments,
the
genetically engineered bacteria comprise a mutation and/or deletion in the
endogenous
ldhA and rdA genes. In some embodiments, the genetically engineered bacteria
comprise a
mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some
embodiments, the genetically engineered bacteria comprise a mutation and/or
deletion in
the endogenous frdA and adhE genes. In some embodiments, the genetically
engineered
bacteria comprise a mutation and/or deletion in the endogenous ldhA, the frdA,
and adhE
genes.
[0231] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more acetate than unmodified bacteria of the same
bacterial subtype
under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the the genetically engineered bacteria
produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than
unmodified
bacteria of the same bacterial subtype under the same conditions.
[0232] In certain situations, the need may arise to prevent and/or reduce
acetate
production by of an engineered or naturally occurring strain, e.g., E. coli
Nissle. Without
wishing to be bound by theory, one or more mutations and/or deletions in one
or more
gene(s) encoding one or more enzyme(s) which function in the acetate producing
metabolic arm of fermentation should reduce and/or prevent production of
acetate.
[0233] Phosphate acetyltransferase (Pta) catalyzes the reversible conversion
between acetyl-CoA and acetylphosphate, a step in the metabolism of acetate
(Campos-
Bermudez et al., Functional dissection of Escherichia coli
phosphotransacetylase structural
domains and analysis of key compounds involved in activity regulation; FEBS J.
2010
Apr;277(8):1957-66). Both pyruvate and phosphoenolpyruvate activate the enzyme
in the
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direction of acetylphosphate synthesis and inhibit the enzyme in the direction
of acetyl-
CoA synthesis. The acetate formation from acetyl-CoA I pathway has been the
target of
metabolic engineering to reduce the flux to acetate and increase the
production of
commercially desired end products (see, e.g., Singh, et al., Manipulating
redox and ATP
balancing for improved production of succinate in E. coli; Metab Eng. 2011
Jan;13(1):76-
81). A pta mutant does not grow on acetate as the sole source of carbon (Brown
et al., The
enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli;
J Gen
Microbiol. 1977 Oct;102(2):327-36).
[0234] In some embodiments, the genetically engineered bacteria comprise a
mutation and/or deletion in the endogenous pta gene. In some embodiments, the
gentically
engineered bacteria produce butyrate.In some embodiments, the genetically
engineered
bacteria comprise a mutation and/or deletion in the endogenous pta gene and
also in one or
more endogenous genes selected from the ldhA gene, the frdA gene and the adhE
gene. In
some embodiments, the genetically engineered bacteria comprise a mutation
and/or
deletion in the endogenous pta and adhE genes. In some embodiments, the
genetically
engineered bacteria comprise a mutation and/or deletion in the endogenous pta
and ldhA
genes. In some embodiments, the genetically engineered bacteria comprise a
mutation
and/or deletion in the endogenous pta and frdA genes. In some embodiments, the
genetically engineered bacteria comprise a mutation and/or deletion in the
endogenous pta,
ldhA and frdA genes. In some embodiments, the genetically engineered bacteria
comprise
a mutation and/or deletion in the endogenous pta, ldhA, and adhE genes. In
some
embodiments, the genetically engineered bacteria comprise a mutation and/or
deletion in
the endogenous pta, frdA and adhE genes. In some embodiments, the genetically
engineered bacteria comprise a mutationand/or deletion in the endogenous pta,
ldhA, frdA,
and adhE genes. In some embodiments, the gentically engineered bacteris
produce
butyrate.
[0235] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% less acetate than unmodified bacteria of the same
bacterial subtype
under the same conditions. In yet another embodiment, the genetically
engineered
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bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold less acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the the genetically engineered bacteria
produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than
unmodified bacteria
of the same bacterial subtype under the same conditions.
Butyrate
[0236] In some embodiments, the genetically engineered bacteria of the
invention
comprise a butyrogenic gene cassette and are capable of producing butyrate
under
particular exogenous environmental conditions. The genetically engineered
bacteria may
include any suitable set of butyrogenic genes (see, e.g., Table 2 and Table
3).
Unmodified bacteria comprising butyrate biosynthesis genes are known and
include, but
are not limited to, Peptoclostridium, Clostridium, Fusobacterium,
Butyrivibrio,
Eubacterium, and Treponema. In some embodiments, the genetically engineered
bacteria
of the invention comprise butyrate biosynthesis genes from a different
species, strain, or
substrain of bacteria. In some embodiments, the genetically engineered
bacteria comprise
the eight genes of the butyrate biosynthesis pathway from Peptoclostridium
difficile, e.g.,
Peptoclostridium difficile strain 630: bcd2, e1J133, etfA3, thiAl, hbd, crt2,
pbt, and buk
(Aboulnaga et al., 2013) and are capable of producing butyrate.
Peptoclostridium difficile
strain 630 and strain 1296 are both capable of producing butyrate, but
comprise different
nucleic acid sequences for etfA3, thiAl, hbd, crt2, pbt, and buk. In some
embodiments, the
genetically engineered bacteria comprise a combination of butyrogenic genes
from
different species, strains, and/or substrains of bacteria and are capable of
producing
butyrate. For example, in some embodiments, the genetically engineered
bacteria
comprise bcd2, etf733, effA3, and thiAl from Peptoclostridium difficile strain
630, and hbd,
crt2, pbt, and buk from Peptoclostridium difficile strain 1296. Alternatively,
a single gene
from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) is
capable of
functionally replacing all three of the bcd2, etfB3, and etfA3 genes from
Peptoclostridium
difficile. Thus, a butyrogenic gene cassette may comprise thiAl, hbd, crt2,
pbt, and buk
from Peptoclostridium difficile and ter from Treponema denticola. In another
example of
a butyrate gene cassette, the pbt and buk genes are replaced with tesB (e.g.,
from E coli).
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Thus a butyrogenic gene cassette may comprise ter, thiAl, hbd, crt2, and
tesB.n some
embodiments, the genetically engineered bacteria are capable of expressing the
butyrate
biosynthesis cassette and producing butyrate in low-oxygen conditions, in the
presence of
certain molecules or metabolites, in the presence of molecules or metabolites
associated
with inflammation or an inflammatory response, or in the presence of some
other
metabolite that may or may not be present in the gut, such as arabinose. One
or more of
the butyrate biosynthesis genes may be functionally replaced or modified,
e.g., codon
optimized.
[0237] In some embodiments, additional genes may be mutated or knocked out, to
further increase the levels of butyrate production. Production under anaerobic
conditions
depends on endogenous NADH pools. Therefore, the flux through the butyrate
pathway
may be enhanced by eliminating competing routes for NADH utilization. Non-
limiting
examples of such competing routes are frdA (converts phosphoenolpyruvate to
succinate),
ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol).
Thus, in
certain embodiments, the genetically engineered bacteria further comprise
mutations
and/or deletions in one or more of frdA, ldhA, and adhE.
[0238] Table 2 depicts the nucleic acid sequences of exemplary genes in
exemplary butyrate biosynthesis gene cassettes.
Table 2. Exemplary Butyrate Cassette Sequences
Description Sequence
ATGGATTTAAATTCTAAAAAATATCAGATGCTTAAAGAGCTATATGTAAG
CTTCGCTGAAAATGAAGTTAAACCTTTAGCAACAGAACTTGATGAAGAAG
AAAGATTTCCTTATGAAACAGTGGAAAAAATGGCAAAAGCAGGAATGATG
GGTATACCATATCCAAAAGAATATGGTGGAGAAGGTGGAGACACTGTAGG
ATATATAATGGCAGTTGAAGAATTGTCTAGAGTTTGTGGTACTACAGGAG
TTATATTATCAGCTCATACATCTCTTGGCTCATGGCCTATATATCAATAT
GGTAATGAAGAACAAAAACAAAAATTCTTAAGACCACTAGCAAGTGGAGA
AAAATTAGGAGCATTTGGTCTTACTGAGCCTAATGCTGGTACAGATGCGT
bcd2 CTGGCCAACAAACAACTGCTGTTTTAGACGGGGATGAATACATACTTAAT
SEQ ID NO: 1 GGCTCAAAAATATTTATAACAAACGCAATAGCTGGTGACATATATGTAGT
AATGGCAATGACTGATAAATCTAAGGGGAACAAAGGAATATCAGCATTTA
TAGTTGAAAAAGGAACTCCTGGGTTTAGCTTTGGAGTTAAAGAAAAGAAA
ATGGGTATAAGAGGTTCAGCTACGAGTGAATTAATATTTGAGGATTGCAG
AATACCTAAAGAAAATTTACTTGGAAAAGAAGGTCAAGGATTTAAGATAG
CAATGTCTACTCTTGATGGTGGTAGAATTGGTATAGCTGCACAAGCTTTA
GGTTTAGCACAAGGTGCTCTTGATGAAACTGTTAAATATGTAAAAGAAAG
AGTACAATTTGGTAGACCATTATCAAAATTCCAAAATACACAATTCCAAT
TAGCTGATATGGAAGTTAAGGTACAAGCGGCTAGACACCTTGTATATCAA
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Description Sequence
GCAGCTATAAATAAAGACT TAGGAAAACCT TAT GGAGTAGAAGCAGCAAT
GGCAAAAT TAT T T GCAGC T GAAACAGC TAT GGAAGT TACTACAAAAGCTG
TACAACT T CAT GGAGGATAT GGATACAC T CGT GAC TAT CCAGTAGAAAGA
AT GAT GAGAGAT GC TAAGATAAC T GAAATATAT GAAGGAAC TAGT GAAGT
TCAAAGAATGGT TAT T T CAGGAAAAC TAT TAAAATAG
AT GAATATAGT CGT T TGTATAAAACAAGT TCCAGATACAACAGAAGT TAA
AC TAGAT CC TAATACAGGTAC T T TAAT TAGAGATGGAGTACCAAGTATAA
TAAACCC T GAT GATAAAGCAGGT T TAGAAGAAGCTATAAAAT TAAAAGAA
GAAAT GGGT GC T CAT GTAAC T GT TATAACAAT GGGACC T CC T CAAGCAGA
TAT GGC T T TAAAAGAAGCT T TAGCAATGGGTGCAGATAGAGGTATAT TAT
TAACAGATAGAGCAT T T GCGGGT GC T GATAC T TGGGCAACT T CAT CAGCA
T TAGCAGGAGCAT TAAAAAATATAGAT T T TGATAT TATAATAGCTGGAAG
etfB3 ACAGGCGATAGATGGAGATACTGCACAAGT TGGACCTCAAATAGCTGAAC
SEQ ID NO: 2 AT T TAAATCT T CCAT CAATAACATAT GC T GAAGAAATAAAAAC T GAAGGT
GAATATGTAT TAGTAAAAAGACAAT T TGAAGAT T GT TGCCATGACT TAAA
AGT TAAAATGCCATGCCT TATAACAACTCT TAAAGATATGAACACACCAA
GATACATGAAAGT T GGAAGAATATAT GAT GC T T T CGAAAAT GAT GTAGTA
GAAACATGGACTGTAAAAGATATAGAAGT TGACCCT TCTAAT T TAGGTCT
TAAAGGT TCTCCAACTAGTGTAT T TAAAT CAT T TACAAAATCAGT TAAAC
CAGCTGGTACAATATACAATGAAGATGCGAAAACATCAGCTGGAAT TAT C
ATAGATAAAT TAAAAGAGAAGTATATCATATAA
AT GGGTAACGT T T TAGTAGTAATAGAACAAAGAGAAAATGTAAT TCAAAC
T GT TTCTT TAGAAT TACTAGGAAAGGCTACAGAAATAGCAAAAGAT TAT G
ATACAAAAGT TTCTGCAT TACT T T TAGGTAGTAAGGTAGAAGGT T TAATA
GATACAT TAGCACAC TAT GGT GCAGAT GAGGTAATAGTAGTAGAT GAT GA
AGCT T TAGCAGTGTATACAACTGAACCATATACAAAAGCAGCT TAT GAAG
CAATAAAAGCAGCTGACCCTATAGT TGTAT TAT T TGGTGCAACT TCAATA
GGTAGAGAT T TAGCGCCTAGAGT TTC T GC TAGAATACATACAGGT C T TAC
T GC T GAC T GTACAGGT C T TGCAGTAGCTGAAGATACAAAAT TAT TAT TAA
TGACAAGACCTGCCT T TGGTGGAAATATAATGGCAACAATAGT T TGTAAA
GAT T T CAGACC T CAAAT GT C TACAGT TAGACCAGGGGT TAT GAAGAAAAA
etfA3
T GAACC T GAT GAAAC TAAAGAAGC T GTAAT TAACCGT T TCAAGGTAGAAT
SEQ ID NO: 3
T TAAT GAT GC T GATAAAT TAGT TCAAGT TGTACAAGTAATAAAAGAAGCT
AAAAAACAAGT TAAAATAGAAGAT GC TAAGATAT TAGT TTCT GC T GGACG
TGGAATGGGTGGAAAAGAAAACT TAGACATACT T TAT GAAT TAGCTGAAA
T TATAGGTGGAGAAGT TTCTGGT TCTCGTGCCACTATAGATGCAGGT TGG
T TAGATAAAGCAAGACAAGT TGGTCAAACTGGTAAAACTGTAAGACCAGA
CC T T TATATAGCAT GT GGTATAT C T GGAGCAATACAACATATAGC T GGTA
T GGAAGAT GC T GAGT T TATAGT T GC TATAAATAAAAAT CCAGAAGC T CCA
ATAT T TAAATAT GC T GAT GT TGGTATAGT T GGAGAT GT T CATAAAGT GC T
TCCAGAACT TAT CAGT CAGT TAAGT GT TGCAAAAGAAAAAGGTGAAGT T T
TAGCTAACTAA
AT GAGAGAAGTAGTAAT TGCCAGTGCAGCTAGAACAGCAGTAGGAAGT T T
TGGAGGAGCAT T TAAATCAGT T TCAGCGGTAGAGT TAGGGGTAACAGCAG
thiAl
CTAAAGAAGCTATAAAAAGAGCTAACATAACTCCAGATATGATAGATGAA
SEQ ID NO: 4
TCTCTTT TAGGGGGAGTACT TACAGCAGGTCT TGGACAAAATATAGCAAG
ACAAATAGCAT TAGGAGCAGGAATACCAGTAGAAAAACCAGC TAT GAC TA
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Description Sequence
TAAATATAGTTTGTGGTTCTGGATTAAGATCTGTTTCAATGGCATCTCAA
CTTATAGCATTAGGTGATGCTGATATAATGTTAGTTGGTGGAGCTGAAAA
CATGAGTATGTCTCCTTATTTAGTACCAAGTGCGAGATATGGTGCAAGAA
TGGGTGATGCTGCTTTTGTTGATTCAATGATAAAAGATGGATTATCAGAC
ATATTTAATAACTATCACATGGGTATTACTGCTGAAAACATAGCAGAGCA
ATGGAATATAACTAGAGAAGAACAAGATGAATTAGCTCTTGCAAGTCAAA
ATAAAGCTGAAAAAGCTCAAGCTGAAGGAAAATTTGATGAAGAAATAGTT
CCTGTTGTTATAAAAGGAAGAAAAGGTGACACTGTAGTAGATAAAGATGA
ATATATTAAGCCTGGCACTACAATGGAGAAACTTGCTAAGTTAAGACCTG
CATTTAAAAAAGATGGAACAGTTACTGCTGGTAATGCATCAGGAATAAAT
GATGGTGCTGCTATGTTAGTAGTAATGGCTAAAGAAAAAGCTGAAGAACT
AGGAATAGAGCCTCTTGCAACTATAGTTTCTTATGGAACAGCTGGTGTTG
ACCCTAAAATAATGGGATATGGACCAGTTCCAGCAACTAAAAAAGCTTTA
GAAGCTGCTAATATGACTATTGAAGATATAGATTTAGTTGAAGCTAATGA
GGCATTTGCTGCCCAATCTGTAGCTGTAATAAGAGACTTAAATATAGATA
TGAATAAAGTTAATGTTAATGGTGGAGCAATAGCTATAGGACATCCAATA
GGATGCTCAGGAGCAAGAATACTTACTACACTTTTATATGAAATGAAGAG
AAGAGATGCTAAAACTGGTCTTGCTACACTTTGTATAGGCGGTGGAATGG
GAACTACTTTAATAGTTAAGAGATAG
ATGAAATTAGCTGTAATAGGTAGTGGAACTATGGGAAGTGGTATTGTACA
AACTTTTGCAAGTTGTGGACATGATGTATGTTTAAAGAGTAGAACTCAAG
GTGCTATAGATAAATGTTTAGCTTTATTAGATAAAAATTTAACTAAGTTA
GTTACTAAGGGAAAAATGGATGAAGCTACAAAAGCAGAAATATTAAGTCA
TGTTAGTTCAACTACTAATTATGAAGATTTAAAAGATATGGATTTAATAA
TAGAAGCATCTGTAGAAGACATGAATATAAAGAAAGATGTTTTCAAGTTA
CTAGATGAATTATGTAAAGAAGATACTATCTTGGCAACAAATACTTCATC
hbd ATTATCTATAACAGAAATAGCTTCTTCTACTAAGCGCCCAGATAAAGTTA
TAGGAATGCATTTCTTTAATCCAGTTCCTATGATGAAATTAGTTGAAGTT
SEQ ID NO: 5
ATAAGTGGTCAGTTAACATCAAAAGTTACTTTTGATACAGTATTTGAATT
ATCTAAGAGTATCAATAAAGTACCAGTAGATGTATCTGAATCTCCTGGAT
TTGTAGTAAATAGAATACTTATACCTATGATAAATGAAGCTGTTGGTATA
TATGCAGATGGTGTTGCAAGTAAAGAAGAAATAGATGAAGCTATGAAATT
AGGAGCAAACCATCCAATGGGACCACTAGCATTAGGTGATTTAATCGGAT
TAGATGTTGTTTTAGCTATAATGAACGTTTTATATACTGAATTTGGAGAT
ACTAAATATAGACCTCATCCACTTTTAGCTAAAATGGTTAGAGCTAATCA
ATTAGGAAGAAAAACTAAGATAGGATTCTATGATTATAATAAATAA
ATGAGTACAAGTGATGTTAAAGTTTATGAGAATGTAGCTGTTGAAGTAGA
TGGAAATATATGTACAGTGAAAATGAATAGACCTAAAGCCCTTAATGCAA
TAAATTCAAAGACTTTAGAAGAACTTTATGAAGTATTTGTAGATATTAAT
AATGATGAAACTATTGATGTTGTAATATTGACAGGGGAAGGAAAGGCATT
TGTAGCTGGAGCAGATATTGCATACATGAAAGATTTAGATGCTGTAGCTG
crt2
CTAAAGATTTTAGTATCTTAGGAGCAAAAGCTTTTGGAGAAATAGAAAAT
SEQ ID NO: 6 AGTAAAAAAGTAGTGATAGCTGCTGTAAACGGATTTGCTTTAGGTGGAGG
ATGTGAACTTGCAATGGCATGTGATATAAGAATTGCATCTGCTAAAGCTA
AATTTGGTCAGCCAGAAGTAACTCTTGGAATAACTCCAGGATATGGAGGA
ACTCAAAGGCTTACAAGATTGGTTGGAATGGCAAAAGCAAAAGAATTAAT
CTTTACAGGTCAAGTTATAAAAGCTGATGAAGCTGAAAAAATAGGGCTAG
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Description Sequence
TAAATAGAGTCGT TGAGCCAGACAT TI TAATAGAAGAAGT TGAGAAAT TA
GC TAAGATAATAGC TAAAAAT GC T CAGC T TGCAGT TAGATACTCTAAAGA
AGCAATACAACT T GGT GC T CAAAC T GATATAAATAC T GGAATAGATATAG
AATCTAAT T TAT T T GGT CT T T GT TTTTCAACTAAAGACCAAAAAGAAGGA
AT GT CAGC T T TCGT TGAAAAGAGAGAAGCTAACT T TATAAAAGGGTAA
AT GAGAAGT II TGAAGAAGTAAT TAAGT T TGCAAAAGAAAGAGGACCTAA
AAC TATAT CAGTAGCAT GT TGCCAAGATAAAGAAGT II TAATGGCAGT TG
AAATGGCTAGAAAAGAAAAAATAGCAAATGCCAT II TAGTAGGAGATATA
GAAAAGACTAAAGAAAT TGCAAAAAGCATAGACATGGATATCGAAAAT TA
TGAACTGATAGATATAAAAGAT T TAGCAGAAGCAT C T C TAAAAT C T GT TG
AAT TAGT T TCACAAGGAAAAGCCGACATGGTAATGAAAGGCT TAGTAGAC
ACATCAATAATACTAAAAGCAGT II TAAATAAAGAAGTAGGTCT TAGAAC
TGGAAATGTAT TAAGTCACGTAGCAGTAT T T GAT GTAGAGGGATAT GATA
GAT TAT T T T T CGTAAC T GACGCAGC TAT GAAC T TAGC T CC T GATACAAAT
pbt
AC TAAAAAGCAAAT CATAGAAAAT GC T TGCACAGTAGCACAT T CAT TAGA
SEQ ID NO: 7
TATAAGTGAACCAAAAGT T GC T GCAATAT GCGCAAAAGAAAAAGTAAAT C
CAAAAATGAAAGATACAGT TGAAGCTAAAGAACTAGAAGAAATGTATGAA
AGAGGAGAAATCAAAGGT TGTATGGT TGGTGGGCCTTTTGCAAT TGATAA
TGCAGTATCT T TAGAAGCAGC TAAACATAAAGGTATAAAT CAT CC T GTAG
CAGGACGAGCTGATATAT TAT TAGCCCCAGATAT TGAAGGTGGTAACATA
T TATATAAAGCT T TGGTAT TCTTCTCAAAATCAAAAAATGCAGGAGT TAT
AGT TGGGGCTAAAGCACCAATAATAT TAACT TCTAGAGCAGACAGTGAAG
AAACTAAACTAAACTCAATAGCT T TAGGT GT T T TAATGGCAGCAAAGGCA
TAA
AT GAGCAAAATAT T TAAAATCT TAACAATAAAT CC T GGT TCGACATCAAC
TAAAATAGCTGTAT T TGATAATGAGGAT T TAGTAT T TGAAAAAACT T TAA
GACAT TCT T CAGAAGAAATAGGAAAATAT GAGAAGGT GT C T GACCAAT T T
GAAT T TCGTAAACAAGTAATAGAAGAAGCTCTAAAAGAAGGTGGAGTAAA
AACATCTGAAT TAGAT GC T GTAGTAGGTAGAGGAGGAC T TCT TAAACC TA
TAAAAGGTGGTACT TAT T CAGTAAGT GC T GC TAT GAT TGAAGAT T TAAAA
GT GGGAGT TI TAGGAGAACACGCT TCAAACCTAGGTGGAATAATAGCAAA
ACAAATAGGT GAAGAAGTAAAT GT T CC T T CATACATAGTAGACCC T GT TG
T TGTAGATGAAT TAGAAGAT GI T GC TAGAAT TTCTGGTATGCCTGAAATA
AGTAGAGCAAGT GTAGTACAT GC T T TAAATCAAAAGGCAATAGCAAGAAG
buk ATAT GC TAGAGAAATAAACAAGAAATAT GAAGATATAAAT C T TATAGT TG
SEQ ID NO: 8 CACACATGGGTGGAGGAGT T TC T GT TGGAGCTCATAAAAATGGTAAAATA
GTAGAT GT TGCAAACGCAT TAGATGGAGAAGGACCTTTCTCTCCAGAAAG
AAGTGGTGGACTACCAGTAGGTGCAT TAGTAAAAAT GT GC T T TAGTGGAA
AATATACTCAAGATGAAAT TAAAAAGAAAATAAAAGGTAAT GGCGGAC TA
GI TGCATACT TAAACAC TAAT GAT GC TAGAGAAGT TGAAGAAAGAAT T GA
AGC T GGT GAT GAAAAAGC TAAAT TAGTATAT GAAGC TAT GGCATAT CAAA
TCTC TAAAGAAATAGGAGC TAGT GC T GCAGT TCT TAAGGGAGATGTAAAA
GCAATAT TAT TAACTGGTGGAATCGCATAT T CAAAAAT GT T TACAGAAAT
GAT TGCAGATAGAGT TAAAT T TATAGCAGATGTAAAAGT T TAT CCAGGT G
AAGAT GAAAT GAT TGCAT TAGCTCAAGGTGGACT TAGAGT T T TAACTGGT
GAAGAAGAGGCTCAAGT T TAT GATAAC TAA
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Description Sequence
ATGATCGTAAAACCTATGGTACGCAACAATATCTGCCTGAACGCCCATCC
TCAGGGCTGCAAGAAGGGAGTGGAAGATCAGATTGAATATACCAAGAAAC
GCATTACCGCAGAAGTCAAAGCTGGCGCAAAAGCTCCAAAAAACGTTCTG
GTGCTTGGCTGCTCAAATGGTTACGGCCTGGCGAGCCGCATTACTGCTGC
GTTCGGATACGGGGCTGCGACCATCGGCGTGTCCTTTGAAAAAGCGGGTT
CAGAAACCAAATATGGTACACCGGGATGGTACAATAATTTGGCATTTGAT
GAAGCGGCAAAACGCGAGGGTCTTTATAGCGTGACGATCGACGGCGATGC
GTTTTCAGACGAGATCAAGGCCCAGGTAATTGAGGAAGCCAAAAAAAAAG
GTATCAAATTTGATCTGATCGTATACAGCTTGGCCAGCCCAGTACGTACT
GATCCTGATACAGGTATCATGCACAAAAGCGTTTTGAAACCCTTTGGAAA
AACGTTCACAGGCAAAACAGTAGATCCGTTTACTGGCGAGCTGAAGGAAA
ter TCTCCGCGGAACCAGCAAATGACGAGGAAGCAGCCGCCACTGTTAAAGTT
SEQ ID NO: 9 ATGGGGGGTGAAGATTGGGAACGTTGGATTAAGCAGCTGTCGAAGGAAGG
CCTCTTAGAAGAAGGCTGTATTACCTTGGCCTATAGTTATATTGGCCCTG
AAGCTACCCAAGCTTTGTACCGTAAAGGCACAATCGGCAAGGCCAAAGAA
CACCTGGAGGCCACAGCACACCGTCTCAACAAAGAGAACCCGTCAATCCG
TGCCTTCGTGAGCGTGAATAAAGGCCTGGTAACCCGCGCAAGCGCCGTAA
TCCCGGTAATCCCTCTGTATCTCGCCAGCTTGTTCAAAGTAATGAAAGAG
AAGGGCAATCATGAAGGTTGTATTGAACAGATCACGCGTCTGTACGCCGA
GCGCCTGTACCGTAAAGATGGTACAATTCCAGTTGATGAGGAAAATCGCA
TTCGCATTGATGATTGGGAGTTAGAAGAAGACGTCCAGAAAGCGGTATCC
GCGTTGATGGAGAAAGTCACGGGTGAAAACGCAGAATCTCTCACTGACTT
AGCGGGGTACCGCCATGATTTCTTAGCTAGTAACGGCTTTGATGTAGAAG
GTATTAATTATGAAGCGGAAGTTGAACGCTTCGACCGTATCTGA
ATGAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGAAAAAAT
TGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAGGTTTACGCCAGG
TGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTGTATGCTGCAAAAGAGACC
GTCCCTGAAGAGCGGCTGGTACATTCGTTTCACAGCTACTTTCTTCGCCC
TGGCGATAGTAAGAAGCCGATTATTTATGATGTCGAAACGCTGCGTGACG
GTAACAGCTTCAGCGCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCG
ATTTTTTATATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGAACA
TCAAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTCGGAAA
tesB CGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGTGCTGAAAGAT
SEQ ID NO: 10 AAATTCATCTGCGATCGTCCGCTGGAAGTCCGTCCGGTGGAGTTTCATAA
CCCACTGAAAGGTCACGTCGCAGAACCACATCGTCAGGTGTGGATCCGCG
CAAATGGTAGCGTGCCGGATGACCTGCGCGTTCATCAGTATCTGCTCGGT
TACGCTTCTGATCTTAACTTCCTGCCGGTAGCTCTACAGCCGCACGGCAT
CGGTTTTCTCGAACCGGGGATTCAGATTGCCACCATTGACCATTCCATGT
GGTTCCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTGGAG
AGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAGTTTTATAC
CCAAGACGGCGTACTGGTTGCCTCGACCGTTCAGGAAGGGGTGATGCGTA
ATCACAATTAA
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[0239] Exemplary polypeptide sequences for the production of butyrate by the
genetically engineered bacteria are provided in Table 3.
Table 3. Exemplary Polypeptide Sequences for Butyrate Production
Description Sequence
Bcd2 MDLNS KKYQMLKELYVSFAENEVKPLATELDEEER
SEQ ID NO: 11 FPYETVEKMAKAGMMGIPYPKEYGGEGGDTVGYIM
AVEELSRVC GTTGVILS AHTSLGS WPIYQYGNEEQK
QKFLRPLAS GE KLGAFGLTEPNAGTD AS GQQTTAVL
DGDEYILNGS KIFITNAIAGDIYVVMAMTD KS KGNK
GIS AFIVEKGTPGFS FGVKEKKMGIRGS AT S ELIFEDC
RIPKENLLGKEGQGFKIAMS TLDGGRIGIAAQALGLA
QGALDETVKYVKERVQFGRPLS KFQNTQFQLADME
VKVQAARHLVYQAAINKDLGKPYGVEAAMAKLFA
AETAMEVTTKAVQLHGGYGYTRDYPVERMMRDAK
ITEIYEGTSEVQRMVIS GKLLK
etfB 3 MNIVVCIKQVPDTTEVKLDPNTGTLIRDGVPSIINPDD
SEQ ID NO: 12 KAGLEEAIKLKEEMGAHVTVITMGPPQADMALKEA
LAMGADRGILLTDRAFAGADTWATS S ALAGALKNI
DFDIIIAGRQAIDGDTAQVGPQIAEHLNLPSITYAEEIK
TEGEYVLVKRQFEDCCHDLKVKMPCLITTLKDMNT
PRYMKVGRIYDAFENDVVETWTVKDIEVDPSNLGL
KGS PT S VFKS FT KS VKPAGTIYNEDAKTS AGIIIDKLK
EKYII
etfA3 MGNVLVVIEQRENVIQTVSLELLGKATEIAKDYDTK
SEQ ID NO: 13 VS ALLLGS KVEGLIDTLAHYGADEVIVVDDEALAVY
TTEPYT KAAYE AI KAADPIVVLFGAT S IGRDLAPRVS
ARIHTGLTADCTGLAVAEDTKLLLMTRPAFGGNIMA
TIVCKDFRPQMS TVRPGVMKKNEPDETKEAVINRFK
VEFND AD KLVQVVQVI KE AKKQV KIEDAKILVS AGR
GMGGKENLDILYELAEIIGGEVS GS RATIDAGWLD K
ARQVGQT GKTVRPDLYIAC GIS GAIQHIAGMEDAEFI
VAIN KNPE APIFKYADVGIVGD VH KVLPELIS QLS VA
KEKGEVLAN
Ter MIVKPMVRNNICLNAHPQGCKKGVEDQIEYTKKRIT
SEQ ID NO: 14 AEVKAGAKAPKNVLVLGCSNGYGLASRITAAFGYG
AATIGVS FE KAGS ET KYGTPGWYNNLAFDEAAKRE
GLYS VTIDGDAFSDEIKAQVIEEAKKKGIKFDLIVYSL
AS PVRTDPDT GIMH KS VLKPFG KTFT G KTVD PFT GEL
KEIS AEPANDEEAAATVKVMGGEDWERWIKQLS KE
GLLEEGCITLAYS YIGPEATQALYRKGTIGKAKEHLE
AT AHRLN KENPS IRAFVS VNKGLVTRAS AVIPVIPLY
LAS LFKVM KE KGNHE GCIE QITRLYAERLYRKD GTIP
VDEENRIRIDDWELEEDVQKAVS ALMEKVT GENAES
LTDLAGYRHDFLASNGFDVEGINYEAEVERFDRI
ThiA MREVVIAS AARTAVGS FGGAFKS VS AVELGVTAAK
SEQ ID NO: 15 EAIKRANITPDMIDESLLGGVLTAGLGQNIARQIALG
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AGIPVEKPAMTINIVCGSGLRSVSMASQLIALGDADI
MLVGGAENMSMSPYLVPSARYGARMGDAAFVDSM
IKDGLSDIFNNYHMGITAENIAEQWNITREEQDELAL
AS QNKAEKAQAEGKFDEEIVPVVIKGRKGDTVVDK
DEYIKPGTTMEKLAKLRPAFKKDGTVTAGNASGIND
GAAMLVVMAKEKAEELGIEPLATIVSYGTAGVDPKI
MGYGPVPATKKALEAANMTIEDIDLVEANEAFAAQ
SVAVIRDLNIDMNKVNVNGGAIAIGHPIGCSGARILT
TLLYEMKRRDAKTGLATLCIGGGMGTTLIVKR
Hbd MKLAVIGSGTMGSGIVQTFASCGHDVCLKSRTQGAI
SEQ ID NO: 16 DKCLALLDKNLT KLVT KGKMDEAT KAEILS HVS S TT
NYEDLKDMDLIIEASVEDMNIKKDVFKLLDELCKED
TILATNTSSLSITEIASSTKRPDKVIGMHFFNPVPMMK
LVEVISGQLTSKVTFDTVFELSKSINKVPVDVSESPGF
VVNRILIPMINEAVGIYADGVASKEEIDEAMKLGAN
HPMGPLALGDLIGLDVVLAIMNVLYTEFGDTKYRPH
PLLAKMVRANQLGRKTKIGFYDYNK
Crt2 MS TSDVKVYENVAVEVDGNICTVKMNRPKALNAIN
SEQ ID NO: 17 SKTLEELYEVFVDINNDETIDVVILTGEGKAFVAGAD
IAYMKDLDAVAAKDFSILGAKAFGEIENSKKVVIAA
VNGFALGGGCELAMACDIRIASAKAKFGQPEVTLGI
TPGYGGTQRLTRLVGMAKAKELIFTGQVIKADEAEK
IGLVNRVVEPDILIEEVEKLAKIIAKNAQLAVRYSKE
AIQLGAQTDINTGIDIESNLFGLCFSTKDQKEGMSAF
VEKREANFIKG
Pbt MRSFEEVIKFAKERGPKTISVACCQDKEVLMAVEMA
SEQ ID NO: 18 RKEKIANAILVGDIEKTKEIAKSIDMDIENYELIDIKD
LAEASLKSVELVSQGKADMVMKGLVDTSIILKAVLN
KEVGLRTGNVLSHVAVFDVEGYDRLFFVTDAAMNL
APDTNTKKQIIENACTVAHSLDISEPKVAAICAKEKV
NPKMKDTVEAKELEEMYERGEIKGCMVGGPFAIDN
AVSLEAAKHKGINHPVAGRADILLAPDIEGGNILYKA
LVFFS KS KNAGVIVGAKAPIILTSRADSEETKLNSIAL
GVLMAAKA
Buk MS KIFKILTINPGSTSTKIAVFDNEDLVFEKTLRHS SE
SEQ ID NO: 19 EIGKYEKVSDQFEFRKQVIEEALKEGGVKTSELDAV
VGRGGLLKPIKGGTYS VS AAMIEDLKVGVLGEHASN
LGGIIAKQIGEEVNVPS YIVDPVVVDELEDVARIS GM
PEISRASVVHALNQKAIARRYAREINKKYEDINLIVA
HMGGGVSVGAHKNGKIVDVANALDGEGPFSPERSG
GLPVGALVKMCFSGKYTQDEIKKKIKGNGGLVAYL
NTNDAREVEERIEAGDEKAKLVYEAMAYQISKEIGA
SAAVLKGDVKAILLTGGIAYSKMFTEMIADRVKFIA
DVKVYPGEDEMIALAQGGLRVLTGEEEAQVYDN
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TesB MS QALKNLLTLINLEKIEEGLFRGQS EDLGLRQVFG
SEQ ID NO: 20 GQV VGQALYAAKETV PEERLVHSFES YFLRYGDSKK
PIINDVETLRDGNS FS ARR VA AIQNGKPIFYMTASFQ
APEAGFEHQKTMPSAPAPDGLPSETQIAQSLAHLLPP
KD K FICD IZPLE RPV EFfi NPLKGH AE PH RQVW1
RANGS VPDDLR VIIQYLLGY ASDLNFLPV ALQPII GIG
FLEPGIQIATIDEISMVVETIRPFN LNEWLLYS VESTSAS
S ARG FV RGEFYTQ DGVLVASTVQEG VM
[0240] The gene products of the bcd2, e03, and etf733 genes in Clostridium
difficile form a complex that converts crotonyl-CoA to butyryl-CoA, which may
function
as an oxygen-dependent co-oxidant. In some embodiments, because the
genetically
engineered bacteria of the invention are designed to produce butyrate in a
microaerobic or
oxygen-limited environment, e.g., the mammalian gut, oxygen dependence could
have a
negative effect on butyrate production in the gut. It has been shown that a
single gene
from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) can
functionally
replace this three-gene complex in an oxygen-independent manner. In some
embodiments, the genetically engineered bacteria comprise a ter gene, e.g.,
from
Treponema denticola, which can functionally replace all three of the bcd2,
etf733, and
etfA3 genes, e.g., from Peptoclostridium difficile. In this embodiment, the
genetically
engineered bacteria comprise thiAl, hbd, crt2, pbt, and buk, e.g., from
Peptoclostridium
difficile, and ter, e.g., from Treponema denticola, and produce butyrate in
low-oxygen
conditions, in the presence of certain molecules or metabolites , in the
presence of
molecules or metabolites associated with inflammation or an inflammatory
response, or in
the presence of some other metabolite that may or may not be present in the
gut, such as
arabinose..
[0241] In some embodiments, the genetically engineered bacteria of the
invention
comprise thiAl, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium
difficile; ter, e.g.,
from Treponema denticola; one or more of bcd2, etf733, and etfA3, e.g., from
Peptoclostridium difficile; and produce butyrate in low-oxygen conditions, in
the presence
of certain molecules or metabolites , in the presence of molecules or
metabolites
associated with inflammation or an inflammatory response, or in the presence
of some
other metabolite that may or may not be present in the gut, such as arabinose.
In some
embodiments, one or more of the butyrate biosynthesis genes is functionally
replaced,
modified, and/or mutated in order to enhance stability and/or increase
butyrate production
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in low-oxygen conditions, in the presence of certain molecules or metabolites,
in the
presence of molecules or metabolites associated with inflammation or an
inflammatory
response, or in the presence of some other metabolite that may or may not be
present in
the gut, such as arabinose.
[0242] The gene products of pbt and buk convert butyrylCoA to Butyrate. In
some
embodiments, the pbt and buk genes can be replaced by a tesB gene. tesB can be
used to
cleave off the CoA from butyryl-coA. In one embodiment, the genetically
engineered
bacteria comprise bcd2, etf733, e03, thiAl, hbd, and crt2, e.g., from
Peptoclostridium
chfficile, and tesB from E. Coli and produce butyrate in low-oxygen
conditions, in the
presence of molecules or metabolites, in the presence of molecules or
metabolites
associated with inflammation or an inflammatory response, or in the presence
of some
other metabolite that may or may not be present in the gut, such as arabinose.
In one
embodiment, the genetically engineered bacteria comprise ter gene (encoding
trans-2-
enoynl-CoA reductase) e.g., from Treponema denticola, thiAl, hbd, crt2, pbt,
and buk,
e.g., from Peptoclostridium chfficile, and tesB from E. Coli , and produce
butyrate in low-
oxygen conditions,in the presence of specific molecules or metabolites, in the
presence of
molecules or metabolites associated with inflammation or an inflammatory
response, or in
the presence of some other metabolite that may or may not be present in the
gut, such as
arabinose. In some embodiments, one or more of the butyrate biosynthesis genes
is
functionally replaced, modified, and/or mutated in order to enhance stability
and/or
increase butyrate production in low-oxygen conditions or in the presence of
specific
molecules or metabolites, or molecules or metabolites associated with
condition(s) such as
inflammation or an inflammatory response, or in the presence of some other
metabolite
that may or may not be present in the gut, such as arabinose.
[0243] In some embodiments, the local production of butyrate induces the
differentiation of regulatory T cells in the gut and/or promotes the barrier
function of
colonic epithelial cells. In some embodiments, the genetically engineered
bacteria
comprise genes for aerobic butyrate biosynthesis and/or genes for anaerobic or
microaerobic butyrate biosynthesis. In some embodiments, local butyrate
production
reduces gut inflammation, a symptom of IBD and other gut related disorders.
[0244] In one embodiment, the bcd2 gene has at least about 80% identity with
SEQ ID NO: 1. In another embodiment, the bcd2 gene has at least about 85%
identity
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with SEQ ID NO: 1. In one embodiment, the bcd2 gene has at least about 90%
identity
with SEQ ID NO: 1. In one embodiment, the bcd2 gene has at least about 95%
identity
with SEQ ID NO: 1. In another embodiment, the bcd2 gene has at least about
96%, 97%,
98%, or 99% identity with SEQ ID NO: 1. Accordingly, in one embodiment, the
bcd2
gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. In
another embodiment, the bcd2 gene comprises the sequence of SEQ ID NO: 1. In
yet
another embodiment the bcd2 gene consists of the sequence of SEQ ID NO: 1.
[0245] In one embodiment, the eff133 gene has at least about 80% identity with
SEQ ID NO: 2. In another embodiment, the eff133 gene has at least about 85%
identity
with SEQ ID NO: 2. In one embodiment, the etf733 gene has at least about 90%
identity
with SEQ ID NO: 2. In one embodiment, the etf733 gene has at least about 95%
identity
with SEQ ID NO: 2. In another embodiment, the etfB3 gene has at least about
96%, 97%,
98%, or 99% identity with SEQ ID NO: 2. Accordingly, in one embodiment, the
effB3
gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2. In
another embodiment, the etf733 gene comprises the sequence of SEQ ID NO: 2. In
yet
another embodiment the effB3 gene consists of the sequence of SEQ ID NO: 2.
[0246] In one embodiment, the effA3 gene has at least about 80% identity with
SEQ ID NO: 3. In another embodiment, the effA3 gene has at least about 85%
identity
with SEQ ID NO: 3. In one embodiment, the etfA3 gene has at least about 90%
identity
with SEQ ID NO: 3. In one embodiment, the etfA3 gene has at least about 95%
identity
with SEQ ID NO: 3. In another embodiment, the etfA3 gene has at least about
96%, 97%,
98%, or 99% identity with SEQ ID NO: 3. Accordingly, in one embodiment, the
effA3
gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. In
another embodiment, the etfA3 gene comprises the sequence of SEQ ID NO: 3. In
yet
another embodiment the effA3 gene consists of the sequence of SEQ ID NO: 3.
[0247] In one embodiment, the thiAl gene has at least about 80% identity with
SEQ ID NO: 4. In another embodiment, the thiAl gene has at least about 85%
identity
with SEQ ID NO: 4. In one embodiment, the thiAl gene has at least about 90%
identity
with SEQ ID NO: 4. In one embodiment, the thiAl gene has at least about 95%
identity
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with SEQ ID NO: 4. In another embodiment, the thiAl gene has at least about
96%, 97%,
98%, or 99% identity with SEQ ID NO: 4. Accordingly, in one embodiment, the
thiAl
gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. In
another embodiment, the thiAl gene comprises the sequence of SEQ ID NO: 4. In
yet
another embodiment the thiAl gene consists of the sequence of SEQ ID NO: 4.
[0248] In one embodiment, the hbd gene has at least about 80% identity with
SEQ
ID NO: 5. In another embodiment, the hbd gene has at least about 85% identity
with SEQ
ID NO: 5. In one embodiment, the hbd gene has at least about 90% identity with
SEQ ID
NO: 5. In one embodiment, the hbd gene has at least about 95% identity with
SEQ ID
NO: 5. In another embodiment, the hbd gene has at least about 96%, 97%, 98%,
or 99%
identity with SEQ ID NO: 5. Accordingly, in one embodiment, the hbd gene has
at least
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. In another
embodiment, the hbd gene comprises the sequence of SEQ ID NO: 5. In yet
another
embodiment the hbd gene consists of the sequence of SEQ ID NO: 5.
[0249] In one embodiment, the crt2 gene has at least about 80% identity with
SEQ
ID NO: 6. In another embodiment, the crt2 gene has at least about 85% identity
with
SEQ ID NO: 6. In one embodiment, the crt2 gene has at least about 90% identity
with
SEQ ID NO: 6. In one embodiment, the crt2 gene has at least about 95% identity
with
SEQ ID NO: 6. In another embodiment, the crt2 gene has at least about 96%,
97%, 98%,
or 99% identity with SEQ ID NO: 6. Accordingly, in one embodiment, the crt2
gene has
at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. In another
embodiment, the crt2 gene comprises the sequence of SEQ ID NO: 6. In yet
another
embodiment the crt2 gene consists of the sequence of SEQ ID NO: 6.
[0250] In one embodiment, the pbt gene has at least about 80% identity with
SEQ
ID NO: 7. In another embodiment, the pbt gene has at least about 85% identity
with SEQ
ID NO: 7. In one embodiment, the pbt gene has at least about 90% identity with
SEQ ID
NO: 7. In one embodiment, the pbt gene has at least about 95% identity with
SEQ ID
NO: 7. In another embodiment, the pbt gene has at least about 96%, 97%, 98%,
or 99%
identity with SEQ ID NO: 7. Accordingly, in one embodiment, the pbt gene has
at least
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about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7. In another
embodiment, the pbt gene comprises the sequence of SEQ ID NO: 7. In yet
another
embodiment the pbt gene consists of the sequence of SEQ ID NO: 7.
[0251] In one embodiment, the buk gene has at least about 80% identity with
SEQ
ID NO: 8. In another embodiment, the buk gene has at least about 85% identity
with SEQ
ID NO: 8. In one embodiment, the buk gene has at least about 90% identity with
SEQ ID
NO: 8. In one embodiment, the buk gene has at least about 95% identity with
SEQ ID
NO: 8. In another embodiment, the buk gene has at least about 96%, 97%, 98%,
or 99%
identity with SEQ ID NO: 8. Accordingly, in one embodiment, the buk gene has
at least
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8. In another
embodiment, the buk gene comprises the sequence of SEQ ID NO: 8. In yet
another
embodiment the buk gene consists of the sequence of SEQ ID NO: 8.
[0252] In one embodiment, the ter gene has at least about 80% identity with
SEQ
ID NO: 9. In another embodiment, the ter gene has at least about 85% identity
with SEQ
ID NO: 9. In one embodiment, the ter gene has at least about 90% identity with
SEQ ID
NO: 9. In one embodiment, the ter gene has at least about 95% identity with
SEQ ID
NO: 9. In another embodiment, the ter gene has at least about 96%, 97%, 98%,
or 99%
identity with SEQ ID NO: 9. Accordingly, in one embodiment, the ter gene has
at least
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9. In another
embodiment, the ter gene comprises the sequence of SEQ ID NO: 9. In yet
another
embodiment the ter gene consists of the sequence of SEQ ID NO: 9.
[0253] In one embodiment, the tesB gene has at least about 80% identity with
SEQ
ID NO: 10. In another embodiment, the tesB gene has at least about 85%
identity with
SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 90%
identity with
SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 95%
identity with
SEQ ID NO: 10. In another embodiment, the tesB gene has at least about 96%,
97%,
98%, or 99% identity with SEQ ID NO: 10. Accordingly, in one embodiment, the
tesB
gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. In
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another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 10. In
yet
another embodiment the tesB gene consists of the sequence of SEQ ID NO: 10.
[0254] In one embodiment, one or more polypeptides encoded by the butyrate
circuits and expressed by the genetically engineered bacteria have at least
about 80%
identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In another
embodiment, one or more polypeptides encoded by the butyrate circuits and
expressed by
the genetically engineered bacteria have at least about 85% identity with one
or more of
SEQ ID NO: 11 through SEQ ID NO: 20. In one embodiment, one or more
polypeptides
encoded by the butyrate circuits and expressed by the genetically engineered
bacteria have
at least about 90% identity with one or more of SEQ ID NO: 11 through SEQ ID
NO: 20.
In one embodiment, one or more polypeptides encoded by the butyrate circuits
and
expressed by the genetically engineered bacteria have at least about 95%
identity with one
or more of SEQ ID NO: 11 through SEQ ID NO: 20. In another embodiment, one or
more
polypeptides encoded by the butyrate circuits and expressed by the genetically
engineered
bacteria have at least about 96%, 97%, 98%, or 99% identity with one or more
of SEQ ID
NO: 11 through SEQ ID NO: 20. Accordingly, in one embodiment, one or more
polypeptides encoded by the butyrate circuits and expressed by the genetically
engineered
bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more
of
SEQ ID NO: 11 through SEQ ID NO: 20. In another embodiment, one or more
polypeptides encoded by the butyrate circuits and expressed by the genetically
engineered
bacteria one or more polypeptides encoded by the butyrate circuits and
expressed by the
genetically engineered bacteria comprise the sequence of with one or more of
SEQ ID
NO: 11 through SEQ ID NO: 20. In yet another embodiment one or more
polypeptides
encoded by the butyrate circuits and expressed by the genetically engineered
bacteria
consist of the sequence of with one or more of SEQ ID NO: 11 through SEQ ID
NO: 20.
[0255] In some embodiments, one or more of the butyrate biosynthesis genes is
a
synthetic butyrate biosynthesis gene. In some embodiments, one or more of the
butyrate
biosynthesis genes is a Treponema denticola butyrate biosynthesis gene. In
some
embodiments, one or more of the butyrate biosynthesis genes is a C. glutamicum
butyrate
biosynthesis gene. In some embodiments, one or more of the butyrate
biosynthesis genes
is a Peptoclostridicum difficile butyrate biosynthesis gene. The butyrate gene
cassette may
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comprise genes for the aerobic biosynthesis of butyrate and/or genes for the
anaerobic or
microaerobic biosynthesis of butyrate.
[0256] To improve acetate production, while maintaining high levels of
butyrate
production, one or more targeted deletions can be introduced in competing
metabolic
arms of mixed acid fermentation to prevent the production of alternative
metabolic
fermentative byproducts (thereby simultaneously increasing butyrate and
acetate
production). Non-limiting examples of such competing metabolic arms are frdA
(converts
phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and
adhE (converts
Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include
deletion of
adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered
bacteria
comprise one or more butyrate-producing cassette(s) and further comprise
mutations
and/or deletions in one or more of frdA, ldhA, and adhE genes.
[0257] In some embodiments, the genetically engineered bacteria comprise one
or
more butyrate producing cassette(s) described herein and one or more
mutation(s) and/or
deletion(s) in one or more genes selected from the ldhA gene, the frdA gene
and the adhE
gene.
[0258] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) encoding one or more enzymes for the production of
butyrate and
further comprise a mutation and/or deletion in one or more endogenous genes
selected
from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments,
the
genetically engineered bacteria comprise one or more gene sequence(s) encoding
one or
more enzymes for the production of butyrate and further comprise a mutation
and/or
deletion in the endogenous ldhA gene. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) encoding one or more enzymes
for the
production of butyrate and further comprise a mutation and/or deletion in the
endogenous
adhE gene. In some embodiments, the genetically engineered bacteria comprise
one or
more gene sequence(s) encoding one or more enzymes for the production of
butyrate and
further comprise a mutation and/or deletion in the endogenous frdA gene. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes for the production of butyrate and further
comprise a
mutation and/or deletion in the endogenous ldhA and rdA genes. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
encoding one
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or more enzymes for the production of butyrate and further comprise a mutation
and/or
deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s) encoding
one or
more enzymes for the production of butyrate and further comprise a mutation
and/or
deletion in the endogenous frdA and adhE genes. In some embodiments, the
genetically
engineered bacteria comprise one or more gene sequence(s) encoding one or more
enzymes for the production of butyrate and further comprise a mutation and/or
deletion in
the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the
genetically
engineered bacteria comprise one or more gene sequence(s) encoding one or more
enzymes for the production of butyrate and further comprise a mutation and/or
deletion in
one or more endogenous genes selected from in the ldhA gene, the frdA gene and
the adhE
genes.
[0259] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and
further
comprise a mutation and/or deletion in the endogenous ldhA gene. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
comprising
one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a
mutation
and/or deletion in the endogenous ldhA gene. In some embodiments, the
genetically
engineered bacteria comprise one or more gene sequence(s) selected from ter,
thiAl, hbd,
crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the
endogenous
adhE gene. In some embodiments, the genetically engineered bacteria comprise
one or
more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene
cassette(s) and further comprise a mutation and/or deletion in the endogenous
adhE gene.
In some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further
comprise a
mutation and/or deletion in the endogenous frdA gene. In some embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s)
comprising one or
more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a
mutation and/or
deletion in the endogenous frdA gene. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd,
crt2, pbt,
and/or buk and further comprise a mutation and/or deletion in the endogenous
ldhA and
frdA genes. In some embodiments, the genetically engineered bacteria comprise
one or
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more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene
cassette(s) and further comprise a mutation and/or deletion in the endogenous
ldhA and
frdA genes. In some embodiments, the genetically engineered bacteria comprise
one or
more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and
further
comprise a mutation and/or deletion in the endogenous ldhA genes and adhE
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s)
and
further comprise a mutation and/or deletion in the endogenous ldhA genes and
adhE
genes. In some embodiments, the genetically engineered bacteria comprise one
or more
gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and
further comprise
a mutation and/or deletion in the endogenous frdA and adhE genes. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
comprising
one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a
mutation
and/or deletion in the endogenous frdA and adhE genes. In some embodiments,
the
genetically engineered bacteria comprise one or more gene sequence(s) selected
from ter,
thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or
deletion in the
endogenous ldhA, the frdA, and adhE genes. In some embodiments, the
genetically
engineered bacteria comprise one or more gene sequence(s) comprising one or
more ter-
thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or
deletion in
the endogenous ldhA, the frdA, and adhE genes.
[0260] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further
comprise a
mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s)
comprising one or
more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation
and/or
deletion in the endogenous ldhA gene. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd,
crt2, tesB
and further comprise a mutation and/or deletion in the endogenous adhE gene.
In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
comprising one or more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further
comprise a
mutation and/or deletion in the endogenous adhE gene. In some embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s) selected
from ter,
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thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the
endogenous
frdA gene. In some embodiments, the genetically engineered bacteria comprise
one or
more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene
cassette(s)
and further comprise a mutation and/or deletion in the endogenous frdA gene.
In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation
and/or deletion in
the endogenous ldhA and frdA genes. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) comprising one or more ter-
thiAl-hbd-
crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in
the
endogenous ldhA and frdA genes. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd,
crt2, tesB
and further comprise a mutation and/or deletion in the endogenous ldhA genes
and adhE
genes. In some embodiments, the genetically engineered bacteria comprise one
or more
gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene
cassette(s) and
further comprise a mutation and/or deletion in the endogenous ldhA genes and
adhE
genes. In some embodiments, the genetically engineered bacteria comprise one
or more
gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further
comprise a mutation
and/or deletion in the endogenous frdA and adhE genes. In some embodiments,
the
genetically engineered bacteria comprise one or more gene sequence(s)
comprising one or
more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation
and/or
deletion in the endogenous frdA and adhE genes. In some embodiments, the
genetically
engineered bacteria comprise one or more gene sequence(s) selected from ter,
thiAl, hbd,
crt2, tesB and further comprise a mutation and/or deletion in the endogenous
ldhA, the
frdA, and adhE genes. In some embodiments, the genetically engineered bacteria
comprise
one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB
gene
cassette(s) and further comprise a mutation and/or deletion in the endogenous
ldhA, the
frdA, and adhE genes.
[0261] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more acetate than unmodified bacteria of the same
bacterial subtype
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under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the the genetically engineered bacteria
produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold, more acetate than
unmodified
bacteria of the same bacterial subtype under the same conditions.
[0262] In some embodiments, the genetically engineered bacteria produce 0% to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more butyrate than unmodified bacteria of the same
bacterial
subtype under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more butyrate than unmodified bacteria of the same bacterial subtype
under the same
conditions. In yet another embodiment, the the genetically engineered bacteria
produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more butyrate than
unmodified
bacteria of the same bacterial subtype under the same conditions.
[0263] In certain situations, the need may arise to prevent and/or reduce
acetate
production of an engineered or naturally occurring strain, e.g., E. coli
Nissle, while
maintaining high levels of butyrate production. Without wishing to be bound by
theory,
one or more mutations and/or deletions in one or more gene(s) encoding in one
or more
enzymes which function in the acetate producing metabolic arm of fermentation
should
reduce and/or prevent production of acetate. A non-limiting example of such an
enzyme is
phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic
arm
converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or
a gene
encoding another enzyme in this metabolic arm may also allow for more acetyl-
CoA to be
used for butyrate production. Additionally, one or more mutations preventing
or reducing
the flow through other metabolic arms of mixed acid fermentaion, such as those
which
produce succinate, lactate, and/or ethanol can increase the production of
acetyl-CoA,
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which is available for butyrate synthesis. Such mutations and/or deletions,
include but are
not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE
genes.
[0264] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) encoding one or more enzymes for the production of
butyrate and
further comprise a mutation and/or deletion in the endogenous pta gene. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes for the production of butyrate and further
comprise a
mutation and/or deletion in the endogenous pta gene and in one or more
endogenous genes
selected from in the ldhA gene, the frdA gene and the adhE gene. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
encoding one
or more enzymes for the production of butyrate and further comprise a mutation
in the
endogenous pta and adhE genes. In some embodiments, the genetically engineered
bacteria comprise one or more gene sequence(s) encoding one or more enzymes
for the
production of butyrate and further comprise a mutation in the endogenous pta
and ldhA
genes. In some embodiments, the genetically engineered bacteria comprise one
or more
gene sequence(s) encoding one or more enzymes for the production of butyrate
and further
comprise a mutation in the endogenous pta and frdA genes. In some embodiments,
the
genetically engineered bacteria comprise one or more gene sequence(s) encoding
one or
more enzymes for the production of butyrate and further comprise a mutation
and/or
deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s) encoding
one or
more enzymes for the production of butyrate and further comprise a mutation in
the
endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) encoding one or more enzymes
for the
production of butyrate and further comprise a mutation in the endogenous pta,
frdA and
adhE genes. In some embodiments, the genetically engineered bacteria comprise
one or
more gene sequence(s) encoding one or more enzyme(s) for the production of
butyrate and
further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA,
and adhE
genes.
[0265] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and
further
comprise a mutation and/or deletion in the endogenous pta gene. In some
embodiments,
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the genetically engineered bacteria comprise one or more gene sequence(s)
comprising
one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further
comprise a
mutation and/or deletion in the endogenous pta gene. In some embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s) selected
from ter,
thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or
deletion in the
endogenous pta gene and in one or more endogenous genes selected from in the
ldhA
gene, the frdA gene and the adhE gene. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) comprising one or more ter-
thiAl-hbd-
crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or
deletion in the
endogenous pta gene and in one or more endogenous genes selected from in the
ldhA
gene, the frdA gene and the adhE gene. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd,
crt2, pbt,
and/or buk and further comprise a mutation in the endogenous pta and adhE
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate
cassette(s) and
further comprise a mutation in the endogenous pta and adhE genes. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
selected from
ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the
endogenous
pta and ldhA genes.
[0266] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk
butyrate
cassette(s) and further comprise a mutation in the endogenous pta and ldhA
genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a
mutation in the
endogenous pta and frdA genes. In some embodiments, the genetically engineered
bacteria
comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-
crt2-pbt-
buk butyrate cassette(s) and further comprise a mutation in the endogenous pta
and frdA
genes. In some embodiments, the genetically engineered bacteria comprise one
or more
gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and
further comprise
a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and
further
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comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA
genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a
mutation in the
endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) comprising one or more ter-
thiAl-hbd-
crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the
endogenous pta,
ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria
comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2,
pbt, and/or buk
and further comprise a mutation in the endogenous pta, frdA and adhE genes. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and
further
comprise a mutation in the endogenous pta, frdA and adhE genes. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
selected from
ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the
endogenous
pta, ldhA, frdA, and adhE genes. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) comprising one or more ter-
thiAl-hbd-
crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the
endogenous pta,
ldhA, frdA, and adhE genes.
[0267] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further
comprise a
mutation and/or deletion in the endogenous pta gene. In some embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s)
comprising one or
more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a
mutation and/or
deletion in the endogenous pta gene. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd,
crt2, tesB
and further comprise a mutation and/or deletion in the endogenous pta gene and
in one or
more endogenous genes selected from in the ldhA gene, the frdA gene and the
adhE gene.
In some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate
cassette(s) and
further comprise a mutation and/or deletion in the endogenous pta gene and in
one or more
endogenous genes selected from in the ldhA gene, the frdA gene and the adhE
gene. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
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sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a
mutation in the
endogenous pta and adhE genes. In some embodiments, the genetically engineered
bacteria comprise one or more gene sequence(s) comprising one or more ter-
thiAl-hbd-
crt2-tesB butyrate cassette(s) and further comprise a mutation in the
endogenous pta and
adhE genes. In some embodiments, the genetically engineered bacteria comprise
one or
more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further
comprise a
mutation in the endogenous pta and ldhA genes.
[0268] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate
cassette(s) and further comprise a mutation in the endogenous pta and ldhA
genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation in
the
endogenous pta and frdA genes. In some embodiments, the genetically engineered
bacteria
comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-
crt2-tesB
butyrate cassette(s) and further comprise a mutation in the endogenous pta and
frdA genes.
In some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a
mutation
and/or deletion in the endogenous pta, ldhA and frdA genes. In some
embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s)
comprising one or
more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a
mutation and/or
deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s) selected
from ter,
thiAl, hbd, crt2, tesB and further comprise a mutation in the endogenous pta,
ldhA, and
adhE genes. In some embodiments, the genetically engineered bacteria comprise
one or
more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate
cassette(s) and further comprise a mutation in the endogenous pta, ldhA, and
adhE genes.
In some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a
mutation in the
endogenous pta, frdA and adhE genes. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) comprising one or more ter-
thiAl-hbd-
crt2-tesB butyrate cassette(s) and further comprise a mutation in the
endogenous pta, frdA
and adhE genes. In some embodiments, the genetically engineered bacteria
comprise one
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or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further
comprise a
mutation in the endogenous pta, ldhA, frdA, and adhE genes. In some
embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s)
comprising one or
more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a
mutation in the
endogenous pta, ldhA, frdA, and adhE genes.
[0269] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% less acetate than unmodified bacteria of the same
bacterial subtype
under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold less acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the the genetically engineered bacteria
produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than
unmodified bacteria
of the same bacterial subtype under the same conditions.
[0270] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more butyrate than unmodified bacteria of the same
bacterial
subtype under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more butyrate than unmodified bacteria of the same bacterial subtype
under the same
conditions. In yet another embodiment, the the genetically engineered bacteria
produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more butyrate than
unmodified
bacteria of the same bacterial subtype under the same conditions.
[0271] In some embodiments, the genetically engineered bacteria comprise a
combination of butyrate biosynthesis genes from different species, strains,
and/or
substrains of bacteria, and are capable of producing butyrate. In some
embodiments, one
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or more of the butyrate biosynthesis genes is functionally replaced, modified,
and/or
mutated in order to enhance stability and/or increase butyrate production. In
some
embodiments, the local production of butyrate reduces food intake and
ameliorates
improves gut barrier function and reduces inflammation. In some embodiments,
the
genetically engineered bacteria are capable of expressing the butyrate
biosynthesis cassette
and producing butyrate in low-oxygen conditions, in the presence of certain
molecules or
metabolites, in the presence of molecules or metabolites associated with
inflammation or
an inflammatory response, or in the presence of some other metabolite that may
or may
not be present in the gut, such as arabinose.
[0272] In one embodiment, the butyrate gene cassette is directly operably
linked to
a first promoter. In another embodiment, the butyrate gene cassette is
indirectly operably
linked to a first promoter. In one embodiment, the promoter is not operably
linked with
the butyrate gene cassette in nature.
[0273] In some embodiments, the butyrate gene cassette is expressed under the
control of a constitutive promoter. In another embodiment, the butyrate gene
cassette is
expressed under the control of an inducible promoter. In some embodiments, the
butyrate
gene cassette is expressed under the control of a promoter that is directly or
indirectly
induced by exogenous environmental conditions. In one embodiment, the butyrate
gene
cassette is expressed under the control of a promoter that is directly or
indirectly induced
by low-oxygen or anaerobic conditions, wherein expression of the butyrate gene
cassette is
activated under low-oxygen or anaerobic environments, such as the environment
of the
mammalian gut. Inducible promoters are described in more detail infra.
[0274] The butyrate gene cassette may be present on a plasmid or chromosome in
the bacterial cell. In one embodiment, the butyrate gene cassette is located
on a plasmid in
the bacterial cell. In another embodiment, the butyrate gene cassette is
located in the
chromosome of the bacterial cell. In yet another embodiment, a native copy of
the
butyrate gene cassette is located in the chromosome of the bacterial cell, and
a butyrate
gene cassette from a different species of bacteria is located on a plasmid in
the bacterial
cell. In yet another embodiment, a native copy of the butyrate gene cassette
is located on a
plasmid in the bacterial cell, and a butyrate gene cassette from a different
species of
bacteria is located on a plasmid in the bacterial cell. In yet another
embodiment, a native
copy of the butyrate gene cassette is located in the chromosome of the
bacterial cell, and a
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butyrate gene cassette from a different species of bacteria is located in the
chromosome of
the bacterial cell.
[0275] In some embodiments, the butyrate gene cassette is expressed on a low-
copy plasmid. In some embodiments, the butyrate gene cassette is expressed on
a high-
copy plasmid. In some embodiments, the high-copy plasmid may be useful for
increasing
expression of butyrate.
Propionate
[0276] In alternate embodiments, the genetically engineered bacteria of the
invention are capable of producing an anti-inflammatory or gut barrier
enhancer molecule,
e.g., propionate, that is synthesized by a biosynthetic pathway requiring
multiple genes
and/or enzymes.
[0277] In some embodiments, the genetically engineered bacteria of the
invention
comprise a propionate gene cassette and are capable of producing propionate
under
particular exogenous environmental conditions. The genetically engineered
bacteria may
express any suitable set of propionate biosynthesis genes (see, e.g., Table 4,
Table 5,
Table 6, Table 7). Unmodified bacteria that are capable of producing
propionate via an
endogenous propionate biosynthesis pathway include, but are not limited to,
Clostridium
propionicum, Megasphaera elsdenii, and Prevotella ruminicola. In some
embodiments,
the genetically engineered bacteria of the invention comprise propionate
biosynthesis
genes from a different species, strain, or substrain of bacteria. In some
embodiments, the
genetically engineered bacteria comprise the genes pct, lcd, and acr from
Clostridium
propionicum. In some embodiments, the genetically engineered bacteria comprise
acrylate pathway genes for propionate biosynthesis, e.g., pct, lcdA, lcdB,
lcdC, e0, acrB,
and acrC. In some embodiments, the rate limiting step catalyzed by the Acr
enzyme, is
replaced by the AcuI from R. sphaeroides, which catalyzes the NADPH-dependent
acrylyl-CoA reduction to produce propionyl-CoA. Thus the propionate cassette
comprises
pct, lcdA, lcdB, lcdC, and acuI. In another embodiment, the homolog of AcuI in
E coli,
yhdH is used. This the propionate cassette comprises pct, lcdA, lcdB, lcdC,
and yhdH. In
alternate embodiments, the genetically engineered bacteria comprise pyruvate
pathway
genes for propionate biosynthesis, e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE,
aceF, and 1pd, and
optionally further comprise tesB. In another embodiment, the propionate gene
cassette
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comprises the genes of the Sleepting Beauty Mutase operon, e.g., from E. coli
(sbm, ygfD,
ygfG, ygfH). The SBM pathway is cyclical and composed of a series of
biochemical
conversions forming propionate as a fermentative product while regenerating
the starting
molecule of succinyl-CoA. Sbm converts succinyl CoA to L-methylmalonylCoA,
ygfG
converts L-methylmalonylCoA into PropionylCoA, and ygfH converts propionylCoA
into
propionate and succinate into succinylCoA.
[0278] This pathway is very similar to the oxidative propionate pathway of
Propionibacteria, which also converts succinate to propionate. Succinyl-CoA is
converted
to R-methylmalonyl-CoA by methymalonyl-CoA mutase (mutAB). This is in turn
converted to S-methylmalonyl-CoA via methymalonyl-CoA epimerase (GI:18042134).
There are three genes which encode methylmalonyl-CoA carboxytransferase (mmdA,
PFREUD 18870, bccp) which converts methylmalonyl-CoA to propionyl-CoA.
[0279] The genes may be codon-optimized, and translational and transcriptional
elements may be added. Table 4-6 lists the nucleic acid sequences of exemplary
genes in
the propionate biosynthesis gene cassette. Table 7 lists the polypeptide
sequences
expressed by exemplary propionate biosynthesis genes.
Table 4. Propionate Cassette Sequences (Acrylate Pathway)
Gene sequence Description
ATGCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCAAAAC
TGATCAAGGACGGCGACACCGTGACAACTAGCGGCTTTGTGGGT
AACGCGATCCCTGAGGCCCTTGACCGTGCAGTCGAAAAGCGTTT
CCTGGAAACGGGCGAACCGAAGAACATTACTTATGTATATTGCG
GCAGTCAGGGCAATCGCGACGGTCGTGGCGCAGAACATTTCGC
GCATGAAGGCCTGCTGAAACGTTATATCGCTGGCCATTGGGCGA
CCGTCCCGGCGTTAGGGAAAATGGCCATGGAGAATAAAATGGA
GGCCTACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCGA
TATTGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAGGAATCG
GCACCTTCATTGATCCACGTAACGGTGGTGGGAAGGTCAACGAT
pct
ATTACCAAGGAAGATATCGTAGAACTGGTGGAAATTAAAGGGC
SEQ ID NO: 21
AGGAATACCTGTTTTATCCGGCGTTCCCGATCCATGTCGCGCTG
ATTCGTGGCACCTATGCGGACGAGAGTGGTAACATCACCTTTGA
AAAAGAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGTCAAGCGG
TGAAGAACTCGGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTC
GTCAAAGCAGGCACGCTGGATCCGCGCCATGTGAAAGTTCCGG
GTATCTATGTAGATTACGTAGTCGTCGCGGATCCGGAGGACCAT
CAACAGTCCCTTGACTGCGAATATGATCCTGCCCTTAGTGGAGA
GCACCGTCGTCCGGAGGTGGTGGGTGAACCACTGCCTTTATCCG
CGAAGAAAGTCATCGGCCGCCGTGGCGCGATTGAGCTCGAGAA
AGACGTTGCAGTGAACCTTGGGGTAGGTGCACCTGAGTATGTGG
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Gene sequence Description
CCTCCGTGGCCGATGAAGAAGGCATTGTGGATTTTATGACTCTC
ACAGCGGAGTCCGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGT
TCGCTTTGGGGCGAGCTACAATGCTGACGCCTTGATCGACCAGG
GCTACCAATTTGATTATTACGACGGTGGGGGTCTGGATCTTTGTT
ACCTGGGTTTAGCTGAATGCGACGAAAAGGGTAATATCAATGTT
AGCCGCTTCGGTCCTCGTATCGCTGGGTGCGGCGGATTCATTAA
CATTACCCAAAACACGCCGAAAGTCTTCTTTTGTGGGACCTTTA
CAGCCGGGGGGCTGAAAGTGAAAATTGAAGATGGTAAGGTGAT
TATCGTTCAGGAAGGGAAACAGAAGAAATTCCTTAAGGCAGTG
GAGCAAATCACCTTTAATGGAGACGTGGCCTTAGCGAACAAGC
AACAAGTTACCTACATCACGGAGCGTTGCGTCTTCCTCCTCAAA
GAAGACGGTTTACACCTTTCGGAAATCGCGCCAGGCATCGATCT
GCAGACCCAGATTTTGGATGTTATGGACTTTGCCCCGATCATTG
ATCGTGACGCAAACGGGCAGATTAAACTGATGGACGCGGCGTT
ATTCGCAGAAGGGCTGATGGGCTTGAAAGAAATGAAGTCTTAA
ATGAGCTTAACCCAAGGCATGAAAGCTAAACAACTGTTAGCAT
ACTTTCAGGGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGC
CCGCGGTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCCGG
AATTTTGCGTAACAATGGGCATTGCCATGATCTACCCGGAGACT
CATGCAGCGGGCATCGGTGCCCGCAAAGGTGCGATGGACATGC
TGGAAGTTGCGGACCGCAAAGGCTACAACGTGGATTGTTGTTCC
TACGGCCGTGTAAATATGGGTTACATGGAATGTTTAAAAGAAGC
CGCCATCACGGGCGTCAAGCCGGAAGTTTTGGTTAATTCCCCTG
CTGCTGACGTTCCGCTTCCCGATTTGGTGATTACGTGTAATAATA
TCTGTAACACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGA
ACTCGATATTCCTTGCATCGTGATCGACGTACCGTTTAATCATAC
CATGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGACCAGT
TCCGCAATGCAATTTCTCAGCTGGAAGTTATTTGTGGCCGTCCGT
TCGATTGGAAGAAATTTAAGGAGGTCAAAGATCAGACCCAGCG
lcdA =TAGCGTATACCACTGGAACCGCATTGCCGAGATGGCGAAATAC
SEQ ID NO: 22
AAGCCTAGCCCGCTGAACGGCTTCGATCTGTTCAATTACATGGC
GTTAATCGTGGCGTGCCGCAGCCTGGATTATGCAGAAATTACCT
TTAAAGCGTTCGCGGACGAATTAGAAGAGAATTTGAAGGCGGG
TATCTACGCCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGG
AAGGTATCGCGGTGTGGCCACATTTAGGTCACACGTTTAAATCT
ATGAAGAATCTGAATTCGATTATGACCGGTACGGCATACCCCGC
CCTTTGGGACCTGCACTATGACGCTAACGACGAATCTATGCACT
CTATGGCTGAAGCGTACACCCGTATTTATATTAATACTTGTCTGC
AGAACAAAGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCA
GGTGGATGGTACCGTATATCATCTGAATCGCAGCTGCAAACTGA
TGAGTTTCCTGAACGTGGAAACGGCTGAAATTATTAAAGAGAA
GAACGGTCTTCCTTACGTCTCCATTGATGGCGATCAGACCGATC
CTCGCGTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAGGCCC
TGGTTGAGATGATGGAGGCCAATATGGCGGCAGCGGAATAA
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Gene sequence Description
ATGTCACGCGTGGAGGCAATCCTGTCGCAGCTGAAAGATGTCGC
CGCGAATCCGAAAAAAGCCATGGATGACTATAAAGCTGAAACA
GGTAAGGGCGCGGTTGGTATCATGCCGATCTACAGCCCCGAAG
AAATGGTACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGC
GCCCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCCTGC
TTTTGCCTGCAGCGTAATGCAGCAGGTTATGGAATTACAGTGCG
AGGGCGCGTATGATGACCTGTCCGCAGTTATTTTTAGCGTACCG
TGCGACACTCTCAAATGTCTTAGCCAGAAATGGAAAGGTACGTC
CCCAGTGATTGTATTTACGCATCCGCAGAACCGCGGATTAGAAG
CGGCGAACCAATTCTTGGTTACCGAGTATGAACTGGTAAAAGCA
CAACTGGAATCAGTTCTGGGTGTGAAAATTTCAAACGCCGCCCT
GGAAAATTCGATTGCAATTTATAACGAGAATCGTGCCGTGATGC
lcdB GTGAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATTGAC
SEQ ID NO: 23 GCAGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAGTTTATGCT
TAAGGAAAAACATACCGCACTTGTGAAAGAACTGATCGCTGAG
ATTAAAGCAACGCCAGTCCAGCCGTGGGACGGAAAAAAGGTTG
TAGTGACGGGCATTCTGTTGGAACCGAATGAGTTATTAGATATC
TTTAATGAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCA
GGAAAGCCGTCAGATCCGTGTTGACGTTCTGGACGGAGAAGGC
GGACCGCTCTACCGTATGGCTAAAGCGTGGCAGCAAATGTATGG
CTGCTCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATGT
TAATTAACAAAACGATTCAGACCGGTGCGGACGCTATCGTAGTT
GCAATGATGAAGTTTTGCGACCCAGAAGAATGGGATTATCCGGT
AATGTACCGTGAATTTGAAGAAAAAGGGGTCAAATCACTTATG
ATTGAGGTGGATCAGGAAGTATCGTCTTTCGAACAGATTAAAAC
CCGTCTGCAGTCATTCGTCGAAATGCTTTAA
ATGTATACCTTGGGGATTGATGTCGGTTCTGCCTCTAGTAAAGC
GGTGATTCTGAAAGATGGAAAAGATATTGTCGCTGCCGAGGTTG
TCCAAGTCGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGAC
AAAGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAGCTA
CACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAGCGACGCGG
ATAAACAGATTTCGGAAATTAGCTGTCATGCCAAAGGCATTTAT
TTCTTAGTACCAACTGCGCGCACTATTATTGACATTGGCGGCCA
AGATGCGAAAGCCATCCGCCTGGACGACAAGGGGGGTATTAAG
lcdC CAATTCTTCATGAATGATAAATGCGCGGCGGGCACGGGGCGTTT
SEQ ID NO: 24 CCTGGAAGTCATGGCTCGCGTACTTGAAACCACCCTGGATGAAA
TGGCTGAACTGGATGAACAGGCGACTGACACCGCTCCCATTTCA
AGCACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAATT
GAGCAATGGTGTCTCACGCAACAACATCATTAAAGGTGTCCATC
TGAGCGTTGCGTCACGTGCGTGTGGTCTGGCGTATCGCGGCGGT
TTGGAGAAAGATGTTGTTATGACAGGTGGCGTGGCAAAAAATG
CAGGGGTGGTGCGCGCGGTGGCGGGCGTTCTGAAGACCGATGT
TATCGTTGCTCCGAATCCTCAGACGACCGGTGCACTGGGGGCAG
CGCTGTATGCTTATGAGGCCGCCCAGAAGAAGTA
etfA ATGGCCTTCAATAGCGCAGATATTAATTCTTTCCGCGATATTTGG
SEQ ID NO: 25 GTGTTTTGTGAACAGCGTGAGGGCAAACTGATTAACACCGATTT
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Gene sequence Description
CGAATTAATTAGCGAAGGTCGTAAACTGGCTGACGAACGCGGA
AGCAAACTGGTTGGAATTTTGCTGGGGCACGAAGTTGAAGAAA
TCGCAAAAGAATTAGGCGGCTATGGTGCGGACAAGGTAATTGT
GTGCGATCATCCGGAACTTAAATTTTACACTACGGATGCTTATG
CCAAAGTTTTATGTGACGTCGTGATGGAAGAGAAACCGGAGGT
AATTTTGATCGGTGCCACCAACATTGGCCGTGATCTCGGACCGC
GTTGTGCTGCACGCTTGCACACGGGGCTGACGGCTGATTGCACG
CACCTGGATATTGATATGAATAAATATGTGGACTTTCTTAGCAC
CAGTAGCACCTTGGATATCTCGTCGATGACTTTCCCTATGGAAG
ATACAAACCTTAAAATGACGCGCCCTGCATTTGGCGGACATCTG
ATGGCAACGATCATTTGTCCACGCTTCCGTCCCTGTATGAGCAC
AGTGCGCCCCGGAGTGATGAAGAAAGCGGAGTTCTCGCAGGAG
ATGGCGCAAGCATGTCAAGTAGTGACCCGTCACGTAAATTTGTC
GGATGAAGACCTTAAAACTAAAGTAATTAATATCGTGAAGGAA
ACGAAAAAGATTGTGGATCTGATCGGCGCAGAAATTATTGTGTC
AGTTGGTCGTGGTATCTCGAAAGATGTCCAAGGTGGAATTGCAC
TGGCTGAAAAACTTGCGGACGCATTTGGTAACGGTGTCGTGGGC
GGCTCGCGCGCAGTGATTGATTCCGGCTGGTTACCTGCGGATCA
TCAGGTTGGACAAACCGGTAAGACCGTGCACCCGAAAGTCTAC
GTGGCGCTGGGTATTAGTGGGGCTATCCAGCATAAGGCTGGGAT
GCAAGACTCTGAACTGATCATTGCCGTCAACAAAGACGAAACG
GCGCCTATCTTCGACTGCGCCGATTATGGCATCACCGGTGATTT
ATTTAAAATCGTACCGATGATGATCGACGCGATCAAAGAGGGT
AAAAACGCATGA
ATGCGCATCTATGTGTGTGTGAAACAAGTCCCAGATACGAGCGG
CAAGGTGGCCGTTAACCCTGATGGGACCCTTAACCGTGCCTCAA
TGGCAGCGATTATTAACCCGGACGATATGTCCGCGATCGAACAG
GCATTAAAACTGAAAGATGAAACCGGATGCCAGGTTACGGCGC
TTACGATGGGTCCTCCTCCTGCCGAGGGCATGTTGCGCGAAATT
ATTGCAATGGGGGCCGACGATGGTGTGCTGATTTCGGCCCGTGA
ATTTGGGGGGTCCGATACCTTCGCAACCAGTCAAATTATTAGCG
CGGCAATCCATAAATTAGGCTTAAGCAATGAAGACATGATCTTT
TGCGGTCGTCAGGCCATTGACGGTGATACGGCCCAAGTCGGCCC
acrB
TCAAATTGCCGAAAAACTGAGCATCCCACAGGTAACCTATGGCG
SEQ ID NO: 26
CAGGAATCAAAAAATCTGGTGATTTAGTGCTGGTGAAGCGTATG
TTGGAGGATGGTTATATGATGATCGAAGTCGAAACTCCATGTCT
GATTACCTGCATTCAGGATAAAGCGGTAAAACCACGTTACATGA
CTCTCAACGGTATTATGGAATGCTACTCCAAGCCGCTCCTCGTTC
TCGATTACGAAGCACTGAAAGATGAACCGCTGATCGAACTTGAT
ACCATTGGGCTTAAAGGCTCCCCGACGAATATCTTTAAATCGTT
TACGCCGCCTCAGAAAGGCGTTGGTGTCATGCTCCAAGGCACCG
ATAAGGAAAAAGTCGAGGATCTGGTGGATAAGCTGATGCAGAA
ACATGTCATCTAA
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Gene sequence Description
ATGTTCTTACTGAAGATTAAAAAAGAACGTATGAAACGCATGG
ACTTTAGTTTAACGCGTGAACAGGAGATGTTAAAAAAACTGGCG
CGTCAGTTTGCTGAGATCGAGCTGGAACCGGTGGCCGAAGAGA
TTGATCGTGAGCACGTTTTTCCTGCAGAAAACTTTAAGAAGATG
GCGGAAATTGGCTTAACCGGCATTGGTATCCCGAAAGAATTTGG
TGGCTCCGGTGGAGGCACCCTGGAGAAGGTCATTGCCGTGTCAG
AATTCGGCAAAAAGTGTATGGCCTCAGCTTCCATTTTAAGCATT
CATCTTATCGCGCCGCAGGCAATCTACAAATATGGGACCAAAGA
ACAGAAAGAGACGTACCTGCCGCGTCTTACCAAAGGTGGTGAA
CTGGGCGCCTTTGCGCTGACAGAACCAAACGCCGGAAGCGATG
CCGGCGCGGTAAAAACGACCGCGATTCTGGACAGCCAGACAAA
CGAGTACGTGCTGAATGGCACCAAATGCTTTATCAGCGGGGGCG
GGCGCGCGGGTGTTCTTGTAATTTTTGCGCTTACTGAACCGAAA
acrC AAAGGTCTGAAAGGGATGAGCGCGATTATCGTGGAGAAAGGGA
SEQ ID NO: 27 CCCCGGGCTTCAGCATCGGCAAGGTGGAGAGCAAGATGGGGAT
CGCAGGTTCGGAAACCGCGGAACTTATCTTCGAAGATTGTCGCG
TTCCGGCTGCCAACCTTTTAGGTAAAGAAGGCAAAGGCTTTAAA
ATTGCTATGGAAGCCCTGGATGGCGCCCGTATTGGCGTGGGCGC
TCAAGCAATCGGAATTGCCGAGGGGGCGATCGACCTGAGTGTG
AAGTACGTTCACGAGCGCATTCAATTTGGTAAACCGATCGCGAA
TCTGCAGGGAATTCAATGGTATATCGCGGATATGGCGACCAAAA
CCGCCGCGGCACGCGCACTTGTTGAGTTTGCAGCGTATCTTGAA
GACGCGGGTAAACCGTTCACAAAGGAATCTGCTATGTGCAAGCT
GAACGCCTCCGAAAACGCGCGTTTTGTGACAAATTTAGCTCTGC
AGATTCACGGGGGTTACGGTTATATGAAAGATTATCCGTTAGAG
CGTATGTATCGCGATGCTAAGATTACGGAAATTTACGAGGGGAC
ATCAGAAATCCATAAGGTGGTGATTGCGCGTGAAGTAATGAAA
CGCTAA
ATGCGAGTGTTGAAGTTCGGCGGTACATCAGTGGCAAATGCAG
AACGTTTTCTGCGTGTTGCCGATATTCTGGAAAGCAATGCCAGG
CAGGGGCAGGTGGCCACCGTCCTCTCTGCCCCCGCCAAAATCAC
CAACCACCTGGTGGCGATGATTGAAAAAACCATTAGCGGCCAG
GATGCTTTACCCAATATCAGCGATGCCGAACGTATTTTTGCCGA
ACTTTTGACGGGACTCGCCGCCGCCCAGCCGGGGTTCCCGCTGG
CGCAATTGAAAACTTTCGTCGATCAGGAATTTGCCCAAATAAAA
CATGTCCTGCATGGCATTAGTTTGTTGGGGCAGTGCCCGGATAG
thrgbr CATCAACGCTGCGCTGATTTGCCGTGGCGAGAAAATGTCGATCG
SEQ ID NO: 28 CCATTATGGCCGGCGTATTAGAAGCGCGCGGTCACAACGTTACT
GTTATCGATCCGGTCGAAAAACTGCTGGCAGTGGGGCATTACCT
CGAATCTACCGTCGATATTGCTGAGTCCACCCGCCGTATTGCGG
CAAGCCGCATTCCGGCTGATCACATGGTGCTGATGGCAGGTTTC
ACCGCCGGTAATGAAAAAGGCGAACTGGTGGTGCTTGGACGCA
ACGGTTCCGACTACTCTGCTGCGGTGCTGGCTGCCTGTTTACGC
GCCGATTGTTGCGAGATTTGGACGGACGTTGACGGGGTCTATAC
CTGCGACCCGCGTCAGGTGCCCGATGCGAGGTTGTTGAAGTCGA
TGTCCTACCAGGAAGCGATGGAGCTTTCCTACTTCGGCGCTAAA
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Gene sequence Description
GTTCTTCACCCCCGCACCATTACCCCCATCGCCCAGTTCCAGATC
CCTTGCCTGATTAAAAATACCGGAAATCCTCAAGCACCAGGTAC
GCTCATTGGTGCCAGCCGTGATGAAGACGAATTACCGGTCAAGG
GCATTTCCAATCTGAATAACATGGCAATGTTCAGCGTTTCTGGT
CCGGGGATGAAAGGGATGGTCGGCATGGCGGCGCGCGTCTTTG
CAGCGATGTCACGCGCCCGTATTTCCGTGGTGCTGATTACGCAA
TCATCTTCCGAATACAGCATCAGTTTCTGCGTTCCACAAAGCGA
CTGTGTGCGAGCTGAACGGGCAATGCAGGAAGAGTTCTACCTG
GAACTGAAAGAAGGCTTACTGGAGCCGCTGGCAGTGACGGAAC
GGCTGGCCATTATCTCGGTGGTAGGTGATGGTATGCGCACCTTG
CGTGGGATCTCGGCGAAATTCTTTGCCGCACTGGCCCGCGCCAA
TATCAACATTGTCGCCATTGCTCAGAGATCTTCTGAACGCTCAA
TCTCTGTCGTGGTAAATAACGATGATGCGACCACTGGCGTGCGC
GTTACTCATCAGATGCTGTTCAATACCGATCAGGTTATCGAAGT
GTTTGTGATTGGCGTCGGTGGCGTTGGCGGTGCGCTGCTGGAGC
AACTGAAGCGTCAGCAAAGCTGGCTGAAGAATAAACATATCGA
CTTACGTGTCTGCGGTGTTGCCAACTCGAAGGCTCTGCTCACCA
ATGTACATGGCCTTAATCTGGAAAACTGGCAGGAAGAACTGGC
GCAAGCCAAAGAGCCGTTTAATCTCGGGCGCTTAATTCGCCTCG
TGAAAGAATATCATCTGCTGAACCCGGTCATTGTTGACTGCACT
TCCAGCCAGGCAGTGGCGGATCAATATGCCGACTTCCTGCGCGA
AGGTTTCCACGTTGTCACGCCGAACAAAAAGGCCAACACCTCGT
CGATGGATTACTACCATCAGTTGCGTTATGCGGCGGAAAAATCG
CGGCGTAAATTCCTCTATGACACCAACGTTGGGGCTGGATTACC
GGTTATTGAGAACCTGCAAAATCTGCTCAATGCAGGTGATGAAT
TGATGAAGTTCTCCGGCATTCTTTCTGGTTCGCTTTCTTATATCTT
CGGCAAGTTAGACGAAGGCATGAGTTTCTCCGAGGCGACCACG
CTGGCGCGGGAAATGGGTTATACCGAACCGGACCCGCGAGATG
ATCTTTCTGGTATGGATGTGGCGCGTAAACTATTGATTCTCGCTC
GTGAAACGGGACGTGAACTGGAGCTGGCGGATATTGAAATTGA
ACCTGTGCTGCCCGCAGAGTTTAACGCCGAGGGTGATGTTGCCG
CTTTTATGGCGAATCTGTCACAACTCGACGATCTCTTTGCCGCGC
GCGTGGCGAAGGCCCGTGATGAAGGAAAAGTTTTGCGCTATGTT
GGCAATATTGATGAAGATGGCGTCTGCCGCGTGAAGATTGCCGA
AGTGGATGGTAATGATCCGCTGTTCAAAGTGAAAAATGGCGAA
AACGCCCTGGCCTTCTATAGCCACTATTATCAGCCGCTGCCGTT
GGTACTGCGCGGATATGGTGCGGGCAATGACGTTACAGCTGCCG
GTGTCTTTGCTGATCTGCTACGTACCCTCTCATGGAAGTTAGGA
GTCTGA
-130-
CA 03013770 2018-08-03
WO 2017/136792 PCT/US2017/016603
Gene sequence Description
ATGGTTAAAGTTTATGCCCCGGCTTCCAGTGCCAATATGAGCGT
CGGGTTTGATGTGCTCGGGGCGGCGGTGACACCTGTTGATGGTG
CATTGCTCGGAGATGTAGTCACGGTTGAGGCGGCAGAGACATTC
AGTCTCAACAACCTCGGACGCTTTGCCGATAAGCTGCCGTCAGA
ACCACGGGAAAATATCGTTTATCAGTGCTGGGAGCGTTTTTGCC
AGGAACTGGGTAAGCAAATTCCAGTGGCGATGACCCTGGAAAA
GAATATGCCGATCGGTTCGGGCTTAGGCTCCAGTGCCTGTTCGG
TGGTCGCGGCGCTGATGGCGATGAATGAACACTGCGGCAAGCC
GCTTAATGACACTCGTTTGCTGGCTTTGATGGGCGAGCTGGAAG
GCCGTATCTCCGGCAGCATTCATTACGACAACGTGGCACCGTGT
thrB TTTCTCGGTGGTATGCAGTTGATGATCGAAGAAAACGACATCAT
SEQ ID NO: 29 CAGCCAGCAAGTGCCAGGGTTTGATGAGTGGCTGTGGGTGCTGG
CGTATCCGGGGATTAAAGTCTCGACGGCAGAAGCCAGGGCTATT
TTACCGGCGCAGTATCGCCGCCAGGATTGCATTGCGCACGGGCG
ACATCTGGCAGGCTTCATTCACGCCTGCTATTCCCGTCAGCCTG
AGCTTGCCGCGAAGCTGATGAAAGATGTTATCGCTGAACCCTAC
CGTGAACGGTTACTGCCAGGCTTCCGGCAGGCGCGGCAGGCGG
TCGCGGAAATCGGCGCGGTAGCGAGCGGTATCTCCGGCTCCGGC
CCGACCTTGTTCGCTCTGTGTGACAAGCCGGAAACCGCCCAGCG
CGTTGCCGACTGGTTGGGTAAGAACTACCTGCAAAATCAGGAA
GGTTTTGTTCATATTTGCCGGCTGGATACGGCGGGCGCACGAGT
ACTGGAAAACTAA
ATGAAACTCTACAATCTGAAAGATCACAACGAGCAGGTCAGCTT
TGCGCAAGCCGTAACCCAGGGGTTGGGCAAAAATCAGGGGCTG
TTTTTTCCGCACGACCTGCCGGAATTCAGCCTGACTGAAATTGA
TGAGATGCTGAAGCTGGATTTTGTCACCCGCAGTGCGAAGATCC
TCTCGGCGTTTATTGGTGATGAAATCCCACAGGAAATCCTGGAA
GAGCGCGTGCGCGCGGCGTTTGCCTTCCCGGCTCCGGTCGCCAA
TGTTGAAAGCGATGTCGGTTGTCTGGAATTGTTCCACGGGCCAA
CGCTGGCATTTAAAGATTTCGGCGGTCGCTTTATGGCACAAATG
CTGACCCATATTGCGGGTGATAAGCCAGTGACCATTCTGACCGC
GACCTCCGGTGATACCGGAGCGGCAGTGGCTCATGCTTTCTACG
GTTTACCGAATGTGAAAGTGGTTATCCTCTATCCACGAGGCAAA
thrC ATCAGTCCACTGCAAGAAAAACTGTTCTGTACATTGGGCGGCAA
SEQ ID NO: 30 TATCGAAACTGTTGCCATCGACGGCGATTTCGATGCCTGTCAGG
CGCTGGTGAAGCAGGCGTTTGATGATGAAGAACTGAAAGTGGC
GCTAGGGTTAAACTCGGCTAACTCGATTAACATCAGCCGTTTGC
TGGCGCAGATTTGCTACTACTTTGAAGCTGTTGCGCAGCTGCCG
CAGGAGACGCGCAACCAGCTGGTTGTCTCGGTGCCAAGCGGAA
ACTTCGGCGATTTGACGGCGGGTCTGCTGGCGAAGTCACTCGGT
CTGCCGGTGAAACGTTTTATTGCTGCGACCAACGTGAACGATAC
CGTGCCACGTTTCCTGCACGACGGTCAGTGGTCACCCAAAGCGA
CTCAGGCGACGTTATCCAACGCGATGGACGTGAGTCAGCCGAA
CAACTGGCCGCGTGTGGAAGAGTTGTTCCGCCGCAAAATCTGGC
AACTGAAAGAGCTGGGTTATGCAGCCGTGGATGATGAAACCAC
GCAACAGACAATGCGTGAGTTAAAAGAACTGGGCTACACTTCG
-131-
CA 03013770 2018-08-03
WO 2017/136792 PCT/US2017/016603
Gene sequence Description
GAGCCGCACGCTGCCGTAGCTTATCGTGCGCTGCGTGATCAGTT
GAATCCAGGCGAATATGGCTTGTTCCTCGGCACCGCGCATCCGG
CGAAATTTAAAGAGAGCGTGGAAGCGATTCTCGGTGAAACGTT
GGATCTGCCAAAAGAGCTGGCAGAACGTGCTGATTTACCCTTGC
TTTCACATAATCTGCCCGCCGATTTTGCTGCGTTGCGTAAATTGA
TGATGAATCATCAGTAA
ATGAGTGAAACATACGTGTCTGAGAAAAGTCCAGGAGTGATGG
CTAGCGGAGCGGAGCTGATTCGTGCCGCCGACATTCAAACGGC
GCAGGCACGAATTTCCTCCGTCATTGCACCAACTCCATTGCAGT
ATTGCCCTCGTCTTTCTGAGGAAACCGGAGCGGAAATCTACCTT
AAGCGTGAGGATCTGCAGGATGTTCGTTCCTACAAGATCCGCGG
TGCGCTGAACTCTGGAGCGCAGCTCACCCAAGAGCAGCGCGAT
GCAGGTATCGTTGCCGCATCTGCAGGTAACCATGCCCAGGGCGT
GGCCTATGTGTGCAAGTCCTTGGGCGTTCAGGGACGCATCTATG
TTCCTGTGCAGACTCCAAAGCAAAAGCGTGACCGCATCATGGTT
CACGGCGGAGAGTTTGTCTCCTTGGTGGTCACTGGCAATAACTT
CGACGAAGCATCGGCTGCAGCGCATGAAGATGCAGAGCGCACC
GGCGCAACGCTGATCGAGCCTTTCGATGCTCGCAACACCGTCAT
CGGTCAGGGCACCGTGGCTGCTGAGATCTTGTCGCAGCTGACTT
CCATGGGCAAGAGTGCAGATCACGTGATGGTTCCAGTCGGCGGT
i/vAfbr GGCGGACTTCTTGCAGGTGTGGTCAGCTACATGGCTGATATGGC
SEQ ID NO: 31 ACCTCGCACTGCGATCGTTGGTATCGAACCAGCGGGAGCAGCAT
CCATGCAGGCTGCATTGCACAATGGTGGACCAATCACTTTGGAG
ACTGTTGATCCCTTTGTGGACGGCGCAGCAGTCAAACGTGTCGG
AGATCTCAACTACACCATCGTGGAGAAGAACCAGGGTCGCGTG
CACATGATGAGCGCGACCGAGGGCGCTGTGTGTACTGAGATGCT
CGATCTTTACCAAAACGAAGGCATCATCGCGGAGCCTGCTGGCG
CGCTGTCTATCGCTGGGTTGAAGGAAATGTCCTTTGCACCTGGT
TCTGCAGTGGTGTGCATCATCTCTGGTGGCAACAACGATGTGCT
GCGTTATGCGGAAATCGCTGAGCGCTCCTTGGTGCACCGCGGTT
TGAAGCACTACTTCTTGGTGAACTTCCCGCAAAAGCCTGGTCAG
TTGCGTCACTTCCTGGAAGATATCCTGGGACCGGATGATGACAT
CACGCTGTTTGAGTACCTCAAGCGCAACAACCGTGAGACCGGTA
CTGCGTTGGTGGGTATTCACTTGAGTGAAGCATCAGGATTGGAT
TCTTTGCTGGAACGTATGGAGGAATCGGCAATTGATTCCCGTCG
CCTCGAGCCGGGCACCCCTGAGTACGAATACTTGACCTAA
ATGTCAGAACGTTTCCCAAATGACGTGGATCCGATCGAAACTCG
CGACTGGCTCCAGGCGATCGAATCGGTCATCCGTGAAGAAGGT
GTTGAGCGTGCTCAGTATCTGATCGACCAACTGCTTGCTGAAGC
CCGCAAAGGCGGTGTAAACGTAGCCGCAGGCACAGGTATCAGC
aceE AACTACATCAACACCATCCCCGTTGAAGAACAACCGGAGTATCC
SEQ ID NO: 32 GGGTAATCTGGAACTGGAACGCCGTATTCGTTCAGCTATCCGCT
GGAACGCCATCATGACGGTGCTGCGTGCGTCGAAAAAAGACCT
CGAACTGGGCGGCCATATGGCGTCCTTCCAGTCTTCCGCAACCA
TTTATGATGTGTGCTTTAACCACTTCTTCCGTGCACGCAACGAGC
AGGATGGCGGCGACCTGGTTTACTTCCAGGGCCACATCTCCCCG
-132-
CA 03013770 2018-08-03
WO 2017/136792 PCT/US2017/016603
Gene sequence Description
GGCGTGTACGCTCGTGCTTTCCTGGAAGGTCGTCTGACTCAGGA
GCAGCTGGATAACTTCCGTCAGGAAGTTCACGGCAATGGCCTCT
CTTCCTATCCGCACCCGAAACTGATGCCGGAATTCTGGCAGTTC
CCGACCGTATCTATGGGTCTGGGTCCGATTGGTGCTATTTACCA
GGCTAAATTCCTGAAATATCTGGAACACCGTGGCCTGAAAGATA
CCTCTAAACAAACCGTTTACGCGTTCCTCGGTGACGGTGAAATG
GACGAACCGGAATCCAAAGGTGCGATCACCATCGCTACCCGTG
AAAAACTGGATAACCTGGTCTTCGTTATCAACTGTAACCTGCAG
CGTCTTGACGGCCCGGTCACCGGTAACGGCAAGATCATCAACGA
ACTGGAAGGCATCTTCGAAGGTGCTGGCTGGAACGTGATCAAA
GTGATGTGGGGTAGCCGTTGGGATGAACTGCTGCGTAAGGATAC
CAGCGGTAAACTGATCCAGCTGATGAACGAAACCGTTGACGGC
GACTACCAGACCTTCAAATCGAAAGATGGTGCGTACGTTCGTGA
ACACTTCTTCGGTAAATATCCTGAAACCGCAGCACTGGTTGCAG
ACTGGACTGACGAGCAGATCTGGGCACTGAACCGTGGTGGTCA
CGATCCGAAGAAAATCTACGCTGCATTCAAGAAAGCGCAGGAA
ACCAAAGGCAAAGCGACAGTAATCCTTGCTCATACCATTAAAG
GTTACGGCATGGGCGACGCGGCTGAAGGTAAAAACATCGCGCA
CCAGGTTAAGAAAATGAACATGGACGGTGTGCGTCATATCCGC
GACCGTTTCAATGTGCCGGTGTCTGATGCAGATATCGAAAAACT
GCCGTACATCACCTTCCCGGAAGGTTCTGAAGAGCATACCTATC
TGCACGCTCAGCGTCAGAAACTGCACGGTTATCTGCCAAGCCGT
CAGCCGAACTTCACCGAGAAGCTTGAGCTGCCGAGCCTGCAAG
ACTTCGGCGCGCTGTTGGAAGAGCAGAGCAAAGAGATCTCTAC
CACTATCGCTTTCGTTCGTGCTCTGAACGTGATGCTGAAGAACA
AGTCGATCAAAGATCGTCTGGTACCGATCATCGCCGACGAAGCG
CGTACTTTCGGTATGGAAGGTCTGTTCCGTCAGATTGGTATTTAC
AGCCCGAACGGTCAGCAGTACACCCCGCAGGACCGCGAGCAGG
TTGCTTACTATAAAGAAGACGAGAAAGGTCAGATTCTGCAGGA
AGGGATCAACGAGCTGGGCGCAGGTTGTTCCTGGCTGGCAGCG
GCGACCTCTTACAGCACCAACAATCTGCCGATGATCCCGTTCTA
CATCTATTACTCGATGTTCGGCTTCCAGCGTATTGGCGATCTGTG
CTGGGCGGCTGGCGACCAGCAAGCGCGTGGCTTCCTGATCGGCG
GTACTTCCGGTCGTACCACCCTGAACGGCGAAGGTCTGCAGCAC
GAAGATGGTCACAGCCACATTCAGTCGCTGACTATCCCGAACTG
TATCTCTTACGACCCGGCTTACGCTTACGAAGTTGCTGTCATCAT
GCATGACGGTCTGGAGCGTATGTACGGTGAAAAACAAGAGAAC
GTTTACTACTACATCACTACGCTGAACGAAAACTACCACATGCC
GGCAATGCCGGAAGGTGCTGAGGAAGGTATCCGTAAAGGTATC
TACAAACTCGAAACTATTGAAGGTAGCAAAGGTAAAGTTCAGC
TGCTCGGCTCCGGTTCTATCCTGCGTCACGTCCGTGAAGCAGCT
GAGATCCTGGCGAAAGATTACGGCGTAGGTTCTGACGTTTATAG
CGTGACCTCCTTCACCGAGCTGGCGCGTGATGGTCAGGATTGTG
AACGCTGGAACATGCTGCACCCGCTGGAAACTCCGCGCGTTCCG
TATATCGCTCAGGTGATGAACGACGCTCCGGCAGTGGCATCTAC
CGACTATATGAAACTGTTCGCTGAGCAGGTCCGTACTTACGTAC
-133-
CA 03013770 2018-08-03
WO 2017/136792 PCT/US2017/016603
Gene sequence Description
CGGCTGACGACTACCGCGTACTGGGTACTGATGGCTTCGGTCGT
TCCGACAGCCGTGAGAACCTGCGTCACCACTTCGAAGTTGATGC
TTCTTATGTCGTGGTTGCGGCGCTGGGCGAACTGGCTAAACGTG
GCGAAATCGATAAGAAAGTGGTTGCTGACGCAATCGCCAAATT
CAACATCGATGCAGATAAAGTTAACCCGCGTCTGGCGTAA
ATGGCTATCGAAATCAAAGTACCGGACATCGGGGCTGATGAAG
TTGAAATCACCGAGATCCTGGTCAAAGTGGGCGACAAAGTTGA
AGCCGAACAGTCGCTGATCACCGTAGAAGGCGACAAAGCCTCT
ATGGAAGTTCCGTCTCCGCAGGCGGGTATCGTTAAAGAGATCAA
AGTCTCTGTTGGCGATAAAACCCAGACCGGCGCACTGATTATGA
TTTTCGATTCCGCCGACGGTGCAGCAGACGCTGCACCTGCTCAG
GCAGAAGAGAAGAAAGAAGCAGCTCCGGCAGCAGCACCAGCG
GCTGCGGCGGCAAAAGACGTTAACGTTCCGGATATCGGCAGCG
ACGAAGTTGAAGTGACCGAAATCCTGGTGAAAGTTGGCGATAA
AGTTGAAGCTGAACAGTCGCTGATCACCGTAGAAGGCGACAAG
GCTTCTATGGAAGTTCCGGCTCCGTTTGCTGGCACCGTGAAAGA
GATCAAAGTGAACGTGGGTGACAAAGTGTCTACCGGCTCGCTG
ATTATGGTCTTCGAAGTCGCGGGTGAAGCAGGCGCGGCAGCTCC
GGCCGCTAAACAGGAAGCAGCTCCGGCAGCGGCCCCTGCACCA
GCGGCTGGCGTGAAAGAAGTTAACGTTCCGGATATCGGCGGTG
ACGAAGTTGAAGTGACTGAAGTGATGGTGAAAGTGGGCGACAA
AGTTGCCGCTGAACAGTCACTGATCACCGTAGAAGGCGACAAA
GCTTCTATGGAAGTTCCGGCGCCGTTTGCAGGCGTCGTGAAGGA
aceF
ACTGAAAGTCAACGTTGGCGATAAAGTGAAAACTGGCTCGCTG
SEQ ID NO: 33
ATTATGATCTTCGAAGTTGAAGGCGCAGCGCCTGCGGCAGCTCC
TGCGAAACAGGAAGCGGCAGCGCCGGCACCGGCAGCAAAAGCT
GAAGCCCCGGCAGCAGCACCAGCTGCGAAAGCGGAAGGCAAAT
CTGAATTTGCTGAAAACGACGCTTATGTTCACGCGACTCCGCTG
ATCCGCCGTCTGGCACGCGAGTTTGGTGTTAACCTTGCGAAAGT
GAAGGGCACTGGCCGTAAAGGTCGTATCCTGCGCGAAGACGTT
CAGGCTTACGTGAAAGAAGCTATCAAACGTGCAGAAGCAGCTC
CGGCAGCGACTGGCGGTGGTATCCCTGGCATGCTGCCGTGGCCG
AAGGTGGACTTCAGCAAGTTTGGTGAAATCGAAGAAGTGGAAC
TGGGCCGCATCCAGAAAATCTCTGGTGCGAACCTGAGCCGTAAC
TGGGTAATGATCCCGCATGTTACTCACTTCGACAAAACCGATAT
CACCGAGTTGGAAGCGTTCCGTAAACAGCAGAACGAAGAAGCG
GCGAAACGTAAGCTGGATGTGAAGATCACCCCGGTTGTCTTCAT
CATGAAAGCCGTTGCTGCAGCTCTTGAGCAGATGCCTCGCTTCA
ATAGTTCGCTGTCGGAAGACGGTCAGCGTCTGACCCTGAAGAAA
TACATCAACATCGGTGTGGCGGTGGATACCCCGAACGGTCTGGT
TGTTCCGGTATTCAAAGACGTCAACAAGAAAGGCATCATCGAGC
TGTCTCGCGAGCTGATGACTATTTCTAAGAAAGCGCGTGACGGT
-134-
CA 03013770 2018-08-03
WO 2017/136792 PCT/US2017/016603
Gene sequence Description
AAGCTGACTGCGGGCGAAATGCAGGGCGGTTGCTTCACCATCTC
CAGCATCGGCGGCCTGGGTACTACCCACTTCGCGCCGATTGTGA
ACGCGCCGGAAGTGGCTATCCTCGGCGTTTCCAAGTCCGCGATG
GAGCCGGTGTGGAATGGTAAAGAGTTCGTGCCGCGTCTGATGCT
GCCGATTTCTCTCTCCTTCGACCACCGCGTGATCGACGGTGCTG
ATGGTGCCCGTTTCATTACCATCATTAACAACACGCTGTCTGAC
ATTCGCCGTCTGGTGATGTAA
ATGAGTACTGAAATCAAAACTCAGGTCGTGGTACTTGGGGCAG
GCCCCGCAGGTTACTCCGCTGCCTTCCGTTGCGCTGATTTAGGTC
TGGAAACCGTAATCGTAGAACGTTACAACACCCTTGGCGGTGTT
TGCCTGAACGTCGGCTGTATCCCTTCTAAAGCACTGCTGCACGT
AGCAAAAGTTATCGAAGAAGCCAAAGCGCTGGCTGAACACGGT
ATCGTCTTCGGCGAACCGAAAACCGATATCGACAAGATTCGTAC
CTGGAAAGAGAAAGTGATCAATCAGCTGACCGGTGGTCTGGCT
GGTATGGCGAAAGGCCGCAAAGTCAAAGTGGTCAACGGTCTGG
GTAAATTCACCGGGGCTAACACCCTGGAAGTTGAAGGTGAGAA
CGGCAAAACCGTGATCAACTTCGACAACGCGATCATTGCAGCG
GGTTCTCGCCCGATCCAACTGCCGTTTATTCCGCATGAAGATCC
GCGTATCTGGGACTCCACTGACGCGCTGGAACTGAAAGAAGTA
CCAGAACGCCTGCTGGTAATGGGTGGCGGTATCATCGGTCTGGA
AATGGGCACCGTTTACCACGCGCTGGGTTCACAGATTGACGTGG
TTGAAATGTTCGACCAGGTTATCCCGGCAGCTGACAAAGACATC
GTTAAAGTCTTCACCAAGCGTATCAGCAAGAAATTCAACCTGAT
1pd
GCTGGAAACCAAAGTTACCGCCGTTGAAGCGAAAGAAGACGGC
SEQ ID NO: 34
ATTTATGTGACGATGGAAGGCAAAAAAGCACCCGCTGAACCGC
AGCGTTACGACGCCGTGCTGGTAGCGATTGGTCGTGTGCCGAAC
GGTAAAAACCTCGACGCAGGCAAAGCAGGCGTGGAAGTTGACG
ACCGTGGTTTCATCCGCGTTGACAAACAGCTGCGTACCAACGTA
CCGCACATCTTTGCTATCGGCGATATCGTCGGTCAACCGATGCT
GGCACACAAAGGTGTTCACGAAGGTCACGTTGCCGCTGAAGTTA
TCGCCGGTAAGAAACACTACTTCGATCCGAAAGTTATCCCGTCC
ATCGCCTATACCAAACCAGAAGTTGCATGGGTGGGTCTGACTGA
GAAAGAAGCGAAAGAGAAAGGCATCAGCTATGAAACCGCCACC
TTCCCGTGGGCTGCTTCTGGTCGTGCTATCGCTTCCGACTGCGCA
GACGGTATGACCAAGCTGATTTTCGACAAAGAATCTCACCGTGT
GATCGGTGGTGCGATTGTCGGTACTAACGGCGGCGAGCTGCTGG
GTGAAATCGGCCTGGCAATCGAAATGGGTTGTGATGCTGAAGA
CATCGCACTGACCATCCACGCGCACCCGACTCTGCACGAGTCTG
TGGGCCTGGCGGCAGAAGTGTTCGAAGGTAGCATTACCGACCTG
CCGAACCCGAAAGCGAAGAAGAAGTAA
ATGAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGA
AAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTA
tesB GGTTTACGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTT
SEQ ID NO: 10 GTATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGCTGGTACATT
CGTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAAGCCG
ATTATTTATGATGTCGAAACGCTGCGTGACGGTAACAGCTTCAG
-135-
CA 03013770 2018-08-03
WO 2017/136792 PCT/US2017/016603
Gene sequence Description
CGCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCGATTTTTT
ATATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGAACAT
CAAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTC
GGAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCA
GTGCTGAAAGATAAATTCATCTGCGATCGTCCGCTGGAAGTCCG
TCCGGTGGAGTTTCATAACCCACTGAAAGGTCACGTCGCAGAAC
CACATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGAT
GACCTGCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTT
AACTTCCTGCCGGTAGCTCTACAGCCGCACGGCATCGGTTTTCT
CGAACCGGGGATTCAGATTGCCACCATTGACCATTCCATGTGGT
TCCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTG
GAGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGA
GTTTTATACCCAAGACGGCGTACTGGTTGCCTCGACCGTTCAGG
AAGGGGTGATGCGTAATCACAATTAA
ATGCGTGCGGTACTGATCGAGAAGTCCGATGATACACAGTCCGT
CTCTGTCACCGAACTGGCTGAAGATCAACTGCCGGAAGGCGAC
GTTTTGGTAGATGTTGCTTATTCAACACTGAACTACAAAGACGC
CCTGGCAATTACCGGTAAAGCCCCCGTCGTTCGTCGTTTTCCGAT
GGTACCTGGAATCGACTTTACGGGTACCGTGGCCCAGTCTTCCC
ACGCCGACTTCAAGCCAGGTGATCGCGTAATCCTGAATGGTTGG
GGTGTGGGGGAAAAACATTGGGGCGGTTTAGCGGAGCGCGCTC
GCGTGCGCGGAGACTGGCTTGTTCCCTTGCCAGCCCCCCTGGAC
TTACGCCAAGCGGCCATGATCGGTACAGCAGGATACACGGCGA
TGTTGTGCGTTCTGGCGCTTGAACGTCACGGAGTGGTGCCGGGT
AATGGGGAAATCGTGGTGTCCGGTGCAGCAGGCGGCGTCGGCT
acul
CCGTTGCGACGACCCTTCTTGCCGCTAAGGGCTATGAGGTAGCG
SEQ =ID NO: 35
GCAGTGACTGGACGTGCGTCCGAAGCAGAATATCTGCGCGGTTT
GGGGGCGGCGAGCGTAATTGATCGTAACGAATTAACGGGGAAG
GTACGCCCGCTGGGTCAGGAGCGTTGGGCTGGCGGGATTGACGT
GGCGGGATCAACCGTGCTTGCGAACATGCTTTCTATGATGAAGT
ATCGCGGGGTAGTCGCTGCGTGTGGCCTGGCCGCGGGCATGGAT
CTGCCCGCGTCTGTCGCGCCCTTTATTCTTCGTGGGATGACGCTG
GCAGGGGTGGATAGCGTTATGTGCCCAAAGACAGATCGTTTAGC
AGCGTGGGCCCGTTTGGCGTCAGATCTTGACCCTGCCAAGCTGG
AGGAGATGACTACAGAGTTGCCGTTTAGTGAAGTAATCGAGAC
AGCACCCAAATTCTTGGACGGGACGGTTCGTGGCCGCATTGTTA
TCCCCGTAACGCCCTAA
Table 5. Propionate Cassette Sequences Sleeping Beauty Operon
Sbm ATGTCTAACGTGCAGGAGTGGCAACAGCTTGCCAACAAGGAA
SEQ ID NO: 36 TTGAGCCGTCGGGAGAAAACTGTCGACTCGCTGGTTCATCAAA
CCGCGGAAGGGATCGCCATCAAGCCGCTGTATACCGAAGCCG
ATCTCGATAATCTGGAGGTGACAGGTACCCTTCCTGGTTTGCC
GCCCTACGTTCGTGGCCCGCGTGCCACTATGTATACCGCCCAA
CCGTGGACCATCCGTCAGTATGCTGGTTTTTCAACAGCAAAAG
-136-
CA 03013770 2018-08-03
WO 2017/136792 PCT/US2017/016603
AGTCCAACGCTTTTTATCGCCGTAACCTGGCCGCCGGGCAAAA
AGGTCTTTCCGTTGCGTTTGACCTTGCCACCCACCGTGGCTAC
GACTCCGATAACCCGCGCGTGGCGGGCGACGTCGGCAAAGCG
GGCGTCGCTATCGACACCGTGGAAGATATGAAAGTCCTGTTCG
ACCAGATCCCGCTGGATAAAATGTCGGTTTCGATGACCATGAA
TGGCGCAGTGCTACCAGTACTGGCGTTTTATATCGTCGCCGCA
GAAGAGCAAGGTGTTACACCTGATAAACTGACCGGCACCATT
CAAAACGATATTCTCAAAGAGTACCTCTGCCGCAACACCTATA
TTTACCCACCAAAACCGTCAATGCGCATTATCGCCGACATCAT
CGCCTGGTGTTCCGGCAACATGCCGCGATTTAATACCATCAGT
ATCAGCGGTTACCACATGGGTGAAGCGGGTGCCAACTGCGTG
CAGCAGGTAGCATTTACGCTCGCTGATGGGATTGAGTACATCA
AAGCAGCAATCTCTGCCGGACTGAAAATTGATGACTTCGCTCC
TCGCCTGTCGTTCTTCTTCGGCATCGGCATGGATCTGTTTATGA
ACGTCGCCATGTTGCGTGCGGCACGTTATTTATGGAGCGAAGC
GGTCAGTGGATTTGGCGCACAGGACCCGAAATCACTGGCGCT
GCGTACCCACTGCCAGACCTCAGGCTGGAGCCTGACTGAACA
GGATCCGTATAACAACGTTATCCGCACCACCATTGAAGCGCTG
GCTGCGACGCTGGGCGGTACTCAGTCACTGCATACCAACGCCT
TTGACGAAGCGCTTGGTTTGCCTACCGATTTCTCAGCACGCAT
TGCCCGCAACACCCAGATCATCATCCAGGAAGAATCAGAACT
CTGCCGCACCGTCGATCCACTGGCCGGATCCTATTACATTGAG
TCGCTGACCGATCAAATCGTCAAACAAGCCAGAGCTATTATCC
AACAGATCGACGAAGCCGGTGGCATGGCGAAAGCGATCGAAG
CAGGTCTGCCAAAACGAATGATCGAAGAGGCCTCAGCGCGCG
AACAGTCGCTGATCGACCAGGGCAAGCGTGTCATCGTTGGTGT
CAACAAGTACAAACTGGATCACGAAGACGAAACCGATGTACT
TGAGATCGACAACGTGATGGTGCGTAACGAGCAAATTGCTTC
GCTGGAACGCATTCGCGCCACCCGTGATGATGCCGCCGTAACC
GCCGCGTTGAACGCCCTGACTCACGCCGCACAGCATAACGAA
AACCTGCTGGCTGCCGCTGTTAATGCCGCTCGCGTTCGCGCCA
CCCTGGGTGAAATTTCCGATGCGCTGGAAGTCGCTTTCGACCG
TTATCTGGTGCCAAGCCAGTGTGTTACCGGCGTGATTGCGCAA
AGCTATCATCAGTCTGAGAAATCGGCCTCCGAGTTCGATGCCA
TTGTTGCGCAAACGGAGCAGTTCCTTGCCGACAATGGTCGTCG
CCCGCGCATTCTGATCGCTAAGATGGGCCAGGATGGACACGA
TCGCGGCGCGAAAGTGATCGCCAGCGCCTATTCCGATCTCGGT
TTCGACGTAGATTTAAGCCCGATGTTCTCTACACCTGAAGAGA
TCGCCCGCCTGGCCGTAGAAAACGACGTTCACGTAGTGGGCG
CATCCTCACTGGCTGCCGGTCATAAAACGCTGATCCCGGAACT
GGTCGAAGCGCTGAAAAAATGGGGACGCGAAGATATCTGCGT
GGTCGCGGGTGGCGTCATTCCGCCGCAGGATTACGCCTTCCTG
CAAGAGCGCGGCGTGGCGGCGATTTATGGTCCAGGTACACCT
ATGCTCGACAGTGTGCGCGACGTACTGAATCTGATAAGCCAGC
ATCATGATTAA
ygfD ATGATTAATGAAGCCACGCTGGCAGAAAGTATTCGCCGCTTAC
SEQ ID NO: 37 GTCAGGGTGAGCGTGCCACACTCGCCCAGGCCATGACGCTGG
TGGAAAGCCGTCACCCGCGTCATCAGGCACTAAGTACGCAGC
-137-
CA 03013770 2018-08-03
WO 2017/136792 PCT/US2017/016603
TGCTTGATGCCATTATGCCGTACTGCGGTAACACCCTGCGACT
GGGCGTTACCGGCACCCCCGGCGCGGGGAAAAGTACCTTTCTT
GAGGCCTTTGGCATGTTGTTGATTCGAGAGGGATTAAAGGTCG
CGGTTATTGCGGTCGATCCCAGCAGCCCGGTCACTGGCGGTAG
CATTCTCGGGGATAAAACCCGCATGAATGACCTGGCGCGTGCC
GAAGCGGCGTTTATTCGCCCGGTACCATCCTCCGGTCATCTGG
GCGGTGCCAGTCAGCGAGCGCGGGAATTAATGCTGTTATGCG
AAGCAGCGGGTTATGACGTAGTGATTGTCGAAACGGTTGGCG
TCGGGCAGTCGGAAACAGAAGTCGCCCGCATGGTGGACTGTT
TTATCTCGTTGCAAATTGCCGGTGGCGGCGATGATCTGCAGGG
CATTAAAAAAGGGCTGATGGAAGTGGCTGATCTGATCGTTATC
AACAAAGACGATGGCGATAACCATACCAATGTCGCCATTGCC
CGGCATATGTACGAGAGTGCCCTGCATATTCTGCGACGTAAAT
ACGACGAATGGCAGCCACGGGTTCTGACTTGTAGCGCACTGG
AAAAACGTGGAATCGATGAGATCTGGCACGCCATCATCGACT
TCAAAACCGCGCTAACTGCCAGTGGTCGTTTACAACAAGTGCG
GCAACAACAATCGGTGGAATGGCTGCGTAAGCAGACCGAAGA
AGAAGTACTGAATCACCTGTTCGCGAATGAAGATTTCGATCGC
TATTACCGCCAGACGCTTTTAGCGGTCAAAAACAATACGCTCT
CACCGCGCACCGGCCTGCGGCAGCTCAGTGAATTTATCCAGAC
GCAATATTTTGATTAA
ygfG ATGTCTTATCAGTATGTTAACGTTGTCACTATCAACAAAGTGG
SEQ ID NO: 38 CGGTCATTGAGTTTAACTATGGCCGAAAACTTAATGCCTTAAG
TAAAGTCTTTATTGATGATCTTATGCAGGCGTTAAGCGATCTC
AACCGGCCGGAAATTCGCTGTATCATTTTGCGCGCACCGAGTG
GATCCAAAGTCTTCTCCGCAGGTCACGATATTCACGAACTGCC
GTCTGGCGGTCGCGATCCGCTCTCCTATGATGATCCATTGCGT
CAAATCACCCGCATGATCCAAAAATTCCCGAAACCGATCATTT
CGATGGTGGAAGGTAGTGTTTGGGGTGGCGCATTTGAAATGAT
CATGAGTTCCGATCTGATCATCGCCGCCAGTACCTCAACCTTC
TCAATGACGCCTGTAAACCTCGGCGTCCCGTATAACCTGGTCG
GCATTCACAACCTGACCCGCGACGCGGGCTTCCACATTGTCAA
AGAGCTGATTTTTACCGCTTCGCCAATCACCGCCCAGCGCGCG
CTGGCTGTCGGCATCCTCAACCATGTTGTGGAAGTGGAAGAAC
TGGAAGATTTCACCTTACAAATGGCGCACCACATCTCTGAGAA
AGCGCCGTTAGCCATTGCCGTTATCAAAGAAGAGCTGCGTGTA
CTGGGCGAAGCACACACCATGAACTCCGATGAATTTGAACGT
ATTCAGGGGATGCGCCGCGCGGTGTATGACAGCGAAGATTAC
CAGGAAGGGATGAACGCTTTCCTCGAAAAACGTAAACCTAAT
TTCGTTGGTCATTAA
ygfH ATGGAAACTCAGTGGACAAGGATGACCGCCAATGAAGCGGCA
SEQ ID NO: 39 GAAATTATCCAGCATAACGACATGGTGGCATTTAGCGGCTTTA
CCCCGGCGGGTTCGCCGAAAGCCCTACCCACCGCGATTGCCCG
CAGAGCTAACGAACAGCATGAGGCCAAAAAGCCGTATCAAAT
TCGCCTTCTGACGGGTGCGTCAATCAGCGCCGCCGCTGACGAT
GTACTTTCTGACGCCGATGCTGTTTCCTGGCGTGCGCCATATC
AAACATCGTCCGGTTTACGTAAAAAGATCAATCAGGGCGCGG
TGAGTTTCGTTGACCTGCATTTGAGCGAAGTGGCGCAAATGGT
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CAATTACGGTTTCTTCGGCGACATTGATGTTGCCGTCATTGAA
GCATCGGCACTGGCACCGGATGGTCGAGTCTGGTTAACCAGC
GGGATCGGTAATGCGCCGACCTGGCTGCTGCGGGCGAAGAAA
GTGATCATTGAACTCAATCACTATCACGATCCGCGCGTTGCAG
AACTGGCGGATATTGTGATTCCTGGCGCGCCACCGCGGCGCAA
TAGCGTGTCGATCTTCCATGCAATGGATCGCGTCGGTACCCGC
TATGTGCAAATCGATCCGAAAAAGATTGTCGCCGTCGTGGAA
ACCAACTTGCCCGACGCCGGTAATATGCTGGATAAGCAAAAT
CCCATGTGCCAGCAGATTGCCGATAACGTGGTCACGTTCTTAT
TGCAGGAAATGGCGCATGGGCGTATTCCGCCGGAATTTCTGCC
GCTGCAAAGTGGCGTGGGCAATATCAATAATGCGGTAATGGC
GCGTCTGGGGGAAAACCCGGTAATTCCTCCGTTTATGATGTAT
TCGGAAGTGCTACAGGAATCGGTGGTGCATTTACTGGAAACC
GGCAAAATCAGCGGGGCCAGCGCCTCCAGCCTGACAATCTCG
GCCGATTCCCTGCGCAAGATTTACGACAATATGGATTACTTTG
CCAGCCGCATTGTGTTGCGTCCGCAGGAGATTTCCAATAACCC
GGAAATCATCCGTCGTCTGGGCGTCATCGCTCTGAACGTCGGC
CTGGAGTTTGATATTTACGGGCATGCCAACTCAACACACGTAG
CCGGGGTCGATCTGATGAACGGCATCGGCGGCAGCGGTGATT
TTGAACGCAACGCGTATCTGTCGATCTTTATGGCCCCGTCGAT
TGCTAAAGAAGGCAAGATCTCAACCGTCGTGCCAATGTGCAG
CCATGTTGATCACAGCGAACACAGCGTCAAAGTGATCATCACC
GAACAAGGGATCGCCGATCTGCGCGGTCTTTCCCCGCTTCAAC
GCGCCCGCACTATCATTGATAATTGTGCACATCCTATGTATCG
GGATTATCTGCATCGCTATCTGGAAAATGCGCCTGGCGGACAT
ATTCACCACGATCTTAGCCACGTCTTCGACTTACACCGTAATTT
AATTGCAACCGGCTCGATGCTGGGTTAA
Table 6. Sequences of Propionate Cassette from Propioni Bacteria
Description Sequence
mutA ATGAGCAGCACGGATCAGGGGACCAACCCCGCCGACACTGAC
SEQ ID NO: 40 GACCTCACTCCCACCACACTCAGTCTGGCCGGGGATTTCCCCA
AGGCCACTGAGGAGCAGTGGGAGCGCGAAGTTGAGAAGGTAT
TCAACCGTGGTCGTCCACCGGAGAAGCAGCTGACCTTCGCCGA
GTGTCTGAAGCGCCTGACGGTTCACACCGTCGATGGCATCGAC
ATCGTGCCGATGTACCGTCCGAAGGACGCGCCGAAGAAGCTG
GGTTACCCCGGCGTCACCCCCTTCACCCGCGGCACCACGGTGC
GCAACGGTGACATGGATGCCTGGGACGTGCGCGCCCTGCACG
AGGATCCCGACGAGAAGTTCACCCGCAAGGCGATCCTTGAAG
ACCTGGAGCGTGGCGTCACCTCCCTGTTGTTGCGCGTTGATCC
CGACGCGATCGCACCCGAGCACCTCGACGAGGTCCTCTCCGAC
GTCCTGCTGGAAATGACCAAGGTGGAGGTCTTCAGCCGCTACG
ACCAGGGTGCCGCCGCCGAGGCCTTGATGGGCGTCTACGAGC
GCTCCGACAAGCCGGCGAAGGACCTGGCCCTGAACCTGGGCC
TGGATCCCATCGGCTTCGCGGCCCTGCAGGGCACCGAGCCGG
ATCTGACCGTGCTCGGTGACTGGGTGCGCCGCCTGGCGAAGTT
CTCACCGGACTCGCGCGCCGTCACGATCGACGCGAACGTCTAC
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CACAACGCCGGTGCCGGCGACGTGGCAGAGCTCGCTTGGGCA
CTGGCCACCGGCGCGGAGTACGTGCGCGCCCTGGTCGAACAG
GGCTTCAACGCCACAGAGGCCTTCGACACGATCAACTTCCGTG
TCACCGCCACCCACGACCAGTTCCTCACGATCGCCCGTCTTCG
CGCCCTGCGCGAGGCATGGGCCCGCATCGGCGAGGTCTTTGGC
GTGGACGAGGACAAGCGCGGCGCTCGCCAGAATGCGATCACC
AGTTGGCGTGAGCTCACCCGCGAAGACCCCTATGTCAACATCC
TTCGCGGTTCGATTGCCACCTTCTCCGCCTCCGTTGGCGGGGC
CGAGTCGATCACGACGCTGCCCTTCACCCAGGCCCTCGGCCTG
CCGGAGGACGACTTCCCGCTGCGCATCGCGCGCAACACGGGC
ATCGTGCTCGCCGAAGAGGTGAACATCGGCCGCGTCAACGAC
CCGGCCGGTGGCTCCTACTACGTCGAGTCGCTCACTCGCACCC
TGGCCGACGCTGCCTGGAAGGAATTCCAGGAGGTCGAGAAGC
TCGGTGGCATGTCGAAGGCGGTCATGACCGAGCACGTCACCA
AGGTGCTCGACGCCTGCAATGCCGAGCGCGCCAAGCGCCTGG
CCAACCGCAAGCAGCCGATCACCGCGGTCAGCGAGTTCCCGA
TGATCGGGGCCCGCAGCATCGAGACCAAGCCGTTCCCAACCG
CTCCGGCGCGCAAGGGCCTGGCCTGGCATCGCGATTCCGAGGT
GTTCGAGCAGCTGATGGATCGCTCCACCAGCGTCTCCGAGCGC
CCCAAGGTGTTCCTTGCCTGCCTGGGCACCCGTCGCGACTTCG
GTGGCCGCGAGGGCTTCTCCAGCCCGGTATGGCACATCGCCGG
TATCGACACCCCGCAGGTCGAAGGCGGCACCACCGCCGAGAT
CGTCGAGGCGTTCAAGAAGTCGGGCGCCCAGGTGGCCGATCT
CTGCTCGTCCGCCAAGATCTACGCGCAGCAGGGACTTGAGGTT
GCCAAGGCGCTCAAGGCCGCCGGCGCGAAGGCCCTGTATCTG
TCGGGCGCCTTCAAGGAGTTCGGCGATGACGCCGCCGAGGCC
GAGAAGCTGATCGACGGACGCCTGTACATGGGCATGGATGTC
GTCGACACCCTGTCCTCCACCCTTGATATCTTGGGAGTCGCGA
AGTGA
mutB GTGAGCACTCTGCCCCGTTTTGATTCAGTTGACCTGGGCAATG
SEQ ID NO: 41 CCCCGGTTCCTGCTGATGCCGCACAGCGCTTCGAGGAGTTGGC
CGCCAAGGCCGGCACCGAAGAGGCGTGGGAGACGGCTGAGCA
GATTCCGGTTGGCACCCTGTTCAACGAAGACGTCTACAAGGAC
ATGGACTGGCTGGACACCTACGCCGGTATCCCGCCGTTCGTCC
ACGGCCCATATGCAACCATGTACGCGTTCCGTCCCTGGACGAT
TCGCCAGTACGCCGGCTTCTCCACGGCCAAGGAGTCCAACGCC
TTCTACCGCCGCAACCTTGCGGCGGGCCAGAAGGGCCTGTCGG
TTGCCTTCGACCTGCCCACCCACCGCGGCTACGACTCGGACAA
TCCCCGCGTCGCCGGTGACGTCGGCATGGCCGGGGTGGCCATC
GACTCCATCTATGACATGCGCGAGCTGTTCGCCGGCATTCCGC
TGGACCAGATGAGCGTGTCGATGACCATGAACGGCGCCGTGC
TGCCGATCCTGGCCCTCTATGTGGTGACCGCCGAGGAGCAGGG
CGTCAAGCCCGAGCAGCTCGCCGGGACGATCCAGAACGACAT
CCTCAAGGAGTTCATGGTTCGTAACACCTATATCTACCCGCCG
CAGCCGAGTATGCGAATCATCTCCGAGATCTTCGCCTACACGA
GTGCCAATATGCCGAAGTGGAATTCGATTTCCATTTCCGGCTA
CCACATGCAGGAAGCCGGCGCCACGGCCGACATCGAGATGGC
CTACACCCTGGCCGACGGTGTCGACTACATCCGCGCCGGCGAG
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TCGGTGGGCCTCAATGTCGACCAGTTCGCGCCGCGTCTGTCCT
TCTTCTGGGGCATCGGCATGAACTTCTTCATGGAGGTTGCCAA
GCTGCGTGCCGCACGTATGTTGTGGGCCAAGCTGGTGCATCAG
TTCGGGCCGAAGAATCCGAAGTCGATGAGCCTGCGCACCCAC
TCGCAGACCTCCGGTTGGTCGCTGACCGCCCAGGACGTCTACA
ACAACGTCGTGCGTACCTGCATCGAGGCCATGGCCGCCACCCA
GGGCCATACCCAGTCGCTGCACACGAACTCGCTCGACGAGGC
CATTGCCCTACCGACCGATTTCAGCGCCCGCATCGCCCGTAAC
ACCCAGCTGTTCCTGCAGCAGGAATCGGGCACGACGCGCGTG
ATCGACCCGTGGAGCGGCTCGGCATACGTCGAGGAGCTCACC
TGGGACCTGGCCCGCAAGGCATGGGGCCACATCCAGGAGGTC
GAGAAGGTCGGCGGCATGGCCAAGGCCATCGAAAAGGGCATC
CCCAAGATGCGCATTGAGGAAGCCGCCGCCCGCACCCAGGCA
CGCATCGACTCCGGCCGTCAGCCGCTGATCGGCGTGAACAAGT
ACCGCCTGGAGCACGAGCCGCCGCTCGATGTGCTCAAGGTTG
ACAACTCCACGGTGCTCGCCGAGCAGAAGGCCAAGCTGGTCA
AGCTGCGCGCCGAGCGCGATCCCGAGAAGGTCAAGGCCGCCC
TCGACAAGATCACCTGGGCTGCCGCCAACCCCGACGACAAGG
ATCCGGATCGCAACCTGCTGAAGCTGTGCATCGACGCTGGCCG
CGCCATGGCGACGGTCGGCGAGATGAGCGACGCGCTCGAGAA
GGTCTTCGGACGCTACACCGCCCAGATTCGCACCATCTCCGGT
GTGTACTCGAAGGAAGTGAAGAACACGCCTGAGGTTGAGGAA
GCACGCGAGCTCGTTGAGGAATTCGAGCAGGCCGAGGGCCGT
CGTCCTCGCATCCTGCTGGCCAAGATGGGCCAGGACGGTCACG
ACCGTGGCCAGAAGGTCATCGCCACCGCCTATGCCGACCTCGG
TTTCGACGTCGACGTGGGCCCGCTGTTCCAGACCCCGGAGGAG
ACCGCACGTCAGGCCGTCGAGGCCGATGTGCACGTGGTGGGC
GTTTCGTCGCTCGCCGGCGGGCATCTGACGCTGGTTCCGGCCC
TGCGCAAGGAGCTGGACAAGCTCGGACGTCCCGACATCCTCA
TCACCGTGGGCGGCGTGATCCCTGAGCAGGACTTCGACGAGCT
GCGTAAGGACGGCGCCGTGGAGATCTACACCCCCGGCACCGT
CATTCCGGAGTCGGCGATCTCGCTGGTCAAGAAACTGCGGGCT
TCGCTCGATGCCTAG
GI:18042134 ATGAGTAATGAGGATCTTTTCATCTGTATCGATCACGTGGCAT
ATGCGTGCCCCGACGCCGACGAGGCTTCCAAGTACTACCAGG
SEQ ID NO: 42
AGACCTTCGGCTGGCATGAGCTCCACCGCGAGGAGAACCCGG
AGCAGGGAGTCGTCGAGATCATGATGGCCCCGGCTGCGAAGC
TGACCGAGCACATGACCCAGGTTCAGGTCATGGCCCCGCTCAA
CGACGAGTCGACCGTTGCCAAGTGGCTTGCCAAGCACAATGG
TCGCGCCGGACTGCACCACATGGCATGGCGTGTCGATGACATC
GACGCCGTCAGCGCCACCCTGCGCGAGCGCGGCGTGCAGCTG
CTGTATGACGAGCCCAAGCTCGGCACCGGCGGCAACCGCATC
AACTTCATGCATCCCAAGTCGGGCAAGGGCGTGCTCATCGAGC
TCACCCAGTACCCGAAGAACTGA
mmdA ATGGCTGAAAACAACAATTTGAAGCTCGCCAGCACCATGGAA
GGTCGCGTGGAGCAGCTCGCAGAGCAGCGCCAGGTGATCGAA
SEQ ID NO: 43
GCCGGTGGCGGCGAACGTCGCGTCGAGAAGCAACATTCCCAG
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GGTAAGCAGACCGCTCGTGAGCGCCTGAACAACCTGCTCGAT
CCCCATTCGTTCGACGAGGTCGGCGCTTTCCGCAAGCACCGCA
CCACGTTGTTCGGCATGGACAAGGCCGTCGTCCCGGCAGATGG
CGTGGTCACCGGCCGTGGCACCATCCTTGGTCGTCCCGTGCAC
GCCGCGTCCCAGGACTTCACGGTCATGGGTGGTTCGGCTGGCG
AGACGCAGTCCACGAAGGTCGTCGAGACGATGGAACAGGCGC
TGCTCACCGGCACGCCCTTCCTGTTCTTCTACGATTCGGGCGG
CGCCCGGATCCAGGAGGGCATCGACTCGCTGAGCGGTTACGG
CAAGATGTTCTTCGCCAACGTGAAGCTGTCGGGCGTCGTGCCG
CAGATCGCCATCATTGCCGGCCCCTGTGCCGGTGGCGCCTCGT
ATTCGCCGGCACTGACTGACTTCATCATCATGACCAAGAAGGC
CCATATGTTCATCACGGGCCCCCAGGTCATCAAGTCGGTCACC
GGCGAGGATGTCACCGCTGACGAACTCGGTGGCGCTGAGGCC
CATATGGCCATCTCGGGCAATATCCACTTCGTGGCCGAGGACG
ACGACGCCGCGGAGCTCATTGCCAAGAAGCTGCTGAGCTTCCT
TCCGCAGAACAACACTGAGGAAGCATCCTTCGTCAACCCGAA
CAATGACGTCAGCCCCAATACCGAGCTGCGCGACATCGTTCCG
ATTGACGGCAAGAAGGGCTATGACGTGCGCGATGTCATTGCC
AAGATCGTCGACTGGGGTGACTACCTCGAGGTCAAGGCCGGC
TATGCCACCAACCTCGTGACCGCCTTCGCCCGGGTCAATGGTC
GTTCGGTGGGCATCGTGGCCAATCAGCCGTCGGTGATGTCGGG
TTGCCTCGACATCAACGCCTCTGACAAGGCCGCCGAATTCGTG
AATTTCTGCGATTCGTTCAACATCCCGCTGGTGCAGCTGGTCG
ACGTGCCGGGCTTCCTGCCCGGCGTGCAGCAGGAGTACGGCG
GCATCATTCGCCATGGCGCGAAGATGCTGTACGCCTACTCCGA
GGCCACCGTGCCGAAGATCACCGTGGTGCTCCGCAAGGCCTA
CGGCGGCTCCTACCTGGCCATGTGCAACCGTGACCTTGGTGCC
GACGCCGTGTACGCCTGGCCCAGCGCCGAGATTGCGGTGATG
GGCGCCGAGGGTGCGGCAAATGTGATCTTCCGCAAGGAGATC
AAGGCTGCCGACGATCCCGACGCCATGCGCGCCGAGAAGATC
GAGGAGTACCAGAACGCGTTCAACACGCCGTACGTGGCCGCC
GCCCGCGGTCAGGTCGACGACGTGATTGACCCGGCTGATACCC
GTCGAAAGATTGCTTCCGCCCTGGAGATGTACGCCACCAAGCG
TCAGACCCGCCCGGCGAAGAAGCATGGAAACTTCCCCTGCTG
A
PFREUD 18870 ATGAGTCCGCGAGAAATTGAGGTTTCCGAGCCGCGCGAGGTT
SEQ ID NO: 44 GGTATCACCGAGCTCGTGCTGCGCGATGCCCATCAGAGCCTGA
TGGCCACACGAATGGCAATGGAAGACATGGTCGGCGCCTGTG
CAGACATTGATGCTGCCGGGTACTGGTCAGTGGAGTGTTGGGG
TGGTGCCACGTATGACTCGTGTATCCGCTTCCTCAACGAGGAT
CCTTGGGAGCGTCTGCGCACGTTCCGCAAGCTGATGCCCAACA
GCCGTCTCCAGATGCTGCTGCGTGGCCAGAACCTGCTGGGTTA
CCGCCACTACAACGACGAGGTCGTCGATCGCTTCGTCGACAAG
TCCGCTGAGAACGGCATGGACGTGTTCCGTGTCTTCGACGCCA
TGAATGATCCCCGCAACATGGCGCACGCCATGGCTGCCGTCAA
GAAGGCCGGCAAGCACGCGCAGGGCACCATTTGCTACACGAT
CAGCCCGGTCCACACCGTTGAGGGCTATGTCAAGCTTGCTGGT
CAGCTGCTCGACATGGGTGCTGATTCCATCGCCCTGAAGGACA
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TGGCCGCCCTGCTCAAGCCGCAGCCGGCCTACGACATCATCAA
GGCCATCAAGGACACCTACGGCCAGAAGACGCAGATCAACCT
GCACTGCCACTCCACCACGGGTGTCACCGAGGTCTCCCTCATG
AAGGCCATCGAGGCCGGCGTCGACGTCGTCGACACCGCCATC
TCGTCCATGTCGCTCGGCCCGGGCCACAACCCCACCGAGTCGG
TTGCCGAGATGCTCGAGGGCACCGGGTACACCACCAACCTTG
ACTACGATCGCCTGCACAAGATCCGCGATCACTTCAAGGCCAT
CCGCCCGAAGTACAAGAAGTTCGAGTCGAAGACGCTTGTCGA
CACCTCGATCTTCAAGTCGCAGATCCCCGGCGGCATGCTCTCC
AACATGGAGTCGCAGCTGCGCGCCCAGGGCGCCGAGGACAAG
ATGGACGAGGTCATGGCAGAGGTGCCGCGCGTCCGCAAGGCC
GCCGGCTTCCCGCCCCTGGTCACCCCGTCCAGCCAGATCGTCG
GCACGCAGGCCGTGTTCAACGTGATGATGGGCGAGTACAAGA
GGATGACCGGCGAGTTCGCCGACATCATGCTCGGCTACTACGG
CGCCAGCCCGGCCGATCGCGATCCGAAGGTGGTCAAGTTGGC
CGAGGAGCAGTCCGGCAAGAAGCCGATCACCCAGCGCCCGGC
CGATCTGCTGCCCCCCGAGTGGGAGGAGCAGTCCAAGGAGGC
CGCGGCCCTCAAGGGCTTCAACGGCACCGACGAGGACGTGCT
CACCTATGCACTGTTCCCGCAGGTCGCTCCGGTCTTCTTCGAG
CATCGCGCCGAGGGCCCGCACAGCGTGGCTCTCACCGATGCCC
AGCTGAAGGCCGAGGCCGAGGGCGACGAGAAGTCGCTCGCCG
TGGCCGGTCCCGTCACCTACAACGTGAACGTGGGCGGAACCG
TCCGCGAAGTCACCGTTCAGCAGGCGTGA
Bccp ATGAAACTGAAGGTAACAGTCAACGGCACTGCGTATGACGTT
GACGTTGACGTCGACAAGTCACACGAAAACCCGATGGGCACC
SEQ ID NO: 45
ATCCTGTTCGGCGGCGGCACCGGCGGCGCGCCGGCACCGCGC
GCAGCAGGTGGCGCAGGCGCCGGTAAGGCCGGAGAGGGCGA
GATTCCCGCTCCGCTGGCCGGCACCGTCTCCAAGATCCTCGTG
AAGGAGGGTGACACGGTCAAGGCTGGTCAGACCGTGCTCGTT
CTCGAGGCCATGAAGATGGAGACCGAGATCAACGCTCCCACC
GACGGCAAGGTCGAGAAGGTCCTTGTCAAGGAGCGTGACGCC
GTGCAGGGCGGTCAGGGTCTCATCAAGATCGGCTGA
[0280] In some embodiments, the genetically engineered bacteria comprise one
or
more nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21- SEQ ID NO: 35, and
SEQ
ID NO: 10) or a functional fragment thereof. In some embodiments, the
genetically
engineered bacteria comprise a nucleic acid sequence that, but for the
redundancy of the
genetic code, encodes the same polypeptide as one or more nucleic acid s
sequence(s) of
Table 4 (SEQ ID NO: 21- SEQ ID NO: 35, and SEQ ID NO: 10) or a functional
fragment thereof. In some embodiments, genetically engineered bacteria
comprise a
nucleic acid sequence that is at least about 80%, at least about 85%, at least
about 90%, at
least about 95%, or at least about 99% homologous to the DNA sequence of one
or more
nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21- SEQ ID NO: 35, and SEQ ID
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NO: 10) or a functional fragment thereof, or a nucleic acid sequence that, but
for the
redundancy of the genetic code, encodes the same polypeptide as one or more
nucleic acid
sequence(s) of Table 4 (SEQ ID NO: 21- SEQ ID NO: 35, and SEQ ID NO: 10) or a
functional fragment thereof.
[0281] In some embodiments, the genetically engineered bacteria comprise one
or
more nucleic acid sequence(s) of Table 5 (SEQ ID NO: 36- SEQ ID NO: 39) or a
functional fragment thereof. In some embodiments, the genetically engineered
bacteria
comprise a nucleic acid sequence that, but for the redundancy of the genetic
code, encodes
the same polypeptide as one or more nucleic acid s sequence(s) of Table 5 (SEQ
ID NO:
36- SEQ ID NO: 39) or a functional fragment thereof. In some embodiments,
genetically
engineered bacteria comprise a nucleic acid sequence that is at least about
80%, at least
about 85%, at least about 90%, at least about 95%, or at least about 99%
homologous to
the DNA sequence of one or more nucleic acid sequence(s) of Table 5 (SEQ ID
NO: 36-
SEQ ID NO: 39) or a functional fragment thereof, or a nucleic acid sequence
that, but for
the redundancy of the genetic code, encodes the same polypeptide as one or
more nucleic
acid sequence(s) of Table 5 (SEQ ID NO: 36- SEQ ID NO: 39) or a functional
fragment
thereof.
[0282] In some embodiments, the genetically engineered bacteria comprise one
or
more nucleic acid sequence(s) of Table 6 (SEQ ID NO: 40- SEQ ID NO: 45) or a
functional fragment thereof. In some embodiments, the genetically engineered
bacteria
comprise a nucleic acid sequence that, but for the redundancy of the genetic
code, encodes
the same polypeptide as one or more nucleic acid s sequence(s) of Table 6 (SEQ
ID NO:
40- SEQ ID NO: 45) or a functional fragment thereof. In some embodiments,
genetically
engineered bacteria comprise a nucleic acid sequence that is at least about
80%, at least
about 85%, at least about 90%, at least about 95%, or at least about 99%
homologous to
the DNA sequence of one or more nucleic acid sequence(s) of Table 6 (SEQ ID
NO: 40-
SEQ ID NO: 45) or a functional fragment thereof, or a nucleic acid sequence
that, but for
the redundancy of the genetic code, encodes the same polypeptide as one or
more nucleic
acid sequence(s) of Table 6 (SEQ ID NO: 40- SEQ ID NO: 45) or a functional
fragment
thereof.
[0283] Table 7 lists exemplary polypeptide sequences, which may be encoded by
the propionate production gene(s) or cattette(s) of the genetically engineered
bacteria.
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Table 7. Polypeptide Sequences for Propionate Synthesis
Pct MRKVPIITADEAAKLIKDGDTvriTSGINGN AIPEALDR AVEKRFLETGE
SEQ ID PKNITYVYCGS QGNRDGRGAEHFAHEGLLKRYIAGHWATVPALG KM
NO: 46 AMENKMEAYN VSQG ALCHLERDIASHKPGVFTKV GIGTHDPRNGGG
KVNDITKEDIVEINEIKGQEYLFYPAFPIEVAIJRGTYADESGNITFEKE
VAPLEGTSVCQAVKNSGGIVVVQVERVVKAGTLDPRFIVKVPGIYVDY
VVADPEDHQQSLDCEYDPALSGEHRRPEVNIGEPLPLS NKKVIGRRG A
IELEKDVAVNLGVGAPEYVASVADEEGTVDFMTLTAESGAIGGVPAGG
VRFGASYNADALIDQGY-QFDY-YDGGGLDLCYLGLAECDEKGNINVSR
FGPRIAGCGGFINITQNTPKVEFCGTFT AGGLKVKIEDG KVIIVQEGKQK
KFLKAVEQITFNGDVALANKQQVTYITERCVFLLKEDGLHLSEIAPGID
LQTQILDVMDFAPIIDRDANGQIKLMDAALFAEGLMGLKEMKS*
lcdA MSLTQGMKAKQLLAYFQG KADQD AREAKARGELVCWS AS V APPEFC
SEQ ID VI MGIAMIYPEffiAAGIG ARKGAMD NILE V ADRKGYN VDCCS YGRVN-
NO: 47 MGYMECLKEAAITGVKPEVINNSPAADVPLPDLVITCNNICNTLLKWY
ENLAAELDIPCIVIDVPFNITIMPIPEYAKAYIADQFRNAISQLEVICGRPF
DWKKFKEV KDQTQRS V YIIWNRIAENTAKYKPSPLNGFDLFNYMALIV
ACRSLDYAEITFKAFADELEENLKAGIYAFKGAEKTRFQWEGIAVWPH
LGHTFKSMKNILNSIMTGTAYPALWDLHYDANDESMHS MAE AYTRIYI
NTCLQNKVEVIJLGIMEKGQVDGTVYIILNRSCKLMS FLNVETAEIIKEK
NGLPYVSIDGDQTDPRVFSPAQFDTRVQALVEMMEANMAAAE*
lcdB MS RVEAILS QLKDVAANPKKAMDDY KAETGKGAVGIMPIYS PEEMVH
SEQ ID AAGYLPMGIWG AQGKTISKARTYLPAFACSVMQQVMELQCEGAYDD
NO: 48 LS AVMS VPCDTLI(CLSQKWKGTSPVIVFTI-IPQNRGLE AANQFLVTEYE
LV :KAQLES VLGV KIS NAALENSIAIYNENRAVMREFV KV AADYPQVID
AVSRHAVF KARQFMLKE KHT Al VKELIAE IKA1PVQPWDGKKVVVR3
ILLEPNELLDIFNEFKIAIVDDDLAQES RQIRVDVLDGEGGPLYRMAKA
-WQQMYGCS LA'FDTKKGRGRMLINKFIQTGADAIVV AMMKFCDPEEW-
DYPVMYREFEEKGVKSLMIEVDQEVSSFEQIKTRLQSFVEML*
lcdC M Y IIGIDVGS ASS K AVII KDGKINV AAEVVQVG`FGSSGPQRALDKAFEV
SEQ ID S GLKKEDIS YTVATGYGRFNFS DAD KQIS EISCHAKGIYFLVPTARTIIDIG
NO: 49 GQDAKAIRLDD KGGIKQFFMND KC AAGTGRFLE V MARVLEFFLDEMAE
LDEQATDTAPISSTCTVFAESEVISQLSNGVSRNMIKGVHLSVASRACGL
AYRGGLEKDVVMTGGVAKNAGVVRAVAGVLKTDVIVAPNPQTTGALG
AMA AY EAAQ K KX
etfA MAFNS ADINS FRDIW VEVEQREGKLIN'fDFELISEGRKLADERGS KING
SEQ ID ILLGHEVEEIAKELGGYGADKVIVCDIVELKFYTTDAY AKVLCDVVME
NO: 50 EKPEVILIGATNIGRDLGPRCAARLHTGLTADCTHLDIDMN KYVDFLST
SSTLDISSMTFPMEDTNLKMTRPAFGGEILMATIICPRERPCMSTVRPGV
MKKAEFSQEMAQACQVVTRHVNLSDEDLKTKVINIVKETKKIVDLIGA
:EfIVS VGRGISKDVQGGIALAEKLADAFGNGVVGGSRAVIDSGWLPAD
FIQVGQTGKTVI-IPKVYNALGISGAIQI-IKAGMQDSELIIAVNKDETAPIF
DCADYGITGDLFKIVPMMIDAIKEGKNA*
acrB MRIYVCVKQVPDTSGKVAVNPDGTLNRASMAAHNPDDMSAIEQALKL
SEQ ID KDETGCQVTALTMGPPPAEGMLREHAMGADDGVLIS AREFGGSDTFA
NO: 51 TSQRSAAILIKLGLS NEDMIFCGRQAIDGDTAQVGPQIAEKLSIPQVTYG
AGIKKSGDINLVKRMLEDGYMMIEVETPCLITCIQDKAVKPRYMTLN
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GIMECYS KPLLVLDYEALKDEPLIELDTIGLKGS PTNIFKS FTPPQKGVG
VMLQGTDKEKVEDLVDKLMQKHVI*
acrC MFLLKIKKERMKRMDFSLTREQEMLICKLARQFAEIELEPV AEEIDREH
SEQ ID VFRAENFKKNIAEIGLTGICTIPKEFGGSGGGTLEKYIAVSEFG KKCMAS A
NO: 52 S ILSIHLIAPQAIY KYGT KEQKETYLPRLTKGGELGAFALTEPNAGS DAG
AV KTTAILDSQTNEYVIAGT KCHISGGGRAGVINIFALTEPICKGLKGM
SAIIVEKGTPGFSIGKVESKMGIAGSETAELIFEDCRVPAANLLG KEGKG
KIAMEALDGARIGVG AQAIG IAEGAID LS V:KYVHERIQFGKPI AN LQGI
QWYIADM ATKTAA ARALVE FAAYLEDAGKPFTKIES AMCKLNAS EN A
RFVTNLALQIHGGYGYMKDYPLERMYRDAKITEIYEGTSEIHKVVIAR
:EVMKR*
thrAfbr MRVLKFGGTSVANAERFLRV AD1LESNARQGQVATVIS APAKITNHL V
SEQ ID AMIEKTISGQDALPNISDAERIFAELLTGLAAAQPGFPLAQLKTFVDQEF
NO: 53 AQIKHVLHGISLLGQCPDS INAALICRGEKMS IAIMAGVLEARGHNVTV
IDPVEKLLAVGHYLES TVDIAES TRRIA AS RIPADHMVLMAGFTAGNE K
GELVVLGRNGSDYSAAVLAACLRADCCEIWTDVDGVYTCDPRQVPD
ARLLKS MS YQEAMELS YFG AKVLHPRTITPIAQFQIPCLIKNTGNPQAP
GTLIGASRDEDELPV S NLNNM AMES VSGPGMKGM VG MAARVFA
AMSRARIS VLITQS SS EYSISFC VPQS DCVRAER AMQEEFYLEI ,KEGLL
:EPLAVIERLAIISVVGDGMRTLRGIS AKEFAALARANINIV AIAQRS S ER
SIS VVVNNDDATTGVRYTHQMLFNTDQVIEVINIGVGGVGGALLEQL
KRQQS WLKN KHIDLRVCGVANS KALLTNVHGLNLENWQEELAQAKE
PFNLGRLIRLVKEYHLLNPVIVDCTSSQAVADQYADFLREGFHVVTPN
KKANTSSMDYYHQLRYAAEKSRRKFLYDTNVGAGLPVIENLQNLLNA
GDELMKFSGILSGSLSYIFG KLDEGMS FSEATTLAREMGYTEPDPRDDL
SGMD ARKLLIL ARETGRE LELADIEIETVLPMEFN AEGDV AAFM ANLS
QLDDLFAARV AKARDEGKVLRY VGNIDEDGVCRVKIAEVDGNDPLFK
VKNGENALAFYSHYYQPLPLVLRGYGAGND VTAAGVFADLLRTLSW
KLGV*
thrB MVKVYAPASS ANMS VGFDVLGAAVTPVDGALLGDV VrtVEAAErfFSL
SEQ ID NNLGRFADKLPSEPRENIVYQCWERFCQELGKQIPVAMTLEKNMPIGS
NO: 54 GLGSSACSVVAALMAMNEHCGKPLNDTRLLALMGELEGRISGSIHYD
NVAPCF LGGMQLMIEENDIISQQVPGFDE WI WVL AYPGIKVSTAE AR'
ILPAQYRRQDCIAHGRFILAGFIHACYS RQPELAAKLMKDVIAEPYRER
LLPGFRQARQ AVAEIGAVAS GIS GS GPTLFALCD KPETAQRVADWLGK
NYLQNQEGFVHICRLDTAGARVLEN*
thrC IVIKLYMKININEQVSFAQAVIQGLGKNQGLFFPLIDLPEFSUFEIDEML
SEQ ID KLDFVTRSAKILSAFIGDEINEILEERVRAAFAFPAPVANVESDVGCLE
NO: 55 LFHGPTLAFKDFGGRFMAQMLTHIAGDKPVT1LTATSGDTGAAVAHAF
YGLPNVKVVILYPRGKISPLQEKLFCTLGGNIET VAIDGDFDACQALVK
QAFDDEELKVALGLNS ANS INIS RLLAQICYYFEAVAQLPQETRNQLVV
S VPSGNEUDLTAGLLAKSLGLPV KRFIAATNVNDTVPRFLHDGQWSPK
ATQATLSNAMDVSQPNNWPRVEELFRRKIWQLKELGYAAVDDETTQ
QTMRELKELGYTSEPHAAVAYRALRDQLNPGEYGLFLGTAHPAKFKE
S'VEAILGETLDLPKELAERADLPLLSEINLPADFAALR KLMNINHQ*
i/vAfbr MSETYVSEKSTGVMASGAELIRA ADIQTAQARISS VIAPTPLQYCPRLSE
SEQ ID ETGAEIYLKREDLQDVRSYKIRGALNSGAQLTQEQRDAGIVAAS AGNH
NO: 56 AQGVAYVCKSLGVQGRIYVPVQTPKQKRDRIMVHGGEFVSLVVIGNN
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EDE AS AAAHEDAERTGATLIEPFDARNTVIGQGTVAAEILS QLTS MG KS
ADHVMVPVGGGGLLAGVVS YMADMAPRTAIVGIEPAGAASMQAALH
NGGPITLETVDPFVDGAAVKRVGDLNYTIVEKNQGRVIIMMSATEGAV
CTEMLDLYQNEGHAEPAGALSIAGLKEMSFAPGSAVVCIISGGNNDVL
RYAEIAIE:RSLVFIRGL KHYfINNFPQKPGQL RH FLEDILGPDDD1TLFEY
LKRNNRETGTALV GUMS E ASGLDS LLERMEESAIDS RRLEPGT PEYEY
LT*
ace MSERFPNDVDPIETRDWLQAIES VIREEGVERAQYLIDQLLAEARKGGV
SEQ ID NVAAGTGIS NY IINTIPVEEQPE YPGNLELERRIR S AIRWN A1MTVLR AS K
NO: 57 KDLELGGHMASFQSS ATIYDVCFNHFFRARNEQDGGDLVYFQGHISPG
VYARAFLEGRLTQEQLDNFRQEVHGNGLSS YPHPKLMPEFWQFPTVS
MGLGPIGATYQ AKFLKYLEHRGL KDTS KQT V YAFLGDGEMDEPES KG
AITIATREKLDNLVEVINCNLQRLDGPVTGNGKIINELEGIFEGAGWNVI
KVM WGSRVYIDEIL RKDT SGKLIQLMNErtV:DGDYQTF KS KDGAYVREH
FFG KYPET AALVADWT DEQIWALNRGGHDPKKIY AAFKKAQETKGK
AT VfLAHTIKGYGMGDAAEGKNIAHQVKKMNMDGVRHIRDRFNVPVS
DADIEKLPYITFPEGSEEHTYLHAQRQ KLUGYLPSRQPNFTEKLELPS LQ
DFGALLEEQS KEISTTIAFVRALNVMLKN KS IKDRLVPHADE ARTFGME
GLFRQIGIYSPNGQQYTPQDREQVAYIKEDEKGQILQEGINELGAGCS
W LA AATS YS'f NNLPMIPFYIYYSMFGFQRIGDLCAVAAGDQQARGFLIG
GTSGRTTLNGEGLQHEDGHSHIQSLTIPNCISYDPAYAYEVAVIMHDGL
:ERMYGEKQENV YY \I TT LNENYHMPAMPEGAEEGIRKGIYKLETIEGS
KG KV QLLGSGS ILREVREAAEILAKDYGVGSDVYSVTSFTELARDGQD
CERWNMLHPLETPRVPYIAQVMNDAPAVASTDYMKLFAEQVRTYVP
ADDYRVLGTDGFGRSDS RE NLREIHFEVD AS YVVV AALGELAKRGEID
KKVVADAIAKFNIDADKVNPRI ,A*
aceF MAIETKVPDTGADEVEITEILV KV GD KVEAEQS UTVEGDKASME VPSPQ
SEQ ID AGIVKEIKVSVGDKTQFGALIMIEDSADGAADAAPAQAEEKKEAAPAA
NO: 58 APAAAAAKIWNVPDIGSDEVENTEILN KWH) KVEAEQS LITVEGDKAS
MEVPAPFAGTVKEIKVNVGDKVSTGSLIMVFEVAGEAGAAAPAAKQE
AAPAAAPAPAAGVKEVNVPDIGGDEVEVTEVMVKVGD KY ANEQS LIT
VEGDKASMEVPAPFAGVVKELKVNVGD KY KTGRAMIFE VEGAAPAA
APAKQE AAAPAPAAKAEAPAAAPAAKAEG KS EFAENDAYVHATPLIR
RLARE FGVN LAKVKG`FGR KGRILREDV QAY VKE Al KR/ME AAPAATGG
GIPGMLPWPKVDFSKFGEIEEVELGRIQKISGANLSRNWVMIPHVTHFD
KTDITELEAFRKQQNEEAAKRKLDV KITPVVFIMKAVAAALEQMPRFN
SS LS EDGQRLTLICKYINIGVAVDTPNGLVVPVFKDVN KKGIIELSRELM
TISKKARDGKLTAGEMQGGCFTISSIGGLGTTHFAPIVNAPEVAILGVS K
SAMEPVWNGKEEVPRLMLPISLSFDHRVIDGADGARFITIINNTLSDIRR
LVM*
Lpd MSTE1 KTQVVVLGAGPAGYS AA FRCADLGL ETVIVERYNTLGGVUN
SEQ ID VGCIPS KALLHVAKVIEEAKALAEHGIVFGEPKTDID KIRTWKE KVINQ
NO: 59 LTGGLAGMAKGRKVKVVNGLGKFTGANTLEVEGENGKTVINFDNAII
NAGS RPIQLPFIPHEDPRIWDSTDALELKEVPERLLV MGGGIIGLEMGTV
YHALGSQIDVVEMFDQVIPAADKDIVKVFTKRISKKFNLMLETKVTAV
AKEDGIYVTMEGKKAPAEPQRYD AV LV Al GRVPNGKNLD AGKAGV-
EVDDRGFIRVDKQLRTNVPHIFAIGDIVGQRMLAHKGVHEGHVAAEVI
AG KKELYFDP KV1PS IAYT KPEVAWVGLTEKEA KE KGIS-YETATFPWAA
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SGRAIASDCADGMTKLIFDKESHRVIGGAIVGTNGGELLGEIGLAIENIG
CDAEDIALTIHAHPTLHESVGLAAEVFEGSTTDLPNPKAKKK*
tesB MSQALKNLLTLLNLEKIEEGLFRGQSEDLGLRQVFGGQVVGQALYAA
SEQ ID KETVPEERLVHSFHS YFLRPGDSKKPHYDVETLRDGNSFSARRVAAIQ
NO: 20 NGKPIFYMTASFQAPEAGFEHQKTMPSAPAPDGLPSETQIAQSLAHLLP
PVL KD KfICDRPLEVRPVEFFINPLKGHVAEPHRQVWIR ANGSVPDDLR
VHQYLLGY ASDLNFLPVALQPHGIGFLEPGI QIATIDEISMWHIRPFNLN
EWLLYSVES'I'SASSARGFVRGEFY-TQDGVLVAS'f VQEGVMRNFIN*
acuI MRAVLIEKSDDTQS VS VTELAEDQLPEGDVINDVAYSTLNYKDALAIT
SEQ ID GKAPVV RRFPM \MGM FTGTVAQSS HADFKPGDRV1LNGWG VGE KIM
NO: 60 GGLAERARVRGDWLVPLPAPLDLRQAANIIGTAGYTAMLCVLALERH
GVVPGNGEIVVSGAAGGVGSVATTLLAAKGYEVAAVTGRASEAEYLR
GLGA AS-VIDRNEUFGKVRPLGQIER`vVAGGIDvAcisTryLANIALS
RGVVAACGLAAGMDLPASVAPFILRGMTLAGVDSVMCPKTDRLAAW
ARLASDLDPAKLEEMYTELPFSEVIETAPKFLDGTVRGRIVIPVTP*
Sbm MSNVQEWQQLANKELSRREKTVDSLVHQTAEGIAIKPLYTEADLDNL
SEQ ID EVTG`FLPGLPPYNRGPRATMYTAQPWFIRQYAGFSTAKESNAFYRRNL
NO: 61 AAGQICGLSVAFDLATHRGYDSDNPRVAGDVGKAGVAIDTVEDMKVL
FDQIPLD KMSVSMTMNGAV LPVLAFYIVAAEEQGVIPDKLTGT IQNDI
LKEYLCRNTYIYPPKRSIVIRIIADIIAWCSGNMPRFNTISISGYHMGEAGA
NCVQQVAFTLADGIEYIKAAISAGLKIDDFAPRLSFFFGIGMDLFMNVA
MLRAARYLWSEAVSGFGAQDPKSLALRTHccgsGwsufEQDPYNNVI
RTTIEALAATLGGTQSUITNAFDEALGLPTDFSARI ARNTQIIIQEESELC
RTVDPLAGSYNIESUTDOVKQARAIIQQ1DEAGGMAKAJEAGLPKRMI
LEASAREQS L1:DQG KRVI VG VN KY KLDHE DET Man:DNA/NI VRNEQIA
SLERIRATRDDAAVTAALNALTHAAQHNENLLAAAVNAARVRATLGE
IS DA-LEV-A FDRY-L VPS QC V TG VI AQS Y FIQS E KS ASEFDAIVAQTEQFLA
DNGRRPRILIAKNIGQDGHDRGAKVIASAYSDLGFDVDLSPNIFSTPEEIA
RLAVENDVHVVG AS S LAAGHKTLIPELVEALKKWGREDICVVAGGVIP
PQDYAFTAXRG V A AIYGPGTPMLDS VRDVLNLISQIIHD*
ygfD MINEATLAES IRRLRQGERATLAQ AMTLVES RHPRHQALSTQLLD AIM
SEQ ID PYCGNTLRLGVTGTPGAGKSTFLEAFGMLIAREGLKVAVIAVDPSSPVT
NO: 62 GGS ILGD KTRMNDLARAEAAFIRPVPS S GHLGGAS QRARELMLLCEAA
GYDV VIVETVGVGQSETEV ARM VDCFIS LQIAGGGDDLQGIKKGLME
VADLIVINKDDGDNHTNVAIARHMYES AI ,HILRRKY DEWQPRVLTCS
ALEKRGIDEIW HAIIDFKTALTASGRLQQVRQQQS VEWLRKQTEEEVL
NHLTANEDFDRYYRQTLLAVKNNTLS PRTGLRQLSEFIQTQYFD*
ygfG MS YQYVNVVIINKV AVM EN YGRKLN ALS KVFIDDL MQ ALS DLNRPEI
SEQ ID RCHLRAPSGS KVFSAGHDIHELPSGGRDPLS YDDPLRQITRMIQKFPKPI
NO: 63 IS MVEGSVWGGAFEMIMSS DLIIAASTSTFSMTPVNLGVPYNLVGIHNL
RDAG Fill V KIELIFT AS PITAQRAL AV GILINI-IV VE VEELEDET LQM AH1-I
ISEIKAPLAIAVIKEELRVLGEAHTMNSDEFERIQGNIRRAVYDSEDYQEG
MNAFLEKRKPNFVGH*
ygfH METQWTRMTANEAAE11QHNDMVAFSGFTPAGSPKALVF AI ARRANEQ
SEQ ID LIE AKKPYQIRLUM AS ISAAADD VLSDADAVSWRAPYQTS SGL RKKIN
NO: 64 QGAVS FVDLHLSEVAQMVNYGFFGDIDVAVIEAS ALAPDGRVWLTS GI
GNAPTWLLRAKKVIIELNHYHDPRVAELADIVIPGAPPRRNSVSIFHAM
DRVGTRYVQIIRKKIVAVVETNLPDAGNMLDKQNPMCQQIADNVVTF
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LLQEMAHGRIPPEFLPLQSGVGNINNANTMARLGENPVIPPFMMYSEVL
QES VNI HELLEMKIS GAS AS S LTI S ADSLR DN YF AS RWLMEIS
NNPEIIRRI ,GVIALNVGI EFDIY Gil ANSTIIVAGVDLMNGIGGSGDPERN
AYLSIFMAPSIAKEGKIST VVPMCSFIVDHSEFIS VKVIITEQGIADLRGLS
PI ,QRART I DNC AI IPMY RD Y I A IRY 114,N APCK3 Ell III I DI SIR I DLL !RNLI
ATGSMI G*
mutA MS S TDQGTNPADTDDLTPTTLS LAGDFP KAT EEQWEREVEKVFNRGRPP
SEQ ID EKQLT FAECLKRLTVHTVDGIDIVPMYRP KD AP KKLGYPGVTPFTRGTT
NO: 65 VRNGDMDAWDVRALHEDPDEKFTRKAILEDLERGVTSLLLRVDPDAIA
PEHLDEVLSDVLLEMTKVEVFSRYDQGAAAEALMGVYERSDKPAKDLA
LNLGLDPIGFAALQGTEPDLTVLGDWVRRLAKFS PDS RAVTIDANVYHN
AGAGDVAELAWALATGAEYVRALVEQGFNATEAFDTINFRVTATHDQF
LTIARLRALREAWARIGEVFGVDEDKRGARQNAITSWRELTREDPYVNI
LRGSIATFS AS VGGAESITTLPFTQALGLPEDDFPLRIARNTGIVLAEEVNI
GRVNDPAGGSYYVESLTRTLADAAWKEFQEVEKLGGMS KAVMTEHVT
KVLDACNAERAKRLANRKQPITAVSEFPMIGARSIETKPFPTAPARKGLA
WHRDSEVFEQLMDRS TS VS ERPKVFLACLGTRRDFGGREGFS SPVWHIA
GIDTPQVEGGTTAEIVEAFKKS GAQVADLCS SAKIYAQQGLEVAKALKA
AGAKALYLS GAFKEFGDDAAEAEKLIDGRLYMGMDVVDTLS STLDILG
VAK
mutB VS TLPRFDS VDLGNAPVPADAAQRFEELAAKAGTEEAWETAEQIPVGTL
SEQ ID FNEDVYKDMDWLDTYAGIPPFVHGPYATMYAFRPWTIRQYAGFS TAKE
NO: 66 SNAFYRRNLAAGQKGLSVAFDLPTHRGYDS DNPRVAGDVGMAGVAIDS
IYDMRELFAGIPLDQMS VS MTMNGAVLPILALYVVTAEEQGVKPEQLA
GTIQNDILKEFMVRNTYIYPPQPS MRIISEIFAYTS ANMP KWNS IS IS GYH
MQEAGATADIEMAYTLADGVDYIRAGES VGLNVDQFAPRLSFFWGIGM
NFFMEVAKLRAARMLWAKLVHQFGPKNPKS MS LRTHS QTS GWS LT AQ
DVYNNVVRTCIEAMAAT QGHT QS LHTNS LDEAIALPTDFS ARIARNTQL
FLQQES GTTRVIDPWS GS AYVEELTWDLARKAWGHIQEVEKVGGMAK
AIEKGIPKMRIEEAAARTQARIDS GRQPLIGVNKYRLEHEPPLDVLKVDN
S TVLAEQKAKLVKLRAERDPEKVKAALDKITWAAANPDDKDPDRNLLK
LCID AGRAMATVGEMS D ALE KVFGRYT AQIRTIS GVYS KEVKNTPEVEE
ARELVEEFEQAEGRRPRILLAKMGQD GHDRGQKVIATAYADLGFDVDV
GPLFQTPEETARQAVEADVHVVGVS S LAGGHLTLVPALRKELDKLGRP
DILITVGGVIPEQDFDELRKDGAVEIYTPGTVIPESAISLVKKLRAS LD A
GI:180421 MSNEDLFICIDHVAYACPDADEAS KYYQETFGWHELHREENPEQGVVEI
34 MMAPAAKLTEHMTQVQVMAPLNDESTVAKWLAKHNGRAGLHHMAW
SEQ ID RVDDIDAVS ATLRERGVQLLYDEPKLGTGGNRINFMHPKS GKGVLIELT
NO: 67 QYPKN
mmdA MAENNNLKLASTMEGRVEQLAEQRQVIEAGGGERRVEKQHS QGKQTA
SEQ ID RERLNNLLDPHS FDEVGAFRKHRTTLFGMDKAVVPADGVVTGRGTILG
NO: 68 RPVHAAS QDFTVMGGS AGET QS TKVVETMEQALLTGTPFLFFYDS GGA
RIQEGIDS LS GYGKMFFANVKLS GVVPQIAIIAGPCAGGASYSPALTDFII
MTKKAHMFITGPQVIKS VT GEDVTADELGGAE AHMAIS GNIHFVAEDD
DAAELIAKKLLS FLPQNNTEEASFVNPNNDVSPNTELRDIVPIDGKKGYD
VRDVIAKIVDWGDYLEVKAGYATNLVTAFARVNGRS VGIVANQPS VMS
GCLDINASDKAAEFVNFCDS FNIPLVQLVDVPGFLPGVQQEYGGIIRHGA
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KMLYAYSEATVPKITVVLRKAYGGSYLAMCNRDLGADAVYAWPS AEI
AVMGAEGAANVIFRKEIKAADDPDAMRAEKIEEYQNAFNTPYVAAARG
QVDDVIDPADTRRKIASALEMYATKRQTRPAKKHGNFPC
PFREUD MSPREIEVSEPREVGITELVLRDAHQSLMATRMAMEDMVGACADIDAA
18870 GYWSVECWGGATYDSCIRFLNEDPWERLRTFRKLMPNSRLQMLLRGQN
SEQ ID LLGYRHYNDEVVDRFVDKSAENGMDVFRVFDAMNDPRNMAHAMAAV
NO: 69 KKAGKHAQGTICYTISPVHTVEGYVKLAGQLLDMGADSIALKDMAALL
KPQPAYDIIKAIKDTYGQKTQINLHCHSTTGVTEVSLMKAIEAGVDVVD
TAISSMSLGPGHNPTESVAEMLEGTGYTTNLDYDRLHKIRDHFKAIRPKY
KKFESKTLVDTSIFKSQIPGGMLSNMESQLRAQGAEDKMDEVMAEVPR
VRKAAGFPPLVTPSSQIVGTQAVFNVMMGEYKRMTGEFADIMLGYYGA
SPADRDPKVVKLAEEQSGKKPITQRPADLLPPEWEEQSKEAAALKGFNG
TDEDVLTYALFPQVAPVFFEHRAEGPHSVALTDAQLKAEAEGDEKSLAV
AGPVTYNVNVGGTVREVTVQQA
Bccp MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPRAAGGA
SEQ ID GAGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKMETEIN
NO: 70 APTDGKVEKVLVKERDAVQGGQGLIKIG
[0284] In some embodiments, the genetically engineered bacteria encode one or
more polypeptide sequences of Table 7 (SEQ ID NO: 46-SEQ ID NO: 70, and SEQ ID
NO: 20) or a functional fragment or variant thereof. In some embodiments,
genetically
engineered bacteria comprise a polypeptide sequence that is at least about
80%, at least
about 85%, at least about 90%, at least about 95%, or at least about 99%
homologous to
the polypeptide sequence of one or more polypeptide sequence of Table 7 (SEQ
ID NO:
46-SEQ ID NO: 70, and SEQ ID NO: 20) or a functional fragment thereof.
[0285] In one embodiment, the bacterial cell comprises a non-native or
heterologous propionate gene cassette. In some embodiments, the disclosure
provides a
bacterial cell that comprises a non-native or heterologous propionate gene
cassette
operably linked to a first promoter. In one embodiment, the first promoter is
an inducible
promoter. In one embodiment, the bacterial cell comprises a propionate gene
cassette
from a different organism, e.g., a different species of bacteria. In another
embodiment, the
bacterial cell comprises more than one copy of a native gene encoding a
propionate gene
cassette. In yet another embodiment, the bacterial cell comprises at least one
native gene
encoding a propionate gene cassette, as well as at least one copy of a
propionate gene
cassette from a different organism, e.g., a different species of bacteria. In
one
embodiment, the bacterial cell comprises at least one, two, three, four, five,
or six copies
of a gene encoding a propionate gene cassette. In one embodiment, the
bacterial cell
comprises multiple copies of a gene or genes encoding a propionate gene
cassette.
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[0286] Multiple distinct propionate gene cassettes are known in the art. In
some
embodiments, a propionate gene cassette is encoded by a gene cassette derived
from a
bacterial species. In some embodiments, a propionate gene cassette is encoded
by a gene
cassette derived from a non-bacterial species. In some embodiments, a
propionate gene
cassette is encoded by a gene derived from a eukaryotic species, e.g., a
fungi. In one
embodiment, the gene encoding the propionate gene cassette is derived from an
organism
of the genus or species that includes, but is not limited to, Clostridium
propionicum,
Megasphaera elsdenii, or Prevotella ruminicola.
[0287] In one embodiment, the propionate gene cassette has been codon-
optimized
for use in the engineered bacterial cell. In one embodiment, the propionate
gene cassette
has been codon-optimized for use in Escherichia coli. In another embodiment,
the
propionate gene cassette has been codon-optimized for use in Lactococcus. When
the
propionate gene cassette is expressed in the engineered bacterial cells, the
bacterial cells
produce more propionate than unmodified bacteria of the same bacterial subtype
under the
same conditions (e.g., culture or environmental conditions). Thus, the
genetically
engineered bacteria comprising a heterologous propionate gene cassette may be
used to
generate propionate to treat autoimmune disease, such as IBD.
[0288] The present disclosure further comprises genes encoding functional
fragments of propionate biosynthesis enzymes or functional variants of a
propionate
biosynthesis enzyme. As used herein, the term "functional fragment thereof' or
"functional variant thereof' relates to an element having qualitative
biological activity in
common with the wild-type enzyme from which the fragment or variant was
derived. For
example, a functional fragment or a functional variant of a mutated propionate
biosynthesis enzyme is one which retains essentially the same ability to
synthesize
propionate as the propionate biosynthesis enzyme from which the functional
fragment or
functional variant was derived. For example a polypeptide having propionate
biosynthesis
enzyme activity may be truncated at the N-terminus or C-terminus, and the
retention of
propionate biosynthesis enzyme activity assessed using assays known to those
of skill in
the art, including the exemplary assays provided herein. In one embodiment,
the
engineered bacterial cell comprises a heterologous gene encoding a propionate
biosynthesis enzyme functional variant. In another embodiment, the engineered
bacterial
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cell comprises a heterologous gene encoding a propionate biosynthesis enzyme
functional
fragment.
[0289] As used herein, the term "percent (%) sequence identity" or "percent
(%)
identity," also including "homology," is defined as the percentage of amino
acid residues
or nucleotides in a candidate sequence that are identical with the amino acid
residues or
nucleotides in the reference sequences after aligning the sequences and
introducing gaps,
if necessary, to achieve the maximum percent sequence identity, and not
considering any
conservative substitutions as part of the sequence identity. Optimal alignment
of the
sequences for comparison may be produced, besides manually, by means of the
local
homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2,482, by means
of
the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48,
443, by
means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl.
Acad.
Sci. USA 85, 2444, or by means of computer programs which use these algorithms
(GAP,
BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).
[0290] The present disclosure encompasses propionate biosynthesis enzymes
comprising amino acids in its sequence that are substantially the same as an
amino acid
sequence described herein. Amino acid sequences that are substantially the
same as the
sequences described herein include sequences comprising conservative amino
acid
substitutions, as well as amino acid deletions and/or insertions. A
conservative amino acid
substitution refers to the replacement of a first amino acid by a second amino
acid that has
chemical and/or physical properties (e.g., charge, structure, polarity,
hydrophobicity/hydrophilicity) that are similar to those of the first amino
acid.
Conservative substitutions include replacement of one amino acid by another
within the
following groups: lysine (K), arginine (R) and histidine (H); aspartate (D)
and glutamate
(E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y),
K, R, H, D and
E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P),
phenylalanine (F),
tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S
and T.
Similarly contemplated is replacing a basic amino acid with another basic
amino acid
(e.g., replacement among Lys, Arg, His), replacing an acidic amino acid with
another
acidic amino acid (e.g., replacement among Asp and Glu), replacing a neutral
amino acid
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with another neutral amino acid (e.g., replacement among Ala, Gly, Ser, Met,
Thr, Leu,
Ile, Asn, Gln, Phe, Cys, Pro, Trp, Tyr, Val).
[0291] In some embodiments, a propionate biosynthesis enzyme is mutagenized;
mutants exhibiting increased activity are selected; and the mutagenized gene
encoding the
propionate biosynthesis enzyme is isolated and inserted into the bacterial
cell of the
disclosure. The gene comprising the modifications described herein may be
present on a
plasmid or chromosome.
[0292] In one embodiment, the propionate biosynthesis gene cassette is from
Clostridium spp. In one embodiment, the Clostridium spp. is Clostridium
propionicum.
In another embodiment, the propionate biosynthesis gene cassette is from a
Megasphaera
spp. In one embodiment, the Megasphaera spp. is Megasphaera elsdenii. In
another
embodiment, the propionate biosynthesis gene cassette is from Prevotella spp.
In one
embodiment, the Prevotella spp. is Prevotella ruminicola. Other propionate
biosynthesis
gene cassettes are well-known to one of ordinary skill in the art.
[0293] In some embodiments, the genetically engineered bacteria comprise the
genes pct, lcd, and acr from Clostridium propionicum. In some embodiments, the
genetically engineered bacteria comprise acrylate pathway genes for propionate
biosynthesis, e.g., pct, lcdA, lcdB, lcdC, e0, acrB, and acrC. In alternate
embodiments,
the genetically engineered bacteria comprise pyruvate pathway genes for
propionate
biosynthesis, e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE, aceF, and 1pd, and
optionally further
comprise tesB. The genes may be codon-optimized, and translational and
transcriptional
elements may be added.
[0294] In one embodiment, the pct gene has at least about 80% identity with
SEQ
ID NO: 21. In another embodiment, the pct gene has at least about 85% identity
with
SEQ ID NO: 21. In one embodiment, the pct gene has at least about 90% identity
with
SEQ ID NO: 21. In one embodiment, the pct gene has at least about 95% identity
with
SEQ ID NO: 21. In another embodiment, the pct gene has at least about 96%,
97%, 98%,
or 99% identity with SEQ ID NO: 21. Accordingly, in one embodiment, the pct
gene has
at least about 80%, 821%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 921%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21. In
another
embodiment, the pct gene comprises the sequence of SEQ ID NO: 21. In yet
another
embodiment the pct gene consists of the sequence of SEQ ID NO: 21.
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[0295] In one embodiment, the lcdA gene has at least about 80% identity with
SEQ ID NO: 22. In another embodiment, the lcdA gene has at least about 85%
identity
with SEQ ID NO: 22. In one embodiment, the lcdA gene has at least about 90%
identity
with SEQ ID NO: 22. In one embodiment, the lcdA gene has at least about 95%
identity
with SEQ ID NO: 22. In another embodiment, the lcdA gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 22. Accordingly, in one embodiment,
the
lcdA gene has at least about 80%, 81%, 822%, 83%, 84%, 85%, 86%, 87%, 88%,
89%,
90%, 91%, 922%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
22. In another embodiment, the lcdA gene comprises the sequence of SEQ ID NO:
22. In
yet another embodiment the lcdA gene consists of the sequence of SEQ ID NO:
22.
[0296] In one embodiment, the lcdB gene has at least about 80% identity with
SEQ ID NO: 23. In another embodiment, the lcdB gene has at least about 85%
identity
with SEQ ID NO: 23. In one embodiment, the lcdB gene has at least about 90%
identity
with SEQ ID NO: 23. In one embodiment, the lcdB gene has at least about 95%
identity
with SEQ ID NO: 23. In another embodiment, the lcdB gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 23. Accordingly, in one embodiment,
the
lcdB gene has at least about 80%, 81%, 82%, 823%, 84%, 85%, 86%, 87%, 88%,
89%,
90%, 91%, 92%, 923%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
23. In another embodiment, the lcdB gene comprises the sequence of SEQ ID NO:
23. In
yet another embodiment the lcdB gene consists of the sequence of SEQ ID NO:
23.
[0297] In one embodiment, the lcdC gene has at least about 80% identity with
SEQ ID NO: 24. In another embodiment, the lcdC gene has at least about 85%
identity
with SEQ ID NO: 24. In one embodiment, the lcdC gene has at least about 90%
identity
with SEQ ID NO: 24. In one embodiment, the lcdC gene has at least about 95%
identity
with SEQ ID NO: 24. In another embodiment, the lcdC gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 24. Accordingly, in one embodiment,
the
lcdA gene has at least about 80%, 81%, 82%, 83%, 824%, 85%, 86%, 87%, 88%,
89%,
90%, 91%, 92%, 93%, 924%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
24. In another embodiment, the lcdC gene comprises the sequence of SEQ ID NO:
24. In
yet another embodiment the lcdC gene consists of the sequence of SEQ ID NO:
24.
[0298] In one embodiment, the e0 gene has at least about 80% identity with SEQ
ID NO: 25. In another embodiment, the etfA gene has at least about 825%
identity with
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SEQ ID NO: 25. In one embodiment, the e0 gene has at least about 90% identity
with
SEQ ID NO: 25. In one embodiment, the e0 gene has at least about 925% identity
with
SEQ ID NO: 25. In another embodiment, the e0 gene has at least about 96%, 97%,
98%, or 99% identity with SEQ ID NO: 25. Accordingly, in one embodiment, the
etfA
gene has at least about 80%, 81%, 82%, 83%, 84%, 825%, 86%, 87%, 88%, 89%,
90%,
91%, 92%, 93%, 94%, 925%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 25.
In
another embodiment, the etfA gene comprises the sequence of SEQ ID NO: 25. In
yet
another embodiment the e0 gene consists of the sequence of SEQ ID NO: 25.
[0299] In one embodiment, the acrB gene has at least about 80% identity with
SEQ ID NO: 26. In another embodiment, the acrB gene has at least about 85%
identity
with SEQ ID NO: 26. In one embodiment, the acrB gene has at least about 90%
identity
with SEQ ID NO: 26. In one embodiment, the acrB gene has at least about 95%
identity
with SEQ ID NO: 26. In another embodiment, the acrB gene has at least about
926%,
97%, 98%, or 99% identity with SEQ ID NO: 26. Accordingly, in one embodiment,
the
acrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 826%, 87%, 88%,
89%,
90%, 91%, 92%, 93%, 94%, 95%, 926%, 97%, 98%, or 99% identity with SEQ ID NO:
26. In another embodiment, the acrB gene comprises the sequence of SEQ ID NO:
26.
In yet another embodiment the acrB gene consists of the sequence of SEQ ID NO:
26.
[0300] In one embodiment, the acrC gene has at least about 80% identity with
SEQ ID NO: 27. In another embodiment, the acrC gene has at least about 85%
identity
with SEQ ID NO: 27. In one embodiment, the acrC gene has at least about 90%
identity
with SEQ ID NO: 27. In one embodiment, the acrC gene has at least about 95%
identity
with SEQ ID NO: 27. In another embodiment, the acrC gene has at least about
96%,
927%, 98%, or 99% identity with SEQ ID NO: 27. Accordingly, in one embodiment,
the
acrC gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 827%, 88%,
89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 927%, 98%, or 99% identity with SEQ ID NO:
27. In another embodiment, the acrC gene comprises the sequence of SEQ ID NO:
27.
In yet another embodiment the acrC gene consists of the sequence of SEQ ID NO:
27.
[0301] In one embodiment, the thrAfbr gene has at least about 280% identity
with
SEQ ID NO: 28. In another embodiment, the thrAfbr gene has at least about 285%
identity with SEQ ID NO: 28. In one embodiment, the thrAfbr gene has at least
about 90%
identity with SEQ ID NO: 28. In one embodiment, the thrAfbr gene has at least
about 95%
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identity with SEQ ID NO: 28. In another embodiment, the thrgbr gene has at
least about
96%, 97%, 928%, or 99% identity with SEQ ID NO: 28. Accordingly, in one
embodiment, the thrgbr gene has at least about 280%, 281%, 282%, 283%, 284%,
285%,
286%, 287%, 2828%, 289%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 928%, or 99%
identity with SEQ ID NO: 28. In another embodiment, the thrgbr gene comprises
the
sequence of SEQ ID NO: 28. In yet another embodiment the thrgbr gene consists
of the
sequence of SEQ ID NO: 28.
[0302] In one embodiment, the thrB gene has at least about 80% identity with
SEQ ID NO: 29. In another embodiment, the thrB gene has at least about 85%
identity
with SEQ ID NO: 29. In one embodiment, the thrB gene has at least about 290%
identity
with SEQ ID NO: 29. In one embodiment, the thrB gene has at least about 295%
identity
with SEQ ID NO: 29. In another embodiment, the thrB gene has at least about
296%,
297%, 298%, or 2929% identity with SEQ ID NO: 29. Accordingly, in one
embodiment,
the thrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
829%,
290%, 291%, 292%, 293%, 294%, 295%, 296%, 297%, 298%, or 2929% identity with
SEQ ID NO: 29. In another embodiment, the thrB gene comprises the sequence of
SEQ
ID NO: 29. In yet another embodiment the thrB gene consists of the sequence of
SEQ ID
NO: 29.
[0303] In one embodiment, the thrC gene has at least about 80% identity with
SEQ ID NO: 30. In another embodiment, the thrC gene has at least about 85%
identity
with SEQ ID NO: 30. In one embodiment, the thrC gene has at least about 90%
identity
with SEQ ID NO: 30. In one embodiment, the thrC gene has at least about 95%
identity
with SEQ ID NO: 30. In another embodiment, the thrC gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 30. Accordingly, in one embodiment,
the
thrC gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
30. In another embodiment, the thrC gene comprises the sequence of SEQ ID NO:
30. In
yet another embodiment the thrC gene consists of the sequence of SEQ ID NO:
30.
[0304] In one embodiment, the i/vAfbr gene has at least about 80% identity
with
SEQ ID NO: 31. In another embodiment, the i/vAfbr gene has at least about 85%
identity
with SEQ ID NO: 31. In one embodiment, the i/vAfbr gene has at least about 90%
identity
with SEQ ID NO: 31. In one embodiment, the i/vAfbr gene has at least about 95%
identity
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with SEQ ID NO: 31. In another embodiment, the i/vAfbr gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 31. Accordingly, in one embodiment,
the
i/vAfbr gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
31. In another embodiment, the i/vAfbr gene comprises the sequence of SEQ ID
NO: 31.
In yet another embodiment the i/vAfbr gene consists of the sequence of SEQ ID
NO: 31.
[0305] In one embodiment, the aceE gene has at least about 80% identity with
SEQ ID NO: 32. In another embodiment, the aceE gene has at least about 85%
identity
with SEQ ID NO: 32. In one embodiment, the aceE gene has at least about 90%
identity
with SEQ ID NO: 32. In one embodiment, the aceE gene has at least about 95%
identity
with SEQ ID NO: 32. In another embodiment, the aceE gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 32. Accordingly, in one embodiment,
the
aceE gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
32. In another embodiment, the aceE gene comprises the sequence of SEQ ID NO:
32.
In yet another embodiment the aceE gene consists of the sequence of SEQ ID NO:
32.
[0306] In one embodiment, the aceF gene has at least about 80% identity with
SEQ ID NO: 33. In another embodiment, the aceF gene has at least about 85%
identity
with SEQ ID NO: 33. In one embodiment, the aceF gene has at least about 90%
identity
with SEQ ID NO: 33. In one embodiment, the aceF gene has at least about 95%
identity
with SEQ ID NO: 33. In another embodiment, the aceF gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 33. Accordingly, in one embodiment,
the
aceF gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
33. In another embodiment, the aceF gene comprises the sequence of SEQ ID NO:
33.
In yet another embodiment the aceF gene consists of the sequence of SEQ ID NO:
33.
[0307] In one embodiment, the 1pd gene has at least about 80% identity with
SEQ
ID NO: 34. In another embodiment, the 1pd gene has at least about 85% identity
with
SEQ ID NO: 34. In one embodiment, the 1pd gene has at least about 90% identity
with
SEQ ID NO: 34. In one embodiment, the 1pd gene has at least about 95% identity
with
SEQ ID NO: 34. In another embodiment, the 1pd gene has at least about 96%,
97%, 98%,
or 99% identity with SEQ ID NO: 34. Accordingly, in one embodiment, the 1pd
gene has
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at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 34. In another
embodiment, the 1pd gene comprises the sequence of SEQ ID NO: 34. In yet
another
embodiment the 1pd gene consists of the sequence of SEQ ID NO: 34.
[0308] In one embodiment, the tesB gene has at least about 80% identity with
SEQ
ID NO: 10. In another embodiment, the tesB gene has at least about 85%
identity with
SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 90%
identity with
SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 95%
identity with
SEQ ID NO: 10. In another embodiment, the tesB gene has at least about 96%,
97%,
98%, or 99% identity with SEQ ID NO: 10. Accordingly, in one embodiment, the
tesB
gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. In
another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 10. In
yet
another embodiment the tesB gene consists of the sequence of SEQ ID NO: 10.
[0309] In one embodiment, the actd gene has at least about 80% identity with
SEQ ID NO: 35. In another embodiment, the actd gene has at least about 85%
identity
with SEQ ID NO: 35. In one embodiment, the actd gene has at least about 90%
identity
with SEQ ID NO: 35. In one embodiment, the actd gene has at least about 95%
identity
with SEQ ID NO: 35. In another embodiment, the actd gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 35. Accordingly, in one embodiment,
the
actd gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
35. In another embodiment, the actd gene comprises the sequence of SEQ ID NO:
35. In
yet another embodiment the actd gene consists of the sequence of SEQ ID NO:
35.
[0310] In one embodiment, the sbm gene has at least about 80% identity with
SEQ
ID NO: 36. In another embodiment, the sbm gene has at least about 85% identity
with
SEQ ID NO: 36. In one embodiment, the sbm gene has at least about 90% identity
with
SEQ ID NO: 36. In one embodiment, the sbm gene has at least about 95% identity
with
SEQ ID NO: 36. In another embodiment, the sbm gene has at least about 96%,
97%,
98%, or 99% identity with SEQ ID NO: 36Ø Accordingly, in one embodiment, the
sbm
gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 36. In
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another embodiment, the sbm gene comprises the sequence of SEQ ID NO: 36. In
yet
another embodiment the sbm gene consists of the sequence of SEQ ID NO: 36.
[0311] In one embodiment, the ygfD gene has at least about 80% identity with
SEQ ID NO: 37. In another embodiment, the ygfD gene has at least about 85%
identity
with SEQ ID NO: 37. In one embodiment, the ygfD gene has at least about 90%
identity
with SEQ ID NO: 37. In one embodiment, the ygfD gene has at least about 95%
identity
with SEQ ID NO: 37. In another embodiment, the ygfD gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 37. Accordingly, in one embodiment,
the
ygfD gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
37. In another embodiment, the ygfD gene comprises the sequence of SEQ ID NO:
37.
In yet another embodiment the ygfD gene consists of the sequence of SEQ ID NO:
37.
[0312] In one embodiment, the ygfG gene has at least about 80% identity with
SEQ ID NO: 38. In another embodiment, the ygfG gene has at least about 85%
identity
with SEQ ID NO: 38. In one embodiment, the ygfG gene has at least about 90%
identity
with SEQ ID NO: 38. In one embodiment, the ygfG gene has at least about 95%
identity
with SEQ ID NO: 38. In another embodiment, the ygfG gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 38.. Accordingly, in one embodiment,
the
ygfG gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
38. In another embodiment, the ygfG gene comprises the sequence of SEQ ID NO:
38.
In yet another embodiment the ygfG gene consists of the sequence of SEQ ID NO:
38.
[0313] In one embodiment, the ygfH gene has at least about 80% identity with
SEQ ID NO: 39. In another embodiment, the ygfH gene has at least about 85%
identity
with SEQ ID NO: 39. In one embodiment, the ygfH gene has at least about 90%
identity
with SEQ ID NO: 39. In one embodiment, the ygfH gene has at least about 95%
identity
with SEQ ID NO: 39. In another embodiment, the ygfH gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 39. .Accordingly, in one embodiment,
the
ygfH gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
39. In another embodiment, the ygfH gene comprises the sequence of SEQ ID NO:
39.
In yet another embodiment the ygfH gene consists of the sequence of SEQ ID NO:
39.
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[0314] In one embodiment, the mutA gene has at least about 80% identity with
SEQ ID NO: 40. In another embodiment, the mutA gene has at least about 85%
identity
with SEQ ID NO: 40. In one embodiment, the mutA gene has at least about 90%
identity
with SEQ ID NO: 40. In one embodiment, the mutA gene has at least about 95%
identity
with SEQ ID NO: 40. In another embodiment, the mutA gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 40. .Accordingly, in one embodiment,
the
mutA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
40. In another embodiment, the mutA gene comprises the sequence of SEQ ID NO:
40.
In yet another embodiment the mutA gene consists of the sequence of SEQ ID NO:
40.
[0315] In one embodiment, the mutB gene has at least about 80% identity with
SEQ ID NO: 41. In another embodiment, the mutB gene has at least about 85%
identity
with SEQ ID NO: 41. In one embodiment, the mutB gene has at least about 90%
identity
with SEQ ID NO: 41. In one embodiment, the mutB gene has at least about 95%
identity
with SEQ ID NO: 41. In another embodiment, the mutB gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 41. .Accordingly, in one embodiment,
the
mutB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
41. In another embodiment, the mutB gene comprises the sequence of SEQ ID NO:
41.
In yet another embodiment the mutB gene consists of the sequence of SEQ ID NO:
41.
[0316] In one embodiment, the GI 18042134 gene has at least about 80% identity
with SEQ ID NO: 42. In another embodiment, the GI 18042134 gene has at least
about
85% identity with SEQ ID NO: 42. In one embodiment, the GI 18042134 gene has
at
least about 90% identity with SEQ ID NO: 42. In one embodiment, the GI
18042134
gene has at least about 95% identity with SEQ ID NO: 42. In another
embodiment, the
GI 18042134 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID
NO:
42..Accordingly, in one embodiment, the GI 18042134 gene has at least about
80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity with SEQ ID NO: 42. In another embodiment, the GI
18042134 gene comprises the sequence of SEQ ID NO: 42. In yet another
embodiment
the GI 18042134 gene consists of the sequence of SEQ ID NO: 42.
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[0317] In one embodiment, the mmdA gene has at least about 80% identity with
SEQ ID NO: 43. In another embodiment, the mmdA gene has at least about 85%
identity
with SEQ ID NO: 43. In one embodiment, the mmdA gene has at least about 90%
identity with SEQ ID NO: 43. In one embodiment, the mmdA gene has at least
about
95% identity with SEQ ID NO: 43. In another embodiment, the mmdA gene has at
least
about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43..Accordingly, in one
embodiment, the mmdA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%,
86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
with
SEQ ID NO: 43. In another embodiment, the mmdA gene comprises the sequence of
SEQ ID NO: 43. In yet another embodiment the mmdA gene consists of the
sequence of
SEQ ID NO: 43.
[0318] In one embodiment, the PFREUD 188870 gene has at least about 80%
identity with SEQ ID NO: 44. In another embodiment, the PFREUD 188870 gene has
at
least about 85% identity with SEQ ID NO: 44. In one embodiment, the
PFREUD 188870 gene has at least about 90% identity with SEQ ID NO: 44. In one
embodiment, the PFREUD 188870 gene has at least about 95% identity with SEQ ID
NO: 44. In another embodiment, the PFREUD 188870 gene has at least about 96%,
97%,
98%, or 99% identity with SEQ ID NO: 44..Accordingly, in one embodiment, the
PFREUD 188870 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with
SEQ
ID NO: 44. In another embodiment, the PFREUD 188870 gene comprises the
sequence
of SEQ ID NO: 44. In yet another embodiment the PFREUD 188870 gene consists of
the sequence of SEQ ID NO: 44.
[0319] In one embodiment, the Bccp gene has at least about 80% identity with
SEQ ID NO: 45. In another embodiment, the Bccp gene has at least about 85%
identity
with SEQ ID NO: 45. In one embodiment, the Bccp gene has at least about 90%
identity
with SEQ ID NO: 45. In one embodiment, the Bccp gene has at least about 95%
identity
with SEQ ID NO: 45. In another embodiment, the Bccp gene has at least about
96%,
97%, 98%, or 99% identity with SEQ ID NO: 45. .Accordingly, in one embodiment,
the
Bccp gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
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45. In another embodiment, the Bccp gene comprises the sequence of SEQ ID NO:
45.
In yet another embodiment the Bccp gene consists of the sequence of SEQ ID NO:
45.
[0320] In one embodiment, one or more polypeptides encoded by the propionate
circuits and expressed by the genetically engineered bacteria have at least
about 80%
identity with one or more of SEQ ID NO: 46 through SEQ ID NO: 70. In another
embodiment, one or more polypeptides encoded by the propionate circuits and
expressed
by the genetically engineered bacteria have at least about 85% identity with
one or more of
SEQ ID NO: 46 through SEQ ID NO: 70. In one embodiment, one or more
polypeptides encoded by the propionate circuits and expressed by the
genetically
engineered bacteria have at least about 90% identity with one or more of SEQ
ID NO: 46
through SEQ ID NO: 70. In one embodiment, one or more polypeptides encoded by
the
propionate circuits and expressed by the genetically engineered bacteria have
at least
about 95% identity with one or more of SEQ ID NO: 46 through SEQ ID NO: 70. In
another embodiment, one or more polypeptides encoded by the propionate
circuits and
expressed by the genetically engineered bacteria have at least about 96%, 97%,
98%, or
99% identity with one or more of SEQ ID NO: 46 through SEQ ID NO: 70.
Accordingly, in one embodiment, one or more polypeptides encoded by the
propionate
circuits and expressed by the genetically engineered bacteria have at least
about 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 46 through SEQ
ID
NO: 70. In another embodiment, one or more polypeptides encoded by the
propionate
circuits and expressed by the genetically engineered bacteria one or more
polypeptides
encoded by the propionate circuits and expressed by the genetically engineered
bacteria
comprise the sequence of one or more of SEQ ID NO: 46 through SEQ ID NO: 70.
In
yet another embodiment one or more polypeptides encoded by the propionate
circuits and
expressed by the genetically engineered bacteria consist of or or more of SEQ
ID NO: 46
through SEQ ID NO: 70.
[0321] In some embodiments, one or more of the propionate biosynthesis genes
is
a synthetic propionate biosynthesis gene. In some embodiments, one or more of
the
propionate biosynthesis genes is an E. coli propionate biosynthesis gene. In
some
embodiments, one or more of the propionate biosynthesis genes is a C.
glutamicum
propionate biosynthesis gene. In some embodiments, one or more of the
propionate
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biosynthesis genes is a C. propionicum propionate biosynthesis gene. In some
embodiments, one or more of the propionate biosynthesis genes is a R.
sphaeroides
propionate biosynthesis gene. The propionate gene cassette may comprise genes
for the
aerobic biosynthesis of propionate and/or genes for the anaerobic or
microaerobic
biosynthesis of propionate.
[0322] To improve acetate production, while maintaining high levels of
propionate
production, targeted one or more deletions can be introduced in competing
metabolic arms
of mixed acid fermentation to prevent the production of alternative metabolic
fermentative
byproducts (thereby increasing acetate production). Non-limiting examples of
competing
such competing metabolic arms are frdA (converts phosphoenolpyruvate to
succinate),
ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol).
Deletions
which may be introduced therefore include deletion of adhE, ldh, and frd.
Thus, in certain
embodiments, the genetically engineered bacteria comprise one or more
propionate
cassette(s) and further comprise mutations and/or deletions in one or more of
frdA, ldhA,
and adhE.
[0323] In some embodiments, the genetically engineered bacteria comprise one
or
more propionate cassette(s) described herein and one or more mutation(s)
and/or
deletion(s) in one or more genes selected from the ldhA gene, the frdA gene
and the adhE
gene.
[0324] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) encoding one or more enzymes for the production of
propionate
and further comprise a mutation and/or deletion in one or more endogenous
genes selected
from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments,
the
genetically engineered bacteria comprise one or more gene sequence(s) encoding
one or
more enzymes for the production of propionate and further comprise a mutation
and/or
deletion in the endogenous ldhA gene. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) encoding one or more enzymes
for the
production of propionate and further comprise a mutation and/or deletion in
the
endogenous adhE gene. In some embodiments, the genetically engineered bacteria
comprise one or more gene sequence(s) encoding one or more enzymes for the
production
of propionate and further comprise a mutation and/or deletion in the
endogenous frdA
gene. In some embodiments, the genetically engineered bacteria comprise one or
more
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gene sequence(s) encoding one or more enzymes for the production of propionate
and
further comprise a mutation and/or deletion in the endogenous ldhA and rdA
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes for the production of propionate and
further
comprise a mutation and/or deletion in the endogenous ldhA genes and adhE
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes for the production of propionate and
further
comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes for the production of propionate and further
comprise a
mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes for the production of propionate and further
comprise a
mutation and/or deletion in one or more endogenous genes selected from in the
ldhA gene,
the frdA gene and the adhE genes.
[0325] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further
comprise
a mutation and/or deletion in the endogenous ldhA gene. In some embodiments,
the
genetically engineered bacteria comprise one or more gene sequence(s)
comprising one or
more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation
and/or
deletion in the endogenous ldhA gene. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG,
and/or
ygfH and further comprise a mutation and/or deletion in the endogenous adhE
gene. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and
further
comprise a mutation and/or deletion in the endogenous adhE gene. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
selected from
sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion
in the
endogenous frdA gene. In some embodiments, the genetically engineered bacteria
comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-
ygfH
gene cassette(s) and further comprise a mutation and/or deletion in the
endogenous frdA
gene. In some embodiments, the genetically engineered bacteria comprise one or
more
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gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further
comprise a
mutation and/or deletion in the endogenous ldhA and frdA genes. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
comprising
one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a
mutation and/or
deletion in the endogenous ldhA and frdA genes. In some embodiments, the
genetically
engineered bacteria comprise one or more gene sequence(s) selected from sbm,
ygfD,
ygfG, and/or ygfH and further comprise a mutation and/or deletion in the
endogenous
ldhA genes and adhE genes. In some embodiments, the genetically engineered
bacteria
comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-
ygfH
gene cassette(s) and further comprise a mutation and/or deletion in the
endogenous ldhA
genes and adhE genes. In some embodiments, the genetically engineered bacteria
comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or
ygfH and
further comprise a mutation and/or deletion in the endogenous frdA and adhE
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and
further
comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation
and/or
deletion in the endogenous ldhA, the frdA, and adhE genes. In some
embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s)
comprising one or
more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation
and/or
deletion in the endogenous ldhA, the frdA, and adhE genes.
[0326] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more acetate than unmodified bacteria of the same
bacterial subtype
under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the the genetically engineered bacteria
produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
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fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold, more acetate than
unmodified
bacteria of the same bacterial subtype under the same conditions.
[0327] In some embodiments, the genetically engineered bacteria produce 0% to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more propionate than unmodified bacteria of the same
bacterial
subtype under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more propionate than unmodified bacteria of the same bacterial subtype
under the
same conditions. In yet another embodiment, the the genetically engineered
bacteria
produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold,
nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more
propionate than
unmodified bacteria of the same bacterial subtype under the same conditions.
[0328] In certain situations, the need may arise to prevent and/or reduce
acetate
production by of an engineered or naturally occurring strain, e.g., E. coli
Nissle, while
maintaining high levels of propionate production. Without wishing to be bound
by theory,
one or more mutations and/or deletions in one or more gene(s) encoding in one
or more
enzymes which function in the acetate producing metabolic arm of fermentation
should
reduce and/or prevent production of acetate. A non-limiting example of such an
enzyme is
phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic
arm
converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or
a gene
encoding another enzyme in this metabolic arm may also allow for more acetyl-
CoA to be
used for propionate production. Additionally, one or more mutations preventing
or
reducing the flow through other metabolic arms of mixed acid fermentaion, such
as those
which produce succinate, lactate, and/or ethanol can increase the production
of acetyl-
CoA, which is available for propionate synthesis. Such mutations and/or
deletions, include
but are not limited to mutations and/or deletions in the frdA, ldhA, and/or
adhE genes.
[0329] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) encoding one or more enzymes for the production of
propionate
and further comprise a mutation and/or deletion in the endogenous pta gene. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
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encoding one or more enzymes for the production of propionate and further
comprise a
mutation and/or deletion in the endogenous pta gene and in one or more
endogenous genes
selected from in the ldhA gene, the frdA gene and the adhE gene. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
encoding one
or more enzymes for the production of propionate and further comprise a
mutation in the
endogenous pta and adhE genes. In some embodiments, the genetically engineered
bacteria comprise one or more gene sequence(s) encoding one or more enzymes
for the
production of propionate and further comprise a mutation in the endogenous pta
and ldhA
genes. In some embodiments, the genetically engineered bacteria comprise one
or more
gene sequence(s) encoding one or more enzymes for the production of propionate
and
further comprise a mutation in the endogenous pta and frdA genes. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
encoding one
or more enzymes for the production of propionate and further comprise a
mutation and/or
deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s) encoding
one or
more enzymes for the production of propionate and further comprise a mutation
in the
endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) encoding one or more enzymes
for the
production of propionate and further comprise a mutation in the endogenous
pta, frdA and
adhE genes. In some embodiments, the genetically engineered bacteria comprise
one or
more gene sequence(s) encoding one or more enzyme(s) for the production of
propionate
and further comprise a mutation and/or deletion in the endogenous pta, ldhA,
frdA, and
adhE genes.
[0330] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further
comprise
a mutation and/or deletion in the endogenous pta gene. In some embodiments,
the
genetically engineered bacteria comprise one or more gene sequence(s)
comprising one or
more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation
and/or
deletion in the endogenous pta gene. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG,
and/or
ygfHand further comprise a mutation and/or deletion in the endogenous pta gene
and in
one or more endogenous genes selected from in the ldhA gene, the frdA gene and
the adhE
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gene. In some embodiments, the genetically engineered bacteria comprise one or
more
gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate
cassette(s) and
further comprise a mutation and/or deletion in the endogenous pta gene and in
one or more
endogenous genes selected from in the ldhA gene, the frdA gene and the adhE
gene. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) selected from sbm, ygfD, ygfG, and/or ygfHand further comprise a
mutation
in the endogenous pta and adhE genes. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-
ygfG-
ygfH propionate cassette(s) and further comprise a mutation in the endogenous
pta and
adhE genes. In some embodiments, the genetically engineered bacteria comprise
one or
more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfHand further
comprise a
mutation in the endogenous pta and ldhA genes.
[0331] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate
cassette(s) and further comprise a mutation in the endogenous pta and ldhA
genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from sbm, ygfD, ygfG, and/or ygfHand further comprise a mutation in
the
endogenous pta and frdA genes. In some embodiments, the genetically engineered
bacteria
comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-
ygfH
propionate cassette(s) and further comprise a mutation in the endogenous pta
and frdA
genes. In some embodiments, the genetically engineered bacteria comprise one
or more
gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfHand further
comprise a
mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further
comprise
a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from sbm, ygfD, ygfG, and/or ygfHand further comprise a mutation in
the
endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-
ygfG-
ygfH propionate cassette(s) and further comprise a mutation in the endogenous
pta, ldhA,
and adhE genes. In some embodiments, the genetically engineered bacteria
comprise one
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or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfHand further
comprise a mutation in the endogenous pta, frdA and adhE genes. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
comprising
one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a
mutation
in the endogenous pta, frdA and adhE genes. In some embodiments, the
genetically
engineered bacteria comprise one or more gene sequence(s) selected from sbm,
ygfD,
ygfG, and/or ygfHand further comprise a mutation in the endogenous pta, ldhA,
frdA, and
adhE genes. In some embodiments, the genetically engineered bacteria comprise
one or
more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate
cassette(s) and further comprise a mutation in the endogenous pta, ldhA, frdA,
and adhE
genes.
[0332] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% less acetate than unmodified bacteria of the same
bacterial subtype
under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold less acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the the genetically engineered bacteria
produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than
unmodified bacteria
of the same bacterial subtype under the same conditions.
[0333] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more propionate than unmodified bacteria of the same
bacterial
subtype under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more propionate than unmodified bacteria of the same bacterial subtype
under the
same conditions. In yet another embodiment, the the genetically engineered
bacteria
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produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold,
nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more
propionate than
unmodified bacteria of the same bacterial subtype under the same conditions.
[0334] In some embodiments, the genetically engineered bacteria comprise a
combination of propionate biosynthesis genes from different species, strains,
and/or
substrains of bacteria, and are capable of producing propionate. In some
embodiments,
one or more of the propionate biosynthesis genes is functionally replaced,
modified,
and/or mutated in order to enhance stability and/or increase propionate
production. In
some embodiments, the local production of propionate reduces food intake and
improves
gut barrier function and reduces inflammation In some embodiments, the
genetically
engineered bacteria are capable of expressing the propionate biosynthesis
cassette and
producing propionate in low-oxygen conditions, in the presence of certain
molecules or
metabolites, in the presence of molecules or metabolites associated with
inflammation or
an inflammatory response, or in the presence of some other metabolite that may
or may
not be present in the gut, such as arabinose.
[0335] In one embodiment, the propionate gene cassette is directly operably
linked
to a first promoter. In another embodiment, the propionate gene cassette is
indirectly
operably linked to a first promoter. In one embodiment, the promoter is not
operably
linked with the propionate gene cassette in nature.
[0336] In some embodiments, the propionate gene cassette is expressed under
the
control of a constitutive promoter. In another embodiment, the propionate gene
cassette is
expressed under the control of an inducible promoter. In some embodiments, the
propionate gene cassette is expressed under the control of a promoter that is
directly or
indirectly induced by exogenous environmental conditions. In one embodiment,
the
propionate gene cassette is expressed under the control of a promoter that is
directly or
indirectly induced by low-oxygen or anaerobic conditions, wherein expression
of the
propionate gene cassette is activated under low-oxygen or anaerobic
environments, such
as the environment of the mammalian gut. Inducible promoters are described in
more
detail infra.
[0337] The propionate gene cassette may be present on a plasmid or chromosome
in the bacterial cell. In one embodiment, the propionate gene cassette is
located on a
plasmid in the bacterial cell. In another embodiment, the propionate gene
cassette is
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located in the chromosome of the bacterial cell. In yet another embodiment, a
native copy
of the propionate gene cassette is located in the chromosome of the bacterial
cell, and a
propionate gene cassette from a different species of bacteria is located on a
plasmid in the
bacterial cell. In yet another embodiment, a native copy of the propionate
gene cassette is
located on a plasmid in the bacterial cell, and a propionate gene cassette
from a different
species of bacteria is located on a plasmid in the bacterial cell. In yet
another
embodiment, a native copy of the propionate gene cassette is located in the
chromosome
of the bacterial cell, and a propionate gene cassette from a different species
of bacteria is
located in the chromosome of the bacterial cell.
[0338] In some embodiments, the propionate gene cassette is expressed on a low-
copy plasmid. In some embodiments, the propionate gene cassette is expressed
on a high-
copy plasmid. In some embodiments, the high-copy plasmid may be useful for
increasing
expression of propionate.
Tryptophan and Tryptophan Metabolism
Kynurenine
[0339] In some embodiments, the genetically engineered bacteria are capable of
producing kynurenine. Kynurenine is a metabolite produced in the first, rate-
limiting step
of tryptophan catabolism. This step involves the conversion of tryptophan to
kynurenine,
and may be catalyzed by the ubiquitously-expressed enzyme indoleamine 2,3-
dioxygenase
(IDO-1), or by tryptophan dioxygenase (TDO), an enzyme which is primarily
localized to
the liver (Alvarado et al., 2015). Biopsies from human patients with IBD show
elevated
levels of IDO- 1 expression compared to biopsies from healthy individuals,
particularly
near sites of ulceration (Ferdinande et al., 2008; Wolf et al., 2004). IDO-1
enzyme
expression is similarly upregulated in trinitrobenzene sulfonic acid- and
dextran sodium
sulfate-induced mouse models of IBD; inhibition of IDO- 1 significantly
augments the
inflammatory response caused by each inducer (Ciorba et al., 2010; Gurtner et
al., 2003;
Matteoli et al., 2010). Kynurenine has also been shown to directly induce
apoptosis in
neutrophils (El-Zaatari et al., 2014). Together, these observations suggest
that IDO-1 and
kynurenine play a role in limiting inflammation. The genetically engineered
bacteria may
comprise any suitable gene for producing kynurenine. In some embodiments, the
genetically engineered bacteria may comprise a gene or gene cassette for
producing a
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tryptophan transporter, a gene or gene cassette for producing IDO-1, and a
gene or gene
cassette for producing TDO. In some embodiments, the gene for producing
kynurenine is
modified and/or mutated, e.g., to enhance stability, increase kynurenine
production, and/or
increase anti-inflammatory potency under inducing conditions. In some
embodiments, the
engineered bacteria have enhanced uptake or import of tryptophan, e.g.,
comprise a
transporter or other mechanism for increasing the uptake of tryptophan into
the bacterial
cell. In some embodiments, the genetically engineered bacteria are capable of
producing
kynurenine under inducing conditions, e.g., under a condition(s) associated
with
inflammation. In some embodiments, the genetically engineered bacteria are
capable of
producing kynurenine in low-oxygen conditions.
[0340] In some embodiments, the genetically engineered bacteria are capable of
producing kynurenic acid. Kynurenic acid is produced from the irreversible
transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine-
oxoglutarate transaminase. Kynurenic acid acts as an antagonist of ionotropic
glutamate
receptors (Turski et al., 2013). While glutamate is known to be a major
excitatory
neurotransmitter in the central nervous system, there is now evidence to
suggest an
additional role for glutamate in the peripheral nervous system. For example,
the activation
of NMDA glutamate receptors in the major nerve supply to the GI tract (i.e.,
the myenteric
plexus) leads to an increase in gut motility (Forrest et al., 2003), but rats
treated with
kynurenic acid exhibit decreased gut motility and inflammation in the early
phase of acute
colitis (Varga et al., 2010). Thus, the elevated levels of kynurenic acid
reported in IBD
patients may represent a compensatory response to the increased activation of
enteric
neurons (Forrest et al., 2003). The genetically engineered bacteria may
comprise any
suitable gene, genes, or gene cassettes for producing kynurenic acid. In some
embodiments, the gene for producing kynurenic acid is modified and/or mutated,
e.g., to
enhance stability, increase kynurenic acid production, and/or increase anti-
inflammatory
potency under inducing conditions. In some embodiments, the genetically
engineered
bacteria are capable of producing kynurenic acid under inducing conditions,
e.g., under a
condition(s) associated with inflammation. In some embodiments, the
genetically
engineered bacteria are capable of producing kynurenic acid in low-oxygen
conditions
Tryptophan, Tryptophan Metabolism, and Tryptophan Metabolites
Tryptophan and the Kynurenine Pathway
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[0341] Tryptophan (TRP) is an essential amino acid that, after consumption, is
either incorporated into proteins via new protein synthesis, or converted a
number of
biologically active metabolites with a number of differing roles in health and
disease
(Perez-De La Cruz et al., 2007 Kynurenine Pathway and Disease: An Overview;
CNS&Neurological Disorders -Drug Targets 2007, 6,398-410). Along one arm of
tryptophan catabolism, trytophan is converted to the neurotransmitter
serotonin (5-
hydroxytryptamine, 5-HT) by tryptophan hydroxylase. Serotonin can further be
converted
into the hormone melatonin. A large share of tryptophan, however, is
metabolized to a
number of bioactive metabolites, collectively called kynurenines, along a
second arm
called the kynurenine pathway (KP). In the first step of catabolism, TRP is
converted to
Kynurenine, (KYN), which has well-documented immune suppressive functions in
several
types of immune cells, and has recently been shown to be an activating ligand
for the
arylcarbon receptor (AhR; also known as dioxin receptor). KYN was initially
shown in the
cancer setting as an endogenous AHR ligand in immune and tumor cells, acting
both in an
autocrine and paracrine manner, and promoting tumor cell survival. In the gut,
kynurenine
pathway metabolism is regulated by gut microbiota, which can regulate
tryptophan
availability for kynurenine pathway metabolism.
[0342] More recently, additional tryptophan metabolites, collectively termed
"indoles", herein, including for example, indole-3 aldehyde, indole-3 acetate,
indole-3
propoinic acid, indole, indole-3 acetaladehyde, indole-3acetonitrile, FICZ,
etc. which are
generated by the microbiota, some by the human host, some from the diet, which
are also
able to function as AhR agonists, see e.g., Table 8 and elsewhere herein, and
Lama et al.,
Nat Med. 2016 Jun;22(6):598-605; CARD9 impacts colitis by altering gut
microbiota
metabolism of tryptophan into aryl hydrocarbon receptor ligands.
[0343] Ahr best known as a receptor for xenobiotics such as polycyclic
aromatic
hydrocarbons AhR is a ligand-dependent cytosolic transcription factor that is
able to
translocate to the cell nucleus after ligand binding. The in additiona to
kynurenine,
tryptophan metabolites L-kynurenine, 6-formylindolcarbazole (FICZ, a
photoproduct of
TRP), and KYNA are have recently been identified as endogenous AhR ligands
mediating
immunosuppressive functions. To induce transcription of AhR target genes in
the nucleus,
AhR partners with proteins such as AhR nuclear translocator (ARNT) or NF-KB
subunit
RelB. Studies on human cancer cells have shown that KYN activates the AhR-ARNT
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associated transcription of IL-6, which induced autocrine activation of IDO1
via STAT3.
This AhR-IL-6-STAT3 loop is associated with a poor prognosis in lung cancer,
supporting
the idea that IDO/kynurenine-mediated immunosuppression enables the immune
escape of
tumor cells.
[0344] In the gut, tryptophan may also be transported across the epithelium by
transport machinery comprising angiotensin I converting enzyme 2 (ACE2), and
converted
to kynurenine, where it functions in the suppression of T cell respononse and
promotion of
Treg cells.
[0345] The rate-limiting conversion of TRP to KYN may be mediated by either of
two forms of indoleamine 2, 3-dioxygenase (IDO) or by tryptophan 2,3-
dioxygenase
(TDO). One characteristic of TRP metabolism is that the rate-limiting step of
the catalysis
from TRP to KYN is generated by both the hepatic enzyme tryptophan 2,3-
dioxygenase
(TDO) and the ubiquitous expressed enzyme ID01. TDO is essential for
homeostasis of
TRP concentrations in organisms and has a lower affinity to TRP than ID01. Its
expression is activated mainly by increased plasma TRP concentrations but can
also be
activated by glucocorticoids and glucagon. The tryptophan kynurenine pathway
is also
expressed in a large number of microbiota, most prominently in
Enterobacteriaceae, and
kynurenine and metabolites may be synthesized in the gut (as shown in the
figures and the
examples, and Sci Transl Med. 2013 July 10; 5(193): 193ra91). In some
embodiments, the
genetically engineered bacteria comprise one or more heterologous bacterially
derived
genes from Enterobacteriaceae, e.g. whose gene products catalyze the
conversion of
TRP:KYN. Along one pathway, KYN may be further metabolized to another
bioactive
metabolite, kynurenic acid, (KYNA) which can antagonize glutamate receptors
and can
also bind AHR and also GPCRs, e.g., GPR35, glutamate receptors, N-methyl D-
aspartate
(NMDA)-receptors, and others. Along a third pathway of the KP, KYN can be
converted
to anthranilic acid (AA) and further downstream quinolinic acid (QUIN), which
is a
glutamate receptor agonist and has a neurotoxic role.
[0346] Therefore, finding a means to upregulate and/or downregulate the levels
of
flux through the KP and to reset relative amounts and/or ratios of tryptophan
and its
various bioactive metabolites may be useful in the prevention, treatment
and/or
management of a number of diseases as described herein. The present disclosure
describes
compositions for modulating, regulating and fine tuning trypophan and
tryptophan
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metabolite levels, e.g., in the serum or in the gastrointestinal system,
through genetically
engineered bacteria which comprise circuitry enabling the synthesis, bacterial
uptake and
catabolism of tryptophan and/or tryptophan metabolites. and provides methods
for using
these compositions in the treatment, management and/or prevention of a number
of
different diseases.
Other Indole Tryptophan Metabolites
[0347] In addition to kynurenine and KYNA, numerous compounds have been
proposed as endogenous AHR ligands, many of which are generated through
pathways
involved in the metabolism of tryptophan and indole (Bittinger et al., 2003;
Chung and
Gadupudi, 2011) A large number of metabolites generated through the tryptophan
indole
pathway are generated by microbiota in the gut. For example, bacteria take up
tryptophan,
which can be converted to mono-substituted indole compounds, such as indole
acetic acid
(IAA) and tryptamine, and other compounds, which have been found to activate
the AHR
(Hubbard et al., 2015, Adaptation of the human aryl hydrocarbon receptor to
sense
microbiota-derived indoles; Nature Scientific Reports 5:12689).
[01] In the gastronintestinal tract, diet derived and bacterially AhR
ligands
promote IL-22 production by innate lymphoid cells, referred to as group 3 ILCs
(Spits et
al., 2013, Zelante et al., Tryptophan Catabolites from Microbiota Engage Aryl
Hydrocarbon Receptor and Balance Mucosal Reactivity via Interleukin-22;
Immunity 39,
372-385, August 22, 2013). AHR is essential for IL-22-production in the
intestinal lamina
propria (Lee et al., Nature Immunology 13, 144-151 (2012); AHR drives the
development
of gut ILC22 cells and postnatal lymphoid tissues via pathways dependent on
and
independent of Notch).
[0348] Through initiation of Jak-STAT signaling pathways, IL-22 expression can
trigger expression of antimicrobial compounds as well as a range of cell
growth related
pathways, both of which enhance tissue repair mechanisms. IL-22 is critical in
promoting
intestinal barrier fidelity and healing, while modulating inflammatory states.
Murine
models have demonstrated improved intestinal inflammation states following
administration of 11-22. Additionally, IL-22 activates STAT3 signaling to
promote
enhanced mucus production to preserve barrier function.
[0349] Table 8 lists exemplary tryptophan metabolites which have been shown to
bind to AhR and which can be produced by the genetically engineered bacteria
of the
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disclosure. Thus, in some embodiments, the engineered bacteria comprises gene
sequence(s) encoding one or more enzymes for the production of one or more
metabolites
listed in Table 8.
Table 8. Indole Tryptophan Metabolites
Origin Compound
Exogenous 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)
Dietary Indole-3-carbinol (I3C)
Dietary Indole-3-acetonitrile (I3ACN)
Dietary 3.3'-Diindolylmethane (DIM)
Dietary 2-(indo1-3-ylmethyl)-3.3'-diindolylmethane (Ltr-1)
Dietary Indolo(3,2-b)carbazole (ICZ)
Dietary 2-(1'H-indole-3'-carbony)-thiazole-4-carboxylic acid methyl
ester (ITE)
Microbial Indole
Microbial Indole-3-acetic acid (IAA)
Microbial Indole-3-aldehyde (IAId)
Microbial Tryptamine
Microbial 3-methyl-indole (Skatole)
Yeast Tryptanthrin
Microbial/Host Indigo
Metabolism
Microbial/Host Indirubin
Metabolism
Microbial/Host Indoxy1-3-sulfate (I3S)
Metabolism
Host Kynurenine (Kyn)
Metabolism
Host Kynurenic acid (KA)
Metabolism
Host Xanthurenic acid
Metabolism
Host Cinnabarinic acid (CA)
Metabolism
UV-Light 6-formylindolo(3,2-b)carbazole (FICZ)
Oxidation
Microbial
metabolism
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[0350] In addition, some indole metabolites may exert their effect through
Pregnane X receptor (PXR), which is thought to play a key role as an essential
regulator of
intestinal barrier function. PXR-deficient (Nr1i2-/-) mice showed a distinctly
"leaky' gut
physiology coupled with upregulation of the Toll-like receptor 4 (TLR4), a
receptor well
known for recognizing LPS and activating the innate immune system (Venkatesh
et al.,
2014 Symbiotic Bacterial Metabolites Regulate Gastrointestinal Barrier
Function via the
Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, August
21,
2014). In particular, indole 3-propionic acid (IPA), produced by microbiota in
the gut, has
been shown to be a ligand for PXR in vivo.
[0351] As a result of PXR agonism, indole levels e.g., produced by commensal
bacteria, or by genetically engineered bacteria, may through the activation of
PXR
regulate and balance the levels of TLR4 expression to promote homeostasis and
gut barrier
health. I.e., low levels of IPA and/or PXR and an excess of TLR4 may lead to
intestinaly
barrier dysfunction, while increasing levels of IPA may promote PXR activation
and
TLR4 downregulation, and improved gut barrier health.
[0352] In other embodiments, IPA producing circuits comprise enzymes depicted
and described in the figures and elsewhere herein. Thus, in some embodiments,
the
engineered bacteria comprise gene sequence(s) encoding one or more enzymes
selected
from TrpDH: tryptophan dehydrogenase (e.g., from from Nostoc punctiforme NIES-
2108); FldHl/F1dH2: indole-3-lactate dehydrogenase (e.g., from Clostridium
sporogenes);
FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase (e.g., from
Clostridium
sporogenes); FldBC: indole-3-lactate dehydratase, (e.g., from Clostridium
sporogenes);
FldD: indole-3-acrylyl-CoA reductase (e.g., from Clostridium sporogenes);
AcuI: acrylyl-
CoA reductase (e.g., from Rhodobacter sphaeroides); 1pdC: Indole-3-pyruvate
decarboxylase (e.g., from Enterobacter cloacae); ladl: Indole-3-acetaldehyde
dehydrogenase (e.g., from Ustilago maydis); and Tdc: Tryptophan decarboxylase
(e.g.,
from Catharanthus roseus or from Clostridium sporogenes). In some embodiments,
the
engineered bacteria comprise gene sequence(s) and/or gene cassette(s) for the
production
of one or more of the following: indole-3-propionic acid (IPA), indole acetic
acid (IAA),
and tryptamine synthesis(TrA).
[0353] Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the
reversible chemical reaction converting L-tryptophan, NAD(P) and water to
(indo1-3-
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yl)pyruvate (IPyA), NH3, NAD(P)H and H. Indole-3-lactate dehydrogenase ((EC
1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) converts
(indo1-
3y1)pyruvate (IpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+. Indole-
3-
propionyl-CoA:indole-3-lactate CoA transferase (F1dA ) converts indole-3-
lactate (ILA)
and indo1-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-
lactate-CoA.
Indole-3-acrylyl-CoA reductase (F1dD ) and acrylyl-CoA reductase (AcuI)
convert indole-
3-acrylyl-CoA to indole-3-propionyl-CoA. Indole-3-lactate dehydratase (FldBC )
converts
indole-3-lactate-CoA to indole-3-acrylyl-CoA. Indole-3-pyruvate decarboxylase
(1pdC:)
converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAA1d) ladl:
Indole-3-
acetaldehyde dehydrogenase coverts Indole-3-acetaldehyde (IAA1d) into Indole-3-
acetic
acid (IAA) Tdc: Tryptophan decarboxylase converts tryptophan (Trp) into
tryptamine
(TrA).
[0354] Although microbial degradation of tryptophan to indole-3-propionate has
been shown in a numver of microorganisms (see, e.g., Elsden et al., The end
products of
the metabolism of aromatic amino acids by Clostridia, Arch Microbiol. 1976 Apr
1;107(3):283-8), to date, the bacterial entire biosynthetic pathway from
tryptophan to IPA
is unknown. In Clostridium sporogenes, tryptophan is catabolized via indole-3-
pyruvate,
indole-3-lactate, and indole-3-acrylate to indole-3-propionate (O'Neill and
DeMoss,
Tryptophan transaminase from Clostridium sporogenes, Arch Biochem Biophys.
1968 Sep
20;127(1):361-9). Two enzymes that have been purified from C. sporogenes are
tryptophan transaminase and indole-3-lactate dehydrogenase (Jean and DeMoss,
Indolelactate dehydrogenase from Clostridium sporogenes, Can J Microbiol. 1968
Apr;14(4):429-35). Lactococcus lactis, catabolizes tryptophan by an
aminotransferase to
indole-3-pyruvate. In Lactobacillus casei and Lactobacillus helveticus
tryptophan is also
catabolized to indole-3-lactate through successive transamination and
dehydrogenation
(see, e.g., Tryptophan catabolism by Lactobacillus casei and Lactobacillus
helveticus
cheese flavor adjuncts Gummalla, S., Broadbent, J. R. J. Dairy Sci 82:2070-
2077, and
references therein).
[0355] L-tryptophan transaminase (e.g., EC 2.6.1.27, e.g., Clostridium
sporogenes
or Lactobacillus casei) converts L-tryptophan and 2-oxoglutarate to (indo1-
3y1)pyruvate
and L-glutamate). Indole-3-lactate dehydrogenase (EC 1.1.1.110, e.g.,
Clostridium
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sporogenes orLactobacillus casei) converts (indo1-3y1) pyruvate and NADH and
H+ to
indole-3 lactate and NAD+.
[0356] In some embodiments, the engineered bacteria comprises gene sequence(s)
encoding one or more enzymes selected from tryptophan transaminase (e.g., from
C.
sporogenes) and/or indole-3-lactate dehydrogenase (e.g., from C. sporogenes),
and/or
indole-3-pyruvate aminotransferase (e.g., from Lactococcus lactis). In other
embodiments,
such enzymes encoded by the bacteria are from Lactobacillus casei and/or
Lactobacillus
helveticus.
[0357] In other embodiments, IPA producing circuits comprise enzymes depicted
and described in FIG. 47 and FIG. 48 and elsewhere herein.
[0358] In some embodiments, the bacteria comprise gene sequence for producing
one or more tryptophan metabolites, e.g., "indoles". In some embodiments, the
bacteria
comprise gene sequence for producing and indole selected from indole-3
aldehyde, indole-
3 acetate, indole-3 propoinic acid, indole, indole-3 acetaladehyde, indole-
3acetonitrile,
FICZ. In some embodiments, the bacteria comprise gene sequence for producing
an
indole that functions as an AhR agonist, see e.g., Table 8..
[0359] In some embodiments, the bacteria comprise any one or more of the
circuits
described and depicted in the figures and examples.
Methoxyindole pathway, Serotonin and Melatonin
[0360] The methoxyindole pathway leads to formation of serotonin (5-HT) and
melatonin. Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic amine
synthesized in a
two-step enzymatic reaction: First, enzymes encoded by one of two tryptophan
hydroxylase genes (Tphl or Tph2) catalyze the rate-limiting conversion of
tryptophan to
5-hydroxytryptophan (5-HTP). Subsequently, 5-HTP undergoes decarboxylation to
serotonin.
[0361] The majority (95%-98%) of total body serotonin is found in the gut
(Berger et al., 2009). Peripheral serotonin acts autonomously on many cells,
tissues, and
organs, including the cardiovascular, gastrointestinal, hematopoietic, and
immune systems
as well as bone, liver, and placenta (Amireault et al., 2013). Serotonin
functions as a
ligand for any of 15 membrane-bound mostly G protein-coupled serotonin
receptors (5-
HTRs) that are involved in various signal transduction pathways in both CNS
and
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periphery. Intestinal serotonin is released by enterochromaffin cells and
neurons and is
regulated via the serotonin re-uptake transporter (SERT). The SERT is located
on
epithelial cells and neurons in the intestine. Gut microbiota are
interconnected with
serotonin signaling and are for example capable of increasing serotonin levels
through
host serotonin production (Jano et al., Cell. 2015 Apr 9;161(2):264-76. doi:
10.1016/j.ce11.2015.02.047. Indigenous bacteria from the gut microbiota
regulate host
serotonin biosynthesis).
[0362] Modulation of tryptophan metabolism, especially serotonin synthesis is
considered a novel potential strategy the treatment of gastrointestinal (GI)
disorders,
including IBD.
[0363] In some embodiments, the engineered bacteria comprise gene sequence
encoding one or more tryptophan hydroxylase genes (Tphl or Tph2). In some
embodiments, the engineered bacteria further comprise gene sequence for
decarboxylating
5-HTP. In some embodiments, the engineered bacteria comprise gene sequence for
the
production of 5-hydroxytryptophan (5-HTP). In some embodiments, the engineered
bacteria comprise gene sequence for the production of seratonin.
[0364] In certain embodiments, the genetically engineered bacteria described
herein may modulate serotonin levels in the gut, e.g., decrease or increase
serotonin levels,
e.g, in the gut and in the circulation. In certain embodiments, the
genetically engineered
bacteria influence serotonin synthesis, release, and/or degradation. In some
embodiments,
the genetically engineered bacteria may modulate the serotonin levels in the
gut to
improve gut barrier function, modulate the inflammatory status, otherwise
ameliorate
symptoms of A gastrointestinal disorder or inflammatory disorder. In some
embodiments,
the genetically engineered bacteria take up serotonin from the environment,
e.g., the gut.
In some embodiments, the genetically engineered bacteria release serotonin
into the
environment, e.g., the gut. In some embodiments, the genetically engineered
modulate or
influence serotonin levels produced by the host. In some embodiments, the
genetically
engineered bacteria counteract microbiota which are responsible for altered
serotonin
function in many metabolic diseases.
[0365] In some embodiments, the genetically engineered bacteria comprise gene
sequence encoding tryptophan hydroxylase (TpH (land/or2)) and/or 1-amino acid
decarboxylase, e.g. for the treatment of constipation-associated metablic
disorders. In
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some embodiments, the genetically engineered bacteria comprise genetic
cassettes which
allow trptophan uptake and catalysis, reducing trptophan availability for
serotonin
synthesis (serotonin depletion). In some embodiments, the genetically
engineered bacteria
comprise cassettes which promote serotonin uptake from the environment, e.g.,
the gut,
and serotonin catalysis.
[0366] Additionally, serotonin also functions a substrate for melatonin
biosynthesis. Melatonin acts as a neurohormone and is associated with the
development of
circadian rhythm and the sleep-wake cycle.
[0367] In bacteria, melatonin is synthesized indirectly with tryptophan as an
intermediate product of the shikimic acid pathway. In these cells, synthesis
starts with d-
erythrose-4-phosphate and phosphoenolpyruvate. In some embodiments, the
genetically
engineered bacteria comprise an endogenous or exogenous cassette for the
production of
melatonin. As a non-limiting example, the cassette is described in Bochkov,
Denis V.;
Sysolyatin, Sergey V.; Kalashnikov, Alexander I.; Surmacheva, Irina A. (2011).
"Shikimic
acid: review of its analytical, isolation, and purification techniques from
plant and
microbial sources". Journal of Chemical Biology 5 (1): 5-17.
doi:10.1007/s12154-011-
0064-8.
[0368] In a non-limiting example, genetically engineered bacteria convert
tryptophan and/or serotonin to melatonin by, e.g., tryptophan hydroxylase
(TPH),
hydroxyl-0-methyltransferase (HIOMT), N-acetyltransferase (NAT), and aromatic
¨
amino acid decarboxylase (AAAD), or equivalents thereof, e.g., bacterial
equivalents.
Exemplary Tryptophan and Tryptophan Metabolite Circuits
Decreasing Exogenous Tryptophan
[0369] In some embodiments, the genetically engineered bacteria are capable of
decreasing the level of tryptophan and/or the level of a tryptophan
metabolite. In some
embodiments, the engineered bacteria comprise gene sequence(s) for encoding
one or
more aromatic amino acid transporter(s). In one embodiment, the amino acid
transporter
is a tryptophan transporter. Tryptophan transporters may be expressed or
modified in the
recombinant bacteria described herein in order to enhance tryptophan transport
into the
cell. Specifically, when the tryptophan transporter is expressed in the
recombinant
bacterial cells described herein, the bacterial cells import more tryptophan
into the cell
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when the transporter is expressed than unmodified bacteria of the same
bacterial subtype
under the same conditions. Thus, the genetically engineered bacteria
comprising a
heterologous gene encoding a tryptophan transporter which may be used to
import
tryptophan into the bacteria.
[0370] The uptake of tryptophan into bacterial cells is mediated by proteins
well
known to those of skill in the art. For example, three different tryptophan
transporters,
distinguishable on the basis of their affinity for tryptophan have been
identified in E. coli
(see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173: 6009-17). The bacterial
genes mtr,
aroP, and tnaB encode tryptophan permeases responsible for tryptophan uptake
in bacteria.
High affinity permease, Mtr, is negatively regulated by the trp repressor and
positively
regulated by the TyR product (see, e.g., Yanofsky et al. (1991) J. Bacteriol.
173: 6009-17
and Heatwole, et al. (1991) J. Bacteriol. 173: 3601-04), while AroP is
negatively regulated
by the tyR product (Chye et al. (1987) J. Bacteriol. 169:386-93).
[0371] In some embodiments, the engineered bacteria comprise gene sequence(s)
for encoding one or more aromatic amino acid transporter(s). In one
embodiment, the
amino acid transporter is a tryptophan transporter. In one embodiment, the at
least one
gene encoding a tryptophan transporter is a gene selected from the group
consisting of mtr,
aroP and tnaB. In one embodiment, the bacterial cell described herein has been
genetically engineered to comprise at least one heterologous gene selected
from the group
consisting of mtr, aroP and tnaB. In one embodiment, the at least one gene
encoding a
tryptophan transporter is the Escherichia coli mtr gene. In one embodiment,
the at least
one gene encoding a tryptophan transporter is the Escherichia coli aroP gene.
In one
embodiment, the at least one gene encoding a tryptophan transporter is the
Escherichia
coli tnaB gene.
[0372] In some embodiments, the tryptophan transporter is encoded by a
tryptophan transporter gene derived from a bacterial genus or species,
including but not
limited to, Escherichia, Corynebacterium, Escherichia coli, Saccharomyces
cerevisiae or
Corynebacterium glutamicum. In some embodiments, the bacterial species is
Escherichia
coli. In some embodiments, the bacterial species is Escherichia coli strain
Nissle.
[0373] Assays for testing the activity of a tryptophan transporter, a
functional
variant of a tryptophan transporter, or a functional fragment of transporter
of tryptophan
are well known to one of ordinary skill in the art. For example, import of
tryptophan may
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be determined using the methods as described in Shang et al. (2013) J.
Bacteriol.
195:5334-42, the entire contents of each of which are expressly incorporated
by reference
herein.
[0374] In one embodiment, when the tryptophan transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells import 10%
more
tryptophan into the bacterial cell when the transporter is expressed than
unmodified
bacteria of the same bacterial subtype under the same conditions. In another
embodiment,
when the tryptophan transporter is expressed in the recombinant bacterial
cells described
herein, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
100%
more tryptophan into the bacterial cell when the transporter is expressed than
unmodified
bacteria of the same bacterial subtype under the same conditions. In yet
another
embodiment, when the tryptophan transporter is expressed in the recombinant
bacterial
cells described herein, the bacterial cells import two-fold more tryptophan
into the cell
when the transporter is expressed than unmodified bacteria of the same
bacterial subtype
under the same conditions. In yet another embodiment, when the tryptophan
transporter is
expressed in the recombinant bacterial cells described herein, the bacterial
cells import
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptophan
into the cell when
the transporter is expressed than unmodified bacteria of the same bacterial
subtype under
the same conditions.
[0375] In addition to the tryptophan uptake transporters, in some embodiments,
the
genetically engineered bacteria further comprise a circuit for the production
of tryptophan
metabolites, as described herein, e.g., for the production of kynurenine,
kynurenine
metabolites, or indole tryptophan metabolites as shown in Table 8.
[0376] In some embodiments, the genetically engineered bacteria are capable of
decreasing the level of tryptophan. In some embodiments, the engineered
bacteria
comprises one or more gene sequences for converting tryptophan to kynurenine.
In some
embodiments, the engineered bacteria comprise gene sequence(s) for encoding
the enzyme
indoleamine 2,3-dioxygenase (IDO-1). In some embodiments, the engineered
bacteria
comprises gene sequence(s) for encoding the enzyme tryptophan dioxygenase
(TDO). In
some embodiments, the engineered bacteria comprise gene sequence(s) for
encoding the
enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme tryptophan
dioxygenase
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(TDO). In some embodiments, the genetically engineered bacteria comprise a
gene
cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52; producing N-
formyl
kynurenine from tryptophan) and Kynurenine formamidase (EC3.5.1.9) producing
kynurenine from n-formylkynurenine). In some embodiments, the enzymes are
bacterially
derived, e.g., as described in Vujkovi-Cvijin et al. 2013.
[0377] In some embodiments, the genetically engineered bacteria are capable of
decreasing the level of tryptophan, e.g., in combination with the production
of indole
metabolites, through expression of gene(s) and gene cassette(s) described
herein. In some
embodiments, expression of the gene sequences(s) is driven by an inducible
promoter,
described in more detail herein. In some embodiments, the expression of the
gene
sequences(s) is driven by a constitutive promoter.
Increasing Kynurenine
[0378] In some embodiments, the genetically engineered bacteria are capable of
producing kynurenine.
[0379] In some embodiments, the genetically engineered bacteria are capable of
decreasing the level of tryptophan. In some embodiments, the engineered
bacteria
comprise one or more gene sequences for converting tryptophan to kynurenine.
In some
embodiments, the engineered bacteria comprise gene sequence(s) for encoding
the enzyme
indoleamine 2,3-dioxygenase (IDO-1). In some embodiments, the engineered
bacteria
comprise gene sequence(s) for encoding the enzyme tryptophan dioxygenase
(TDO). In
some embodiments, the engineered bacteria comprise on or more gene sequence(s)
for
encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme
tryptophan
dioxygenase (TDO). In some embodiments, the genetically engineered bacteria
comprise
a gene cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52;
producing N-
formyl kynurenine from tryptophan) and Kynurenine formamidase (EC3.5.1.9)
producing
kynurenine from n-formylkynurenine). In some embodiments, the enzymes are
bacterially
derived, e.g., as described in Vujkovi-Cvijin et al. 2013.
[0380] In one embodiment, the genetically engineered bacteria comprise one or
more gene sequence(s) which encode one or more tryptophan catabolism enzymes,
which
produce kynurenine from tryptophan. Non-limiting example of such gene
sequence(s) are
shown the figures and described elsewhere herein. In one embodiment, the
genetically
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engineered bacteria comprise one or more gene sequence(s) which encode
ID01(indoleamine 2,3-dioxygenase). In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode IDO1 from homo
sapiens.
In one embodiment, the genetically engineered bacteria comprise one or more
gene
sequence(s) which encode TD02 (tryptophan 2,3-dioxygenase). In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
TD02 from homo sapiens. In one embodiment, the genetically engineered bacteria
comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-
dioxygenase). In one embodiment, the genetically engineered bacteria comprise
one or
more gene sequence(s) which encode BNA2 from S. cerevisiae). In one
embodiment, the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
Afmid: Kynurenine formamidase. In one embodiment, the genetically engineered
bacteria
comprise one or more gene sequence(s) which encode Afmid: Kynurenine
formamidase
from mouse. In one embodiment, the genetically engineered bacteria comprise
one or
more gene sequence(s) which encode Afmid in combination with one or more of
idol
and/or tdo2 and/or bna2. In one embodiment, the genetically engineered
bacteria comprise
one or more gene sequence(s) which encode Afmid in combination with idol. In
one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode BNA2 in combination with tdo2. In one embodiment, the genetically
engineered bacteria comprise one or more gene sequence(s) which encode Afmid
in
combination with bna2.In one embodiment, the genetically engineered bacteria
comprise
one or more gene sequence(s) which encode BNA3 (kynurenine--oxoglutarate
transaminase. In one embodiment, the genetically engineered bacteria comprise
one or
more gene sequence(s) which encode BNA3 from S. cerevisae. In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
BNA2 in combination with one or more of idol and/or tdo2 and/or bna2. In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode BNA2 in combination with idol. In one embodiment, the genetically
engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in
combination with tdo2. In one embodiment, the genetically engineered bacteria
comprise
one or more gene sequence(s) which encode BNA2 in combination with bna2.In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
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which encode one or more of idol and/or tdo2 and/or bna2.In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode one
or more of afmid and/or bna3.
[0381] In one embodiment, the genetically engineered bacteria comprise one or
more gene sequence(s) which encode one or more of idol and/or tdo2 and/or
bna2, in
combination with one or more of afmid and/or bna3.
[0382] In any of these embodiments, the genetically engineered bacteria which
produce kynurenine from tryptophan also optionally comprise one or more gene
sequence(s) comprising one or more enzymes for tryptophan production, and gene
deletions/or mutations as depicted and described in the figures and described
elsewhere
herein. In some embodiments, the genetically engineered bacteria which produce
kynurenine from tryptophan also optionally comprise one or more gene
sequence(s) which
encode one or more transporter(s) as described herein, through which
tryptophan can be
imported. Optionally, in some embodiments, the genetically engineered bacteria
which
produce kynurenine from tryptophan also optionally comprise one or more gene
sequence(s) which encode an exporter as described herein, which can export
tryptophan or
any of its metabolites.
[0383] The genetically engineered bacteria may comprise any suitable gene for
producing kynurenine. In some embodiments, the gene for producing kynurenine
is
modified and/or mutated, e.g., to enhance stability, increase kynurenine
production, and/or
increase anti-inflammatory potency under inducing conditions. In some
embodiments, the
engineered bacteria also have enhanced uptake or import of tryptophan, e.g.,
comprise a
transporter or other mechanism for increasing the uptake of tryptophan into
the bacterial
cell, as discussed in detail above. In some embodiments, the genetically
engineered
bacteria are capable of producing kynurenine under inducing conditions, e.g.,
under a
condition(s) associated with inflammation. In some embodiments, the
genetically
engineered bacteria are capable of producing kynurenine in low-oxygen
conditions, in the
presence of certain molecules or metabolites, in the presence of molecules or
metabolites
associated with inflammation or an inflammatory response, or in the presence
of some
other metabolite that may or may not be present in the gut, such as arabinose
and others
described herein. In some embodiments, the gene sequences(s) are controlled by
an
inducible promoter. In some embodiments, the gene sequences(s) are controlled
by a
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constitutive promoter. In some embodiments, the gene sequences(s) are
controlled by an
inducible and/or constitutive promoter, and are expressed during bacterial
culture in vitro,
e.g., for bacterial expansion, production and/or manufacture, as described
herein.
[0384] In some embodiments, the genetically engineered bacteria comprise one
or
more gene(s) or gene cassette(s) for the consumption of tryptophan and
production of
kynurenine, which are bacterially derived. In some embodiments, the enzymes
for TRP to
KYN conversion are derived from one or more of Pseudomonas, Xanthomonas,
Burkholderia, Stenotrophomonas, Shewanella, and Bacillus, and/or members of
the
families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some
embodiments
the enzymes are derived from the species listed in table S7 of Vujkovic-Cvijin
et al.
(Dysbiosis of the gut microbiota is associated with HIV diseaseprogression and
tryptophan
catabolism Sci Transl Med. 2013 July 10; 5(193): 193ra91), the contents of
which is
herein incorporated by reference in its entirety.
[0385] In some embodiments, the one or more genes for producing kynurenine are
modified and/or mutated, e.g., to enhance stability, increase kynurenine
production, and/or
increase anti-inflammatory potency under inducing conditions. In some
embodiments, the
engineered bacteria have enhanced uptake or import of tryptophan, e.g.,
comprise a
transporter or other mechanism for increasing the uptake of tryptophan into
the bacterial
cell. In some embodiments, the genetically engineered bacteria are capable of
producing
kynurenine under inducing conditions, e.g., under a condition(s) associated
with
inflammation. In some embodiments, the genetically engineered bacteria are
capable of
producing kynurenine in low-oxygen conditions, in the presence of certain
molecules or
metabolites, in the presence of molecules or metabolites associated with
inflammation or
an inflammatory response, or in the presence of some other metabolite that may
or may
not be present in the gut, such as arabinose and others described herein.
[0386] In any of the embodiments described above and elsewhere herein, the
genetically engineered bacteria are capable of expressing any one or more of
the described
circuits in low-oxygen conditions, in the presence of disease or tissue
specific molecules
or metabolites, in the presence of molecules or metabolites associated with
inflammation
or an inflammatory response or immune suppression, liver damage, or metabolic
disease,
or in the presence of some other metabolite that may or may not be present in
the gut, such
as arabinose and others described herein. In some embodiments, any one or more
of the
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described circuits are present on one or more plasmids (e.g., high copy or low
copy) or are
integrated into one or more sites in the bacterial chromosome. Also, in some
embodiments, the genetically engineered bacteria are further capable of
expressing any
one or more of the described circuits and further comprise one or more of the
following:
(1) one or more auxotrophies, such as any auxotrophies known in the art and
provided
herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as
any of the kill-
switches described herein or otherwise known in the art, (3) one or more
antibiotic
resistance circuits, (4) one or more transporters for importing biological
molecules or
substrates, such any of the transporters described herein or otherwise known
in the art, (5)
one or more secretion circuits, such as any of the secretion circuits
described herein and
otherwise known in the art, and (6) combinations of one or more of such
additional
circuits.
Increasing Tryptophan
[0387] In some embodiments, the genetically engineered microorganisms of the
present disclosure are capable of producing tryptophan. Exemplary circuits for
the
production of tryptophan are shown in the figures.
[0388] In some embodiments, the genetically engineered bacteria that produce
tryptophan comprise one or more gene sequences encoding one or more enzymes of
the
tryptophan biosynthetic pathway. In some embodiments, the genetically
engineered
bacteria comprise a tryptophan operon. In some embodiments, the genetically
engineered
bacteria comprise the tryptophan operon of E. coli. (Yanofsky, RNA (2007),
13:1141-
1154). In some embodiments, the genetically engineered bacteria comprise the
tryptophan
operon of B. subtilis. (Yanofsky, RNA (2007), 13:1141-1154). In some
embodiments,
the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-
D, trypC-
F, trypB, and trpA genes. In some embodiments, the genetically engineered
bacteria
comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes
from E.
Coli. In some embodiments, the genetically engineered bacteria comprise
sequence(s)
encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from B. subtilis.
[0389] Also, in any of these embodiments, the genetically engineered bacteria
optionally comprise gene sequence(s) to produce the tryptophan precursor,
chorismate.
Thus, in some embodiments, the genetically engineered bacteria optionally
comprise
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sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequences
encoding one or more enzymes of the tryptophan biosynthetic pathway and one or
more
gene sequences encoding one or more enzymes of the chorismate biosynthetic
pathway.
In some embodiments, the genetically engineered bacteria comprise sequence(s)
encoding
trypE, trypG-D, trypC-F, trypB, and trpA genes from E. Coli and sequence(s)
encoding
aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes. In some embodiments,
the
genetically engineered bacteria comprise sequence(s) encoding trypE, trypD,
trypC, trypF,
trypB, and trpA genes from B. subtilis and sequence(s) encoding aroG, aroF,
aroH, aroB,
aroD, aroE, aroK, and AroC genes.
[0390] In some embodiments, the genetically engineered bacteria comprise
sequence(s) encoding either a wild type or a feedback resistant SerA gene
(Table 10).
Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes
the
first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis.
This step is
an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant
reduction
of NAD+ to NADH. As part of Tryptophan biosynthesis, E. coli uses one serine
for each
tryptophan produced. As a result, by expressing serA, tryptophan production is
improved.
[0391] In any of these embodiments, AroG and TrpE are optionally replaced with
feedback resistant versions to improve tryptophan production (Table 10
[0392] In any of these embodiments, the tryptophan repressor (trpR) optionally
may be deleted, mutated, or modified so as to diminish or obliterate its
repressor function.
[0393] In any of these embodiments the tnaA gene (encoding a tryptophanase
converting Trp into indole) optionally may be deleted to prevent tryptophan
catabolism
along this pathway and to further increase levels of tryptophan produced
(Table 10.
[0394] The inner membrane protein YddG of Escherichia coli, encoded by the
yddG
gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies
have
shown that YddG is capable of exporting aromatic amino acids, including
tryptophan.
Thus, YddG can function as a tryptophan exporter or a tryptophan secretion
system (or
tryptophan secretion protein). Other aromatic amino acid exporters are
described in
Doroshenko et al., FEMS Microbial Lett., 275:312-318 (2007). Thus, in some
embodiments,
the engineered bacteria optionally further comprise gene sequence(s) encoding
YddG. In some
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embodiments, the engineered bacteria can over-express YddG. In some
embodiments, the
engineered bacteria optionally comprise one or more copies of yddG gene.
[0395] In some embodiments, the genetically engineered bacterium or
genetically
engineered microorganism comprises one or more genes for producing tryptophan,
under
the control of a promoter that is activated by low-oxygen conditions, by
inflammatory
conditions, liver damage, and.or metabolic disease, such as any of the
promoters activated
by said conditions and described herein. In some embodiments, the genetically
engineered
bacteria expresses one or more genes for producing tryptophan. In some
embodiments, the
gene sequences(s) are controlled by an inducible promoter. In some
embodiments, the
gene sequences(s) are controlled by a constitutive promoter. In some
embodiments, the
gene sequences(s) are controlled by an inducible and/or constitutive promoter,
and are
expressed during bacterial culture in vitro, e.g., for bacterial expansion,
production and/or
manufacture, as described herein.
[0396] Table 9A and 9B lists exemplary tryptophan synthesis cassettes encoded
by the genetically engineered bacteria of the disclosure.
Table 9A. Tryptophan Synthesis Cassette Sequences
Description Sequence
Tet-regulated
taagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggccgaataagaaggctggctct
Tryptophan
gcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttct
operon
tctttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcatata
SEQ ID NO:
atgcattctctagtgaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcatactgtttttctgtagg
71
ccgtgtacctaaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaa
aaaatcttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcg
tcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacg
ggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagac
a
tcattaattcctaatttttgttgacactctatcattgatagagttattttaccactccctatcagtgatagagaaaagt
g
aactctagaaataattttgtttaactttaagaaggagatatacatatgcaaacacaaaaaccgactctcgaactgct
aacctgcgaaggcgcttatcgcgacaacccgactgcgctttttcaccagttgtgtggggatcgtccggcaacg
ctgctgctggaatccgcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgc
gcattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagccctgttgacactactg
gataacgccttgcctgcgggtgtggaaaatgaacaatcaccaaactgccgcgtactgcgcttcccgcctgtca
gtccactgctggatgaagacgcccgcttatgctccctttcggtttttgacgctttccgcttattacagaatctgttga
atgtaccgaaggaagaacgagaagcaatgttcttcggcggcctgttctcttatgaccttgtggcgggatttgaaa
atttaccgcaactgtcagcggaaaatagctgccctgatttctgtttttatctcgctgaaacgctgatggtgattgac
catcagaaaaaaagcactcgtattcaggccagcctgtttgctccgaatgaagaagaaaaacaacgtctcactgc
tcgcctgaacgaactacgtcagcaactgaccgaagccgcgccgccgctgccggtggtttccgtgccgcatat
gcgttgtgaatgtaaccagagcgatgaagagttcggtggtgtagtgcgtttgttgcaaaaagcgattcgcgccg
gagaaattttccaggtggtgccatctcgccgtttctctctgccctgcccgtcaccgctggcagcctattacgtgct
gaaaaagagtaatcccagcccgtacatgttttttatgcaggataatgatttcaccctgtttggcgcgtcgccggaa
agttcgctcaagtatgacgccaccagccgccagattgagatttacccgattgccggaacacgtccacgcggtc
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155vo5pum515m55o55p5ovp5155Eulo5ERrou55o5515Tr5oo5BETaaroTruo5oo
11515o55115m5m5B5oaro5m5p551E515EE55m5o55EarE5B5oRrol5o5B5o5oaro
Trar5o5B5Bmr5B555155armao5o555o5oamp5m5Eumo5Taum55515arar5
po5515v15EuEvE5E5155501151555o15o515oo5oo5aramaar5Tr000551E5B5o5
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low5o5oaruaruovo55B5o155ERro555551Tro5uroo5o5o5E55prE55E5EaTrui5uo
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5o55.ruar5m5o15oTrEEE5o5EBB5oarum5v5TruE1555E5aro55o55pro5oarol5E5
Ear5omp5oop5515m5o5pri55E5BoB5oarum5o5Truoo5u.ro5plauE5Troo55Tro5
po5o5vEB5Troo5o15Tru5o55o5o15oo5m5EE5Tr0005oo5ar5o55Eum55EuarB5Bo5
arommuar515oarEEE5EE55oararE55o55m551Truo5E55.roaroarlooaroapo5511
BauE5p5oar51Truovio5E5rEBEEE5o55ar5voularaoo5B5oTruar5oo5o5ararur
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BE5oo5p5155prE55ool5mum5155llrup5o55p5oo5ooTro5o55ooarullalTroar55
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Eu5oo5o5Bppom5p15o55EB5E5v55p5o55.roo5o5oTruuTr5oo5arE5vv5Bovul
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E5p5E5o5515ar155155o5uoup5p5maroo5EuE5Euarmo5upo5ar5Earo55.rovi5lo
EuRrE55pBE5oaruo5p5ararmarmo5E5upruauo5m5o555po551o5ouRrarE55
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Eum55aropolTruE5o555o55m155o15vp55555arpo5Eu5o15BE5o55.rovar55opo
511vo55lluivuoo5p5Euo5515o5Bo5ooaropoprE55oo5v151155oo5Eu5o5u5oo515
155000055polopp5v5p5155oovuo5E5Taar5o55po5arallumpar5m5o55oop
ETroaruo5oarmr515515arEvol55Truo5uo5o5B5uov5uo55paruari5ouppopr5ow
vuv5op5p5purar5p55Trupp5ar5E55Earo5TroTro5o5oaroo5urp5o5o5pri51
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BBEB55E155o5o55o55arlogro55o5ar5o15o155Earo5Eu5oo5BEEB5m5Trio5o5ou
15EuE5oaro5155o5Eup5ar5555vvam5po5o5oarpo5aro5poo5ar5olow5aroo5
o5p5E5155B5B5o5000pi5uparo5v515arpopuB5oar5B5ERroaropTr5oo5o15oul
o5oo5m55ooararo5mro5aro55par5vEl50005opv55155p5v5pwarapplo5E
5EuEvoTr5oom5o5v5E55praoTro5oo5Ear5opar5E5Ear55p5o1155v5oo515o15
099I0/LIOZSI1IIDd
Z6L9U/LIOZ OM
0-80-8TOZ OLLETOE0 VD
-Z6T-
o5oo5Ear5opar5E5Ear55p5o1155v5oo515o15o155o5arool5ararE55oo5BE5ooar
mr5E5Baroo5oo5uoaroo5ar5m5Rrop5oB5EuE55oo5o15o5o551115poaromr5Tr
Ev55.ro5vBBB5Troul50005moovul5E5EEEEE5p515arivioo5m551o5oarol50005
poo5plopm5oo5olovoo5155155.roopurEE5E55oo5o5our5o5EuRruo5B5m5o515
E15155155oB5E5EE5Tr5o5amarm5TrE515115o5Trvo5oo515oom55155oo5p5oo5o
o5o5oo5Eu5oar5pruo5uol5oularaaraloo5op5puolo15ourarEEEE5EauE5TrE5
oop5m5loo5uoo55Eolvi5opro5EEEEEEE5uoTroar5BE51551E5p5arEE5p5oplup
1115pmr5poo5p5uTrEEE55o5uol5pruo5oarmrEEE5Bv555o5515Boar5vpopp5
loo55o55opop5Truo5EE5E5arE5EE55Eu5oom5vE5B5pvE5Earivpo5oopp5ar51
BB55oppoop5vpo50005ar5Eav55p5prool5uol5po5000lp5o5pul5o5oo5pr
RroaroTruarE5vEuE55151555o5po5Boo5arEv55pularar5B5poo5EE5E55Truo55
oomo5o55.roovuouol5prou515Euuro5Earivo5o5p5o515Ear5E155p5p5po5Euu VL
Eumr5v5ERro5Ear5ovv5Eo5oovE55p5p5p5oRro55ool5ov55551515115.roarol :ON m Oas
Bpo5o5pr5ooaruar5o5ovip5o55Eu5o5parup5praolopaoarEERrarouRro5Tr .. actil
DDIDDDIVDDIIDOVVDDDIDVDDDDIDDDILLII
DDDDIVDDDVDIDDIVDDDOVVDVDDVVIIVVVDIVDDDVDDDID
VVVIVDDODDDDDVDDVDDOODDDIDDDVDDDDDODDVVDDVVD
DDIIDDVVOLLIVDDDDVDDODDODDIVVVDVDDVIDVDIDDIDI
DODVVOIDDDIDLLIDIIDIDIVILLIDDLLIDDDDDIDVDVVVDD
IDVDIDDOVVVDDVVVVIVVVDIVDDDVDDDIDVVDDDVIDVDV
DDDIVDDDDIDIDODDIDIDVIDDIVDDY5V55V055V050MuuB5u05
o5ar5o55o5uRr5Tr5oaruar151BORrapro55o551o5TuRraaroo5u5vulTuTraruo
5u5ovoTuRrup5ov oo55oB55pmr5o5o555o5p5u55u o5v5m5o5oo5u.rum55u ow
5500005oopv155w555m5uroopo5o5p5aruari5u5Rrap5uu5o5B55pTrovuol
000arp5o5oo5o5oouRrauo5o55oar515o555o5u5o5o15p5Bvioaroup5515o155ar
popo5uTr5uoo5o5p5mr5v5ar5oo5Tru5oo50005mumovioaro5o15vuvol5o5B5
o5oo55.roo5oop0005o5ool5u5uu5515000515v5oo5B55p5155olv5o15o55o15.rua
u5o515u5oo5vBB5u5v5ur o55.r.ruarum5155paruoo5m5v5ppoo55ov000pu oar
5ooaro5Ramo5ouropuo55p5v5u5mo515m5o550000m5u555o55o5mp515o5
praroo5oRrumpaar500055v55o55prooar5oopp0000vo55up5u55p5o5oalo
5155oo5uu5llrulo5ouv5ovivuuu5B5o15.ro5u5Buo555oov5155opoarol5oppop5
ow o5o55.r.r5uRro5o5u55.r.r5B5u0005m5plovu5ario5ouu551r5loTruu5555u5ou
o5u.r.r5Blvv5arop5oaropowar5uuuTr5o55o5o155oopparup55155prio5uo5u5u
Rruu55ooTruuu5o5o5v5Truur5p5o5Tr00055poo5ar000loovu55p5o55000voTr5
55.ravo5loo5Bp5ouRruppo5uu55poo5uu5Taar5oarivp1515oupap5o5ar55
pro5uaruolovi5o5Traruo500055o15ool5000mr55p555oo5plivoopupol5auu5
Buuro555ar5uu5oaruuo5v5Tr5oo5o5uRr5v155oBviovo55515o5o155TrouRrup5
oaro5o55aro5u5o55pruu5ov155aroo55o55po5u5515155Baloo55o15ar5oarua
aruovomr5uo5m5v155oTroo5v.r5o1155155o5511515po5ovp5oo5v55oo5po5o1
55.r.r5u5uRr55p1Tr5uo5o5uur5ouRr5uu5o551v5v55o5uom5u515o5151Troar5000
upooTro500055p5m5oar1555p5vviaro5o5oaruavp5m55oo155pr5o5ario5o
55u5vm5loo5v5Rrapoar5o5oop55o5uTrom55000v515.r.r55o51555vmp5o5Tr
55oolm5oRr0005o15.roo5arap5ar5uRroo51555viumraoo5v.rapo55op5poo
5o5mo5B00055o15o5515o55vo5m155oo5155oaruaoo5BEBERapruum555Tr55
o5u.r5o55pumo55m155op5155.roarupruuuTro5o5o55o55aro5p5mauu5o5o5u
apvi5p5aroararaar555oo5uarmararoo5Trumar5p5o5oarmol5o555oo5Tr
pruuRr5p5par5oRrop5uop55.romuu5pow5uuRruo5o5o5uol5ppo5u.r5uu551o5
uoo5o5pp5po5v5poTruuaroo515m5Tro55o551B5u5155mvi000arupoulTraruo
099I0/LIOZSI1IIDd
Z6L9U/LIOZ OM
0-80-8TOZ OLLETOE0 VD
-E6T-
155oloo55Bpol5aro5ar5ERrov1555oaruo5o15E5m5p5pllummapo555oo5o5p5
5oarEuaro55o5EE5515o5prulauo5o55Elp555555o55p5pB5oRro551p5ovuol5
5vmvpuo155pr5o1B5aruar55o5E555o555.ro155aruar5opm5vvEmE5oTrouo5
uoimr5o5o5o5000poprEE5155o1515Empo55EE55pTroo5o15araro5mo5plo55E5
15o5pwrou5ovvi5p5uov5Eu5vE155vo5pruo515.ro55o55p5ovp5155Eulo5uu
Ear55o5515Tr5oo5BETaaroTruo5oop515o55115v15m5B5oaro5m5p551E515EE55
uo5o55EarE5B5oRrol5o5B5o5oarowar5o5B5Bmr5B555155armao5o555o5ar5
vpo5uo5u.rup5Tr5E.ro55515arar5po5515v15EuEvE5E51555p5B51555o15o515o
o5oo5arapv5ar5Tr000551E5B5o55o1155llappp55arulo5oparoo5EEB5E5o5o5
155.rop5oupouvuoTro55ooTrao5uovE155ar515aruaro5555pruaoo5o5Bo5u5o
5ooari5oarEopiallaol5Bv5o5o5low5o5oaruaruovo55B5o155ERro555551Tro5
uroo5o5o5E55prE55E5Eavul5m15.raoar5p51515551E5E551o15Ear0005o15oo5
uo5Bo5mo5ovTruaraar5v55pri5uopp5vElvip5po5Tr5oo55.roarurio5o5o5
51ovpv5uoarpoov5ovuropar5Eum5vimr5oo5uo500005oTruuoo5uol5oTr0000
loomr5w5E5555.rommuur5E5v5pr5p515.ropwro55opo5arivarumumuoo5o
o5Buo5aro55oov5omr5v515oov515o55EuRrol5oo5o15o5EuRrum515E55pllum5 8L
o55aro5aro5o555.roparo5v5vpmEar5o5aro5u5oo5uop55E5Traumm5mo5510 :ON m Oas
5oaruo5uo5E5ERro5ooarEE5E15551BE5o55.ruar5uo5o15oTrEEE5o5EBB5oaruuo5Tr
Dd.11
Eu1555E5aro55o55pro5oarol5E5Ear5oupo5oop5515m5o5p
E155E5BoB5oaruuo5o5Truoo5u.ro5pw5Eu5voo55Tro5po5o5vEB5Troo5o15Tru5o
55o5o15oo5m5EaTr0005oo5ar5o55EuE155EuarB5Bo5ararupurar515oarEEE5Eu
55oararE55o55m551Truo5E55.roaroarlooaroapo55Bur5Eap5oar5uruovio5E
5EEBEEE5o55ar5Troularaoo5B5oTruar5oo5o5ararivom5EaTr551E555o55o5uo
uo515515m55o5o5oRrov15555015o5o5BoarEE5oo5llaoo5p5155prE55ool5E1
Em5155llrup5o55p5oo5ooTro5o55ooarupauroar555p515oRrop5ppro5ooarE
Eapruo5mo5o1155oo5Tr5o5Troo5oour55oararovi5Eu5oo5o5Bppom5p15o55
EB5E5Tr55p5o55.roo5o5oTrumaoo5aravv5Bovullm55oB5o55o55p5pv55o1
5o1o55oovuE15.roopi5o5E15oaruo55ararEE5o5515EuE5p555o5po55o5oo5o15111
5o515.roo5oomommuoTro5Earuo5m55ar5155o55pri55o15ovv5oo5m5pIEBE5
50005o5000lTroo5o5oo5arEEE55prio5o5oaruo5m5555oo5oTr5E5araooararal
55o5oBEEEE5Tro5E5155p5o55o55o55pruarE55oo5ualo5u5o5515ar155155o5uol
11151o5uoaroo5EuE5Euaruoo5upo5ar5Earo55.rovi5prEEEE55pBE5oaruo5p5aro
Emouroo5E5EprEauo5m5o555po55p5ouRrarE55p5po5op5o555.rooarprpo
uroovE55ooTroop5uomr55151115o5ov5uo5v5arol5o5155o551E5155vo55vEBB
moo5oRrovoarum55oo55oourom5m551155p5opuovp5o5o5515.roo5p5ooarE
EarEBE55oo5m5v5o55m155ar5Traralvo5E5o15o5uum55aropolTruE5o555o55
uo155o15vio55555arpo5Eu5o15BE5o55.roTrou55opo5Bvo55lluivuoo5p5Euo551
5o5Bo5ooaropoprE55oo5m5B55oo5Eu5o5u5oo515155000055poppp5v5p515 9L
5ooTruo5E5Taar5o55po5arallumpar5m5o55oopuTroaruo5oarmr515515armr :ON m Oas
o155Truo5uo5o5B5uoTr5uo55paruari5ouppopr5ovvEv5op5p5purar5p55Tr
ad.11
upp5ou
5E55Earo5TroTro5o5oaroo5llup5o5o5pri5p5o50005uumul5opuraar5oo5EE5
5o15m5oop5pllapool5E15o55p5155oo5u.ro515oaroo5ov155arEEE55155p5o55
op5oov5151vo5parar5olov5o55Tro5o5oarmvp55E155o5o55o55ario5m55o5
ar5o15o155Eauo5Eu5oo5BEEB5m5vio5o5oul5EuE5oaro5155o5EEB5ar5555vw
E5m5po5o5oarpo5aro5poo5ar5opTaaroo5o5p5E5155115115o5000pi5Eloaro5
v515arpopuB5oar5B5ERroaropTr5oo5o15ario5oo5m55ooararo5mro5aro5510
ar5vEl50005opv55155p5v5pvarE5pplo5E5EuEvoTr5oom5o5v5E55prE5ow
099I0/LIOZSI1IIDd
Z6L9U/LIOZ OM
0-80-8TOZ OLLETOE0 VD
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tttccagacgctgcgcgcatattaa
trpB
atgacaacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcg
SEQ ID NO:
ccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacctgctgaaaa
80
actatgccgggcgtccaaccgcgctgaccaaatgccagaacattacagccgggacgaacaccacgctgtatc
tgaagcgcgaagatttgctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcga
agcggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgcca
gcgccctgctcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaacgttttc
cggatgcgcttaatgggtgcggaagtgatcccggtacatagcggttccgcgaccctgaaagatgcctgtaatg
aggcgctacgcgactggtccggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatc
cttacccgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctggaaagaga
aggtcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttcatc
aacgaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaactggcgagcacggcgc
accgttaaaacatggtcgcgtgggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaa
attgaagagtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagc
actggacgcgctgattacgtgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaa
gggatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcgcgaaaatccggaa
aaagagcagctactggtggttaacctttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaa
gcacgaggggaaatctga
trpA
atggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcaccctc
SEQ ID NO:
ggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtgctgacgcgctggagtt
82
aggcatccccttctccgacccactggcggatggcccgacgattcaaaacgccacactgcgtgcttttgcggcg
ggagtaaccccggcgcagtgctttgagatgctggcactcattcgccagaagcacccgaccattcccatcggcc
ttttgatgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtgcgagaaagtcggcgtcga
ttcggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgcgttgcgtcataatgtcg
cacctatctttatttgcccgccgaatgccgacgatgatttgctgcgccagatagcctcttacggtcgtggttacac
ctatttgctgtcgcgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcg
aagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccggatcaggtaaaagccg
cgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgagcaacatattaatgagc
cagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcggcgacgcgcagttaa
Table 9B. Tryptophan Synthesis Polypeptide Sequences
Description Sequence
TrpE MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLL
SEQ ID NO: 75 LES ADIDS KDDLKSLLLVDS ALRITALSDTVTIQALSGN
GEALLTLLDNALPAGVENEQSPNCRVLRFPPVSPLLDE
DARLCS LS VFDAFRLLQNLLNVPKEEREAMFFGGLFS
YDLVAGFENLPQLSAENSCPDFCFYLAETLMVIDHQK
KS TRIQASLFAPNEEEKQRLT ARLNELRQQLTEAAPPL
PVVSVPHMRCECNQSDEEFGGVVRLLQKAIRAGEIFQ
VVPSRRFSLPCPSPLAAYYVLKKSNPSPYMFFMQDND
FTLFGASPESSLKYDATSRQIEIYPIAGTRPRGRRADGS
LDRDLDSRIELEMRTDHKELSEHLMLVDLARNDLARI
CTPGSRYVADLTKVDRYSYVMHLVSRVVGELRHDLD
ALHAYRACMNMGTLSGAPKVRAMQLIAEAEGRRRGS
YGGAVGYFTAHGDLDTCIVIRSALVENGIATVQAGAG
VVLDSVPQSEADETRNKARAVLRAIATAHHAQETF
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TrpD MADILLLDNIDSFTYNLADQLRSNGHNVVIYRNHIPAQ
SEQ ID NO: 77 TLIERLATMSNPVLMLSPGPGVPSEAGCMPELLTRLRG
KLPIIGICLGHQAIVEAYGGYVGQAGEILHGKASSIEHD
GQAMFAGLTNPLPVARYHSLVGSNIPAGLTINAHFNG
MVMAVRHDADRVCGFQFHPESILTTQGARLLEQTLA
WAQQKLEPTNTLQPILEKLYQAQTLS QQESHQLFS AV
VRGELKPEQLAAALVSMKIRGEHPNEIAGAATALLEN
AAPFPRPDYLFADIVGTGGDGSNSINISTASAFVAAAC
GLKVAKHGNRS VS S KS GS SDLLAAFGINLDMNADKSR
QALDELGVCFLFAPKYHTGFRHAMPVRQQLKTRTLF
NVLGPLINPAHPPLALIGVYSPELVLPIAETLRVLGYQR
AAVVHSGGMDEVSLHAPTIVAELHDGEIKSYQLTAED
FGLTPYHQEQLAGGTPEENRDILTRLLQGKGDAAHEA
AVAANVAMLMRLHGHEDLQANAQTVLEVLRS GS AY
DRVTALAARG
TrpC MQTVLAKIVADKAIWVETRKEQQPLASFQNEVQPSTR
SEQ ID NO: 79 HFYDALQGARTAFILECKKASPSKGVIRDDFDPARIAA
IYKHYASAISVLTDEKYFQGSFDFLPIVSQIAPQPILCK
DFIIDPYQIYLARYYQADACLLMLSVLDDEQYRQLAA
VAHSLEMGVLTEVSNEEELERAIALGAKVVGINNRDL
RDLSIDLNRTRELAPKLGHNVTVISESGINTYAQVREL
SHFANGFLIGSALMAHDDLNAAVRRVLLGENKVCGL
TRGQDAKAAYDAGAIYGGLIFVATSPRCVNVEQAQE
VMAAAPLQYVGVFRNHDIADVADKAKVLSLAAVQL
HGNEDQLYIDNLREALPAHVAIWKALSVGETLPARDF
QHIDKYVFDNGQGGSGQRFDWSLLNGQSLGNVLLAG
GLGADNCVEAAQTGCAGLDFNSAVESQPGIKDARLL
AS VFQTLRAY
TrpB MTTLLNPYFGEFGGMYVPQILMPALRQLEEAFVSAQK
SEQ ID NO: 81 DPEFQAQFNDLLKNYAGRPT ALT KCQNITAGTNTTLY
LKREDLLHGGAHKTNQVLGQALLAKRMGKTEIIAET
GAGQHGVAS ALAS ALLGLKCRIYMGAKDVERQSPNV
FRMRLMGAEVIPVHS GS ATLKDACNEALRDWS GS YE
TAHYMLGTAAGPHPYPTIVREFQRMIGEETKAQILERE
GRLPDAVIACVGGGSNAIGMFADFINETDVGLIGVEPG
GHGIETGEHGAPLKHGRVGIYFGMKAPMMQTEDGQI
EESYSISAGLDFPSVGPQHAYLNSTGRADYVSITDDEA
LEAFKTLCLHEGIIPALESSHALAHALKMMRENPEKEQ
LLVVNLSGRGDKDIFTVHDILKARGEI
TrpA MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIID
SEQ ID NO: 83 TLIEAGADALELGIPFSDPLADGPTIQNATLRAFAAGV
TPAQCFEMLALIRQKHPTIPIGLLMYANLVFNKGIDEF
YAECEKVGVDSVLVADVPVEESAPFRQAALRHNVAPI
FICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENR
AALPLNHLVAKLKEYNAAPPLQGFGISAPDQVKAAID
AGAAGAIS GS AIVKIIEQHINEPEKMLAALKAFVQPMK
AATRS
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[0397] In some embodiments, the genetically engineered bacteria comprise one
or
more nucleic acid sequence of Table 9A or a functional fragment thereof. In
some
embodiments, the genetically engineered bacteria comprise a nucleic acid
sequence that,
but for the redundancy of the genetic code, encodes the same polypeptide as
one or more
nucleic acid sequence of Table 9B or a functional fragment thereof. In some
embodiments, genetically engineered bacteria comprise a nucleic acid sequence
that is at
least about 80%, at least about 85%, at least about 90%, at least about 95%,
or at least
about 99% homologous to the DNA sequence of one or more nucleic acid sequence
of
Table 9A or a functional fragment thereof, or a nucleic acid sequence that,
but for the
redundancy of the genetic code, encodes the same polypeptide as one or more
nucleic acid
sequence of Table 9B or a functional fragment thereof.
[0398] In one embodiment, one or more polypeptides and/or polynucleotides
encoded and expressed by the genetically engineered bacteria have at least
about 80%
identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. In one
embodiment, one or more polypeptides and/or polynucleotides encoded and
expressed by
the genetically engineered bacteria have at least about 85% identity with one
or more of
SEQ ID NO: 71 through SEQ ID NO: 83. In one embodiment, one or more
polypeptides and/or polynucleotides encoded and expressed by the genetically
engineered
bacteria have at least about 90% identity with one or more of SEQ ID NO: 71
through
SEQ ID NO: 83. In one embodiment, one or more polypeptides and/or
polynucleotides
encoded and expressed by the genetically engineered bacteria have at least
about 95%
identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. In one
embodiment, one or more polypeptides and/or polynucleotides encoded and
expressed by
the genetically engineered bacteria have have at least about 96%, 97%, 98%, or
99%
identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. Accordingly,
in
one embodiment, one or more polypeptides and/or polynucleotides expressed by
the
genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%,
85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. In another
embodiment, one or more polynucleotides and/or polypeptides encoded and
expressed by
the genetically engineered bacteria comprise the sequence of one or more of
SEQ ID NO:
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71 through SEQ ID NO: 83. In another embodiment, one or more polynucleotides
and/or polypeptides encoded and expressed by the genetically engineered
bacteria consist
of the sequence of one or more of SEQ ID NO: 71 through SEQ ID NO: 83.
[0399] Table 10A depicts exemplary polypeptide sequences feedback resistant
AroG and TrpE. Table 10A also depicts an exemplary TnaA (tryptophanase from E.
coli)
sequence. IN some embodiments, the sequence is encoded in circuits for
tryptophan
catabolism to indole; in other embodimetns, the sequence is deleted from the E
coli
chromosome to increase levels of tryptophan.
Table 10A. Feedback resistant AroG and TrpE and tryptophanase sequences
Description Sequence
AroGfbr: feedback MNYQNDDLRIKEIKELLPPVALLEKFPATENAANTVAHARKAI
resistant 2-dehydro- HKILKGNDDRLLVVIGPCSIHDPVAAKEYATRLLTLREELQDE
3- LEIVMRVYFEKPRTTVGWKGLINDPHMDNSFQINDGLRIARK
deo xypho sphohept LLLDINDS GLPAAGEFLDMITLQYLADLMSWGAIGARTTES Q
o nate aldo las e from VHRELAS GLSCPVGFKNGTDGTIKVAIDAINAAGAPHCFLS VT
E. coli KWGHS AIVNTS GNGDCHIILRGGKEPNYSAKHVAEVKEGLNK
AGLPAQVMIDFS HANS S KQFKKQMDVCTDVCQQIAGGEKAII
SEQ ID NO: 84 GVMVESHLVEGNQSLESGEPLAYGKSITDACIGWDDTDALLR
QLAS AVKARRG
TrpEtbr: feedback MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLLLEFADI
resistant DS KDDLKSLLLVDS ALRIT ALS DTVTIQALS GNGEALLTLLDN
anthranilate ALPAGVENE QS PNCRVLRFPPVS PLLDEDARLC S LS VFDAFRL
synthase LQNLLNVPKEEREAMFFGGLFSYDLVAGFENLPQLS AENS CP
component I from DFCFYLAETLMVIDHQKKSTRIQASLFAPNEEEKQRLTARLNE
E. coli LRQQLTEAAPPLPVVS VPHMRCECNQS DEEFGGVVRLLQ KAI
RAGEIFQVVPSRRFSLPCPSPLAAYYVLKKSNPSPYMFFMQDN
SEQ ID NO: 85 DFTLFGASPES S LKYD AT S RQIEIYPIAGTRPRGRRADGS LDRD
LDSRIELEMRTDHKELSEHLMLVDLARNDLARICTPGSRYVA
DLTKVDRYSYVMHLVSRVVGELRHDLDALHAYRACMNMGT
LS GAPKVRAMQLIAEAEGRRRGSYGGAVGYFTAHGDLDTCIV
IRS ALVENGIATVQAGAGVVLDS VPQSEADETRNKARAVLRA
IATAHHAQETF
SerA: 2- MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGAL
oxoglutarate DDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGT
reductase from E. NQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPE
coli Nissle ANAKAHRGVWNKLAAGS FE ARGKKLGIIGYGHIGT QLGILAE
SLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSDVVSLHVPE
SEQ ID NO: 86 NPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASK
HLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEA
QENIGLEVAGKLIKYSDNGSTLS AVNFPEVSLPLHGGRRLMHI
HENRPGVLTALNKIFAEQGVNIAAQYLQTS AQMGYVVIDIEA
DEDVAEKALQAMKAIPGTIRARLLY
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SerAfbr: feedback MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGAL
resistant 2- DDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGT
oxoglutarate NQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPE
reductase from E. ANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAE
coli Nissle SLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSDVVSLHVPE
NPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASK
SEQ ID NO: 87 HLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEA
QENIGLEVAGKLIKYSDNGSTLSAVNFPEVSLPLHGGRRLMHI
AEARPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEA
DEDVAEKALQAMKAIPGTIRARLLY
TnaA: MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSE
tryptophanase from DVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAE
E. coli SVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRS KM
VAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGN
SEQ ID NO: 88 FDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKVM
YSIAKKYDIPVVMDSARFAENAYFIKQREAEYKDWTIEQITRE
TYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFDVYTECRT
LCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIA
QVQYLVDGLEEIGVVCQQAGGHAAFVDAGKLLPHIPADQFPA
QALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRLTI
PRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRHFT
AKLKEV
Table 10B.
fbrAroG
atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg
cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga
SEQ ID NO: 256 aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca
tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg
aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta
cggtgggctggaaagggctgattaacgatccgcatatggataacagcttccagatcaac
gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg
gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc
gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc
ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta
atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt
gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa
ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc
agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat
ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt
gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct
ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt
acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaa
fbrTrpE atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaa
cccgactgcgctttttcaccagttgtgtggggatcgtccggcaacgctgctgctggaattc
SEQ ID NO: 274 gcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgcg
cattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagccct
-198-
TE 555o 5o OOOE 51E 551OTE 5115 buomuuouu 5501E1510111510551
Imo 5015 51oBEEEE buoboo5oEuom 515ou 5EE 51ou 5101E000E150001E 5o
51005501E0110E00051E 5o boomooluu 5BEEETTEEouu 51E 51E 5510505 5
5EuEouoluEE boluouuoouou11551o5uo515olloo5BEE 5510 5o 55EuEuoo OISI :ON m OHS
Bo 5150 5 5EE 5E15510 51oul 5EETTE 5Euou 5BEE 5E 5510 bolul 5 5EuEo 551E
KIJIvuoS
Eularl5p5p150005o5ouroar1555oourio5Euu
5Truo55.ro5p5o5EEEEE5oo5115ar5Eu5ar5oo5Eu5lluv5BEB5E15Tr
B555Tamoo5oopouRroupTuTruo5o5oo5oTraruoi5o555m5E5oo5
BmurEuarEop5o5prup515o555ool5oarEEE5arooTraro5v5p15o
15o555155aro5proo5p5opi5EE55000parE5155o5pplo5oRrolo5
5Truar5puri5Euov5BEEE1555o5B5EE55105oTuTrE5E55.ro5o5uu
55Eopr5oB55o551Trararoo5ar5lopool5Truar5ollrE51515p5oopi
oarmrooTr5o5uvuoar5o55oarE55ar5000lvi5ar5oTruo55o55555
o551oTrouRro5u5o551o5o5ar515151o5o5molvv5515515pri55o5o
5o1p5vEur5p5p5olo550005EE5Trup5omr5E5EuE5o5o555v5Tr
TruuRroarool5ooTrE5E5Eoari5vo5p15E515515w5o5E5vvE5p5lo
ar5loppluo5rou155.roproo5oRro5551o5oo5pruuarEEE5lluv5vp
Barm5v15TrE555p5oTru5p551olvo555llruo5ar155lluvol55ario
55ovov15551o5EuRruo55o5o5o5EampB555o55o55prEuarE55
1515o5515oTro5o5uRrio5Truoo5Earoo515o55o5o5urp5p5p5pr
E5o551v5155p5E55o5B5plo5o5ouTruEopp5oaro5arumr155000
Tr555o5o5uRro55o55o5v551ov51155.roTruuarE55ovi5p1B5p551
vp5o155prEEEE5m5oo5oRrov515ar5Eapalowooari5000Tr5o
5loo55ovolpr0005v5o5ooTrooTrauuumvuarav5v551o5o5o5
5uRraromraoTrarmaroup551o5m515olloo5EuE551o5o55EuRroo gsz :0N m oas
uo515o55EE5E155p5p1B5EulTr5Euar5EuE5E55p5ov155ERro55Tr 100S
Elo
115ar5E55Epro5TroTro5o5oaroo5llup5o5o5pri5p5o50005EEETru
15opruaar5oo5EE55o15m5oop5lopapool5E15o551o5155oo5u.ro
515oaroo5ov155arEEE55155p5o55olo5oov5151vo5loarar5olow
5o55Tro5o5oarmvp55E155o5o55o55ario5m55o5ar5o15o155EE5
uo5Eu5oo5BEEB5m5vio5o5oul5EuE5oaro5155o5EEB5ar5555vw
avi5loo5o5oarpo5aro5l0005ar5olow5aroo5o5p5E5155115115o5
000pi5Eloaro5v515omplarp5oar5B5ERroarolov5oo5o15ario5o
o5m55ooararo5mro5aro55loar5vEl50005olov551551o5v5low
arapplo5E5EuEvoTr5oari5o5v5E55praoTro5oo5Ear5oloar5E
5Ear55p5o1155v5oo515o15o155o5arool5ararE55oo5BE5000mpu
5E51Tamo5oo5uoaroo5ar5v15E.rop5oB5EuE55oo5o15o5o5511151
ooaroBv5TruTr55.ro5vBBB5Troul50005u000vE15E5EEEEE5p515o
Eurloo5m55p5oarol50005poo5plopm5oo5olowoo5155155.roop
BEEE5E55oo5o5our5o5EuRruo5B5m5o515E15155155oB5E5EE5Tr5
o5E5uoarui5vE515115o5vvo5oo515oom55155oo5p5oo5oo5o5oo
5Eu5oar5pruo5uoi5oularE5arE5loo5olo5prolo15aruarEEEE5EE5
EE5Tru5oolo5B151oo5uoo55.rolvi5opro5EEEEEEE5uovoar5BE515
5v5p5oRrap5oloviBB5lopv5poo5p5ETrEEE55o5uol5pruo5o
armrEEE5mr555o5515Boar5vpolop5po55o55opop5Truo5EE5E
5arE5EE55Eu5oari5vE5B5loTrE5Earivip5oopp5ar5B1B55oppo
op5vpo50005ar5Eav551o5prool5m151oo5000lp5o5pri5o5oo
51ouRroaroTruarE5vEuE55151555o5loo5Boo5arEv55priarar5B5
099I0/LIOZSI1IIDd
Z6L9U/LIOZ OM
0-80-8TOZ OLLETOE0 VD
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cccggtatttaacgcaccgttctcaaatacgcgctctgttgcgg agctggtg attggcg aa
ctgctgctgctattgcgcggcgtgccag aagccaatgctaaagcgcatcgtggcgtgtgg
aacaaactggcggcgggttcttttg aagcgcgcggcaaaaagctgggtatcatcggcta
cggtcatattggtacgcaattgggcattctggctg aatcgctggg aatgtatgtttacttttatg
atattg aaaacaaactgccgctgggcaacgccactcaggtacagcatctttctg acctgc
tg aatatg agcg atgtggtg agtctgcatgtaccag ag aatccgtccaccaaaaatatg a
tgggcgcg aaag ag atttcgctaatg aagcccggctcgctgctg attaatgcttcgcgcg
gtactgtggtgg atattccagcgctgtgtg acgcgctggcg agcaaacatctggcgggg
gcggcaatcg acgtattcccg acgg aaccggcg accaatagcg atccatttacctctcc
gctgtgtg aattcg acaatgtccttctg acgccacacattggcggttcg actcagg aagcg
cagg ag aatatcggcttgg aagttgcgggtaaattg atcaagtattctg acaatggctcaa
cgctctctgcggtg aacttcccgg aagtctcgctgccactgcacggtgggcgtcgtctg at
g cacatcGCTg aaGCTcg tccgggcg tgctaactgcgctcaacaaaatttttgccg a
gcagggcg tcaacatcgccgcgcaatatctacaaacttccgcccag atggg ttatg tag t
tattgatattgaagccgacgaagacgttgccgaaaaagcgctgcaggcaatgaaagct
attccgggtaccattcgcgcccgtctgctgtactaa
[0400] In one embodiment, the genetically engineered bacteria comprise a
sequence which has at least about 80% identity with one or more sequences of
Table 10B.
In another embodiment, the genetically engineered bacteria comprise a sequence
which
has at least about 85% identity with one or more sequences of Table 10B. In
one
embodiment, the genetically engineered bacteria comprise a sequence which has
at least
about 90% identity with one or more sequences of Table 10B. In one embodiment,
the
genetically engineered bacteria comprise a sequence which has at least about
95% identity
with one or more sequences of Table 10B. In another embodiment, the
genetically
engineered bacteria comprise a sequence which has at least about 96%, 97%,
98%, or 99%
identity with one or more sequences of Table 10B. Accordingly, in one
embodiment, the
genetically engineered bacteria comprise a sequence which has at least about
80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity with one or more sequences of Table 10B. In yet
another
embodiment the genetically engineered bacteria comprise a sequence which
consists of the
sequence of with one or more sequences of Table 10B.
[0401] In one embodiment, the genetically engineered bacteria comprise a
sequence which has at least about 80% identity with SEQ ID NO: 256. In another
embodiment, the genetically engineered bacteria comprise a sequence which has
at least
about 85% identity with SEQ ID NO: 256. In one embodiment, the genetically
engineered bacteria comprise a sequence which has at least about 90% identity
with SEQ
ID NO: 256. In one embodiment, the genetically engineered bacteria comprise a
sequence
which has at least about 95% identity with SEQ ID NO: 256. In another
embodiment, the
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bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:
256.
Accordingly, in one embodiment, the genetically engineered bacteria comprise a
sequence
which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 256.
In
another embodiment, the genetically engineered bacteria comprise the sequence
of SEQ
ID NO: 256. In yet another embodiment the genetically engineered bacteria
comprise a
sequence which consists of the sequence of SEQ ID NO: 256.
[0402] In one embodiment, one or more polypeptides and/or polynucleotides
encoded and expressed by the genetically engineered bacteria have at least
about 80%
identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. In one
embodiment, one or more polypeptides and/or polynucleotides encoded and
expressed by
the genetically engineered bacteria have at least about 85% identity with one
or more of
SEQ ID NO: 84 through SEQ ID NO: 87. In one embodiment, one or more
polypeptides and/or polynucleotides encoded and expressed by the genetically
engineered
bacteria have at least about 90% identity with one or more of SEQ ID NO: 84
through
SEQ ID NO: 87. In one embodiment, one or more polypeptides and/or
polynucleotides
encoded and expressed by the genetically engineered bacteria have at least
about 95%
identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. In one
embodiment, one or more polypeptides and/or polynucleotides encoded and
expressed by
the genetically engineered bacteria have have at least about 96%, 97%, 98%, or
99%
identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. Accordingly,
in
one embodiment, one or more polypeptides and/or polynucleotides expressed by
the
genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%,
85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. In another
embodiment, one or more polynucleotides and/or polypeptides encoded and
expressed by
the genetically engineered bacteria comprise the sequence of one or more of
SEQ ID NO:
84 through SEQ ID NO: 87. In another embodiment, one or more polynucleotides
and/or polypeptides encoded and expressed by the genetically engineered
bacteria consist
of the sequence of one or more of SEQ ID NO: 84 through SEQ ID NO: 87.
[0403] In some embodiments, the endogenous TnaA polypeptide comprising SEQ
ID NO: 88 is mutated or deleted.
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[0404] To improve acetate production, while maintaining high levels of
tryptophan
production, targeted one or more deletions can be introduced in competing
metabolic arms
of mixed acid fermentation to prevent the production of alternative metabolic
fermentative
byproducts (thereby increasing acetate production). Non-limiting examples of
competing
such competing metabolic arms are frdA (converts phosphoenolpyruvate to
succinate),
ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol).
Deletions
which may be introduced therefore include deletion of adhE, ldh, and frd.
Thus, in certain
embodiments, the genetically engineered bacteria comprise one or more
tryptophan
production cassette(s) and further comprise mutations and/or deletions in one
or more of
frdA, ldhA, and adhE.
[0405] In some embodiments, the genetically engineered bacteria comprise one
or
more tryptophan production cassette(s) described herein and one or more
mutation(s)
and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA
gene and
the adhE gene.
[0406] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) encoding one or more enzymes described herein for the
production
of tryptophan and further comprise a mutation and/or deletion in one or more
endogenous
genes selected from in the ldhA gene, the frdA gene and the adhE genes. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes described herein for the production of tryptophan
and
further comprise a mutation and/or deletion in the endogenous ldhA gene. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes described herein for the production of tryptophan
and
further comprise a mutation and/or deletion in the endogenous adhE gene. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes described herein for the production of tryptophan
and
further comprise a mutation and/or deletion in the endogenous frdA gene. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes described herein for the production of tryptophan
and
further comprise a mutation and/or deletion in the endogenous ldhA and rdA
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
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tryptophan and further comprise a mutation and/or deletion in the endogenous
ldhA genes
and adhE genes. In some embodiments, the genetically engineered bacteria
comprise one
or more gene sequence(s) encoding one or more enzymes described herein for the
production of tryptophan and further comprise a mutation and/or deletion in
the
endogenous frdA and adhE genes. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) encoding one or more enzymes
described
herein for the production of tryptophan and further comprise a mutation and/or
deletion in
the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the
genetically
engineered bacteria comprise one or more gene sequence(s) encoding one or more
enzymes described herein for the production of tryptophan and further comprise
a
mutation and/or deletion in one or more endogenous genes selected from in the
ldhA gene,
the frdA gene and the adhE genes.
[0407] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and
AtrpR,
AtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA
gene. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA,
and
further comprise a mutation and/or deletion in the endogenous adhE gene. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further
comprise a mutation and/or deletion in the endogenous frdA gene. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
selected from
trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a
mutation
and/or deletion in the endogenous ldhA and frdA genes. In some embodiments,
the
genetically engineered bacteria comprise one or more gene sequence(s) selected
from
trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a
mutation
and/or deletion in the endogenous ldhA genes and adhE genes. In some
embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s) selected
from
trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a
mutation
and/or deletion in the endogenous frdA and adhE genes. In some embodiments,
the
genetically engineered bacteria comprise one or more gene sequence(s) selected
from
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trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a
mutation
and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
[0408] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more acetate than unmodified bacteria of the same
bacterial subtype
under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the the genetically engineered bacteria
produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than
unmodified
bacteria of the same bacterial subtype under the same conditions.
[0409] In some embodiments, the genetically engineered bacteria produce 0% to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more tryptophan than unmodified bacteria of the same
bacterial
subtype under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more tryptophan than unmodified bacteria of the same bacterial subtype
under the
same conditions. In yet another embodiment, the the genetically engineered
bacteria
produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold,
nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more
tryptophan than
unmodified bacteria of the same bacterial subtype under the same conditions.
[0410] In certain situations, the need may arise to prevent and/or reduce
acetate
production by of an engineered or naturally occurring strain, e.g., E. coli
Nissle, while
maintaining high levels of tryptophan production. Without wishing to be bound
by theory,
one or more mutations and/or deletions in one or more gene(s) encoding in one
or more
enzymes described herein which function in the acetate producing metabolic arm
of
fermentation should reduce and/or prevent production of acetate. A non-
limiting example
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of such an enzyme is phosphate acetyltransferase (Pta), which is the first
enzyme in the
metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of
the Pta gene
or a gene encoding another enzyme in this metabolic arm may also allow for
more acetyl-
CoA to be used for tryptophan production. Additionally, one or more mutations
preventing
or reducing the flow through other metabolic arms of mixed acid fermentaion,
such as
those which produce succinate, lactate, and/or ethanol can increase the
production of
acetyl-CoA, which is available for tryptophan synthesis. Such mutations and/or
deletions,
include but are not limited to mutations and/or deletions in the frdA, ldhA,
and/or adhE
genes.
[0411] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) encoding one or more enzymes described herein for the
production
of tryptophan and further comprise a mutation and/or deletion in the
endogenous pta gene.
In some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
tryptophan and further comprise a mutation and/or deletion in the endogenous
pta gene
and in one or more endogenous genes selected from in the ldhA gene, the frdA
gene and
the adhE gene. In some embodiments, the genetically engineered bacteria
comprise one or
more gene sequence(s) encoding one or more enzymes described herein for the
production
of tryptophan and further comprise a mutation in the endogenous pta and adhE
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
tryptophan and further comprise a mutation in the endogenous pta and ldhA
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
tryptophan and further comprise a mutation in the endogenous pta and frdA
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
tryptophan and further comprise a mutation and/or deletion in the endogenous
pta, ldhA
and frdA genes. In some embodiments, the genetically engineered bacteria
comprise one
or more gene sequence(s) encoding one or more enzymes described herein for the
production of tryptophan and further comprise a mutation in the endogenous
pta, ldhA,
and adhE genes. In some embodiments, the genetically engineered bacteria
comprise one
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or more gene sequence(s) encoding one or more enzymes described herein for the
production of tryptophan and further comprise a mutation in the endogenous
pta, frdA and
adhE genes. In some embodiments, the genetically engineered bacteria comprise
one or
more gene sequence(s) encoding one or more enzyme(s) for the production of
tryptophan
and further comprise a mutation and/or deletion in the endogenous pta, ldhA,
frdA, and
adhE genes.
[0412] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and
AtrpR,
AtnaA, and further comprise a mutation and/or deletion in the endogenous pta
gene. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA,
and
further comprise a mutation and/or deletion in the endogenous pta gene and in
one or more
endogenous genes selected from in the ldhA gene, the frdA gene and the adhE
gene. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA,
and
further comprise a mutation in the endogenous pta and adhE genes. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
selected from
trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a
mutation
in the endogenous pta and ldhA genes.
[0413] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and
AtrpR,
AtnaA, and further comprise a mutation in the endogenous pta and frdA genes.
In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further
comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA
genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further
comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
selected from
trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a
mutation
in the endogenous pta, frdA and adhE genes. In some embodiments, the
genetically
engineered bacteria comprise one or more gene sequence(s) selected from
trpEfbr,
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trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation in
the
endogenous pta, ldhA, frdA, and adhE genes.
[0414] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% less acetate than unmodified bacteria of the same
bacterial subtype
under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold less acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the genetically engineered bacteria
produce three-
fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-
fold, fifteen-fold,
twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than
unmodified bacteria of
the same bacterial subtype under the same conditions.
[0415] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more tryptophan than unmodified bacteria of the same
bacterial
subtype under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more tryptophan than unmodified bacteria of the same bacterial subtype
under the
same conditions. In yet another embodiment, the genetically engineered
bacteria produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptophan
than unmodified
bacteria of the same bacterial subtype under the same conditions.
[0416] In some embodiments, the genetically engineered bacteria are capable of
expressing any one or more of the described circuits in low-oxygen conditions,
in the
presence of disease or tissue specific molecules or metabolites, in the
presence of
molecules or metabolites associated with inflammation or an inflammatory
response or
immune suppression, or in the presence of some other metabolite that may or
may not be
present in the gut, such as arabinose and others described herein. In some
embodiments,
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the gene sequences(s) are controlled by a constitutive promoter. In some
embodiments, the
gene sequences(s) are controlled by an inducible and/or constritutive
promoter, and are
expressed during bacterial culture in vitro, e.g., for bacterial expansion,
production and/or
manufacture, as described herein.
[0417] n some embodiments, any one or more of the described circuits are
present
on one or more plasmids (e.g., high copy or low copy) or are integrated into
one or more
sites in the bacterial chromosome. Also, in some embodiments, the genetically
engineered
bacteria are further capable of expressing any one or more of the described
circuits and
further comprise one or more of the following: (1) one or more auxotrophies,
such as any
auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2)
one or more
kill switch circuits, such as any of the kill-switches described herein or
otherwise known
in the art, (3) one or more antibiotic resistance circuits, (4) one or more
transporters for
importing biological molecules or substrates, such any of the transporters
described herein
or otherwise known in the art, (5) one or more secretion circuits, such as any
of the
secretion circuits described herein and otherwise known in the art, and (6)
combinations of
one or more of such additional circuits.
Producing Kynurenic Acid
[0418] In some embodiments, the genetically engineered bacteria are capable of
producing kynurenic acid. Kynurenic acid is produced from the irreversible
transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine-
oxoglutarate transaminase. Kynurenic acid acts as an antagonist of ionotropic
glutamate
receptors (Turski et al., 2013). While glutamate is known to be a major
excitatory
neurotransmitter in the central nervous system, there is now evidence to
suggest an
additional role for glutamate in the peripheral nervous system. For example,
the activation
of NMDA glutamate receptors in the major nerve supply to the GI tract (i.e.,
the myenteric
plexus) leads to an increase in gut motility (Forrest et al., 2003), but rats
treated with
kynurenic acid exhibit decreased gut motility and inflammation in the early
phase of acute
colitis (Varga et al., 2010). Thus, the elevated levels of kynurenic acid
reported in IBD
patients may represent a compensatory response to the increased activation of
enteric
neurons (Forrest et al., 2003). The genetically engineered bacteria may
comprise any
suitable gene or genes for producing kynurenic acid. In some embodiments, the
engineered bacteria comprise gene sequence(s) encoding one or more kynurenine-
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oxoglutarate transaminases (also referred to as kynurenine aminotransferases
(e.g., KAT I,
II, III)).
[0419] In some embodiments, the gene or genes for producing kynurenic acid is
modified and/or mutated, e.g., to enhance stability, increase kynurenic acid
production
under inducing conditions. In some embodiments, the genetically engineered
bacteria are
capable of producing kynurenic acid under inducing conditions, e.g., under a
condition(s)
associated with inflammation. In some embodiments, the genetically engineered
bacteria
are capable of producing kynurenic acid in low-oxygen conditions, in the
presence of
certain molecules or metabolites, in the presence of molecules or metabolites
associated
with inflammation or an inflammatory response, or in the presence of some
other
metabolite that may or may not be present in the gut, such as arabinose. In
some
embodiments, the gene sequences(s) are controlled by a constitutive promoter.
In some
embodiments, the gene sequences(s) are controlled by an inducible promoter. In
some
embodiments, the gene sequences(s) are controlled by an inducible and/or
constitutive
promoter, and are expressed during bacterial culture in vitro, e.g., for
bacterial expansion,
production and/or manufacture, as described herein.
[0420] In some embodiments, the genetically engineered bacteria comprising one
or more gene(s) or gene cassette(s) can alter the TRP:KYNA ratio, e.g. in the
circulation.
In some embodiments the TRP:KYNA ratio is increased. In some embodiments,
TRP:KYNA ratio is decreased.
[0421] In some embodiments, the genetically engineered bacteria comprise one
or
more gene(s) or gene cassette(s) for the consumption of tryptophan and
production of
kynurenic acid, which are bacterially derived. In some embodiments, the
enzymes for
producing kynureic acid are derived from one or more of Pseudomonas,
Xanthomonas,
Burkholderia, Stenotrophomonas, Shewanella, and Bacillus, and/or members of
the
families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some
embodiments
the enzymes are derived from the species listed in table S7 of Vujkovic-Cvijin
et al.
(Dysbiosis of the gut microbiota is associated with HIV diseaseprogression and
tryptophan
catabolism Sci Transl Med. 2013 July 10; 5(193): 193ra91), the contents of
which is
herein incorporated by reference in its entirety.
[0422] In some embodiments, the genetically engineered bacteria comprise gene
sequence(s) encoding one or more tryptophan transporters and gene sequence(s)
encoding
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kynureninase. In some embodiments, the genetically engineered bacteria
comprise gene
sequence(s) encoding one or more tryptophan transporters and gene sequence(s)
encoding
one or more kynurenine-oxoglutarate transaminases (kynurenine
aminotransferases). In
some embodiments, the genetically engineered bacteria comprise gene
sequence(s)
encoding one or more tryptophan transporters, gene sequence(s) encoding
kynureninase,
and gene sequence(s) encoding one or more kynurenine-oxoglutarate
transaminases
(kynurenine aminotransferases). In some embodiments, the genetically
engineered bacteria
comprise gene sequence(s) encoding kynureninase and gene sequence(s) encoding
one or
more kynurenine aminotransferases.
[0423] In one embodiment, the genetically engineered bacteria comprise one or
more gene sequence(s) which encode one or more tryptophan catabolism enzymes,
which
produce kynurenic acid from tryptophan. Non-limiting example of such gene
sequence(s)
are shown in the figures and described elsewhere herein. In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
ID01(indoleamine 2,3-dioxygenase). In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode IDO1 from homo
sapiens.
In one embodiment, the genetically engineered bacteria comprise one or more
gene
sequence(s) which encode TD02 (tryptophan 2,3-dioxygenase). In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
TD02 from homo sapiens. In one embodiment, the genetically engineered bacteria
comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-
dioxygenase). In one embodiment, the genetically engineered bacteria comprise
one or
more gene sequence(s) which encode BNA2 from S. cerevisiae). In one
embodiment, the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
Afmid: Kynurenine formamidase. In one embodiment, the genetically engineered
bacteria
comprise one or more gene sequence(s) which encode Afmid: Kynurenine
formamidase
from mouse. In one embodiment, the genetically engineered bacteria comprise
one or
more gene sequence(s) which encode Afmid in combination with one or more of
idol
and/or tdo2 and/or bna2. In one embodiment, the genetically engineered
bacteria comprise
one or more gene sequence(s) which encode Afmid in combination with ID01. In
one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode BNA2 in combination with TD02. In one embodiment, the genetically
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engineered bacteria comprise one or more gene sequence(s) which encode Afmid
in
combination with bna2. In one embodiment, the genetically engineered bacteria
further
comprise one or more gene sequence(s) which encode cclbl and/or cc1b2 and/or
aadat
and/or got2.In one embodiment, the genetically engineered bacteria comprise
one or more
gene sequence(s) which encode BNA3 (kynurenine--oxoglutarate transaminase. In
one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode BNA3 from S. cerevisae. In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode BNA2 in
combination with
one or more of idol and/or tdo2 and/or bna2. In one embodiment, the
genetically
engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in
combination with idol. In one embodiment, the genetically engineered bacteria
comprise
one or more gene sequence(s) which encode BNA2 in combination with tdo2. In
one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode BNA2 in combination with bna2. In one embodiment, the genetically
engineered bacteria further comprise one or more gene sequence(s) which encode
cclbl
and/or cc1b2 and/or aadat and/or got2.In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode one or more of
idol and/or
tdo2 and/or bna2.
[0424] In one embodiment, the genetically engineered bacteria comprise one or
more gene sequence(s) which encode one or more of afmid and/or bna3.In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode one or more of idol and/or tdo2 and/or bna2, in combination with
one or
more of afmid and/or bna3.In one embodiment, the genetically engineered
bacteria
comprise one or more gene sequence(s) which encode GOT2 (Aspartate
aminotransferase,
mitochondrial). In one embodiment, the genetically engineered bacteria
comprise one or
more gene sequence(s) which encode GOT2 from homo sapiens.In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
AADAT (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial). In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode AADAT from homo sapiens. In one embodiment, the genetically
engineered bacteria comprise one or more gene sequence(s) which encode CCLB1
(Kynurenine--oxoglutarate transaminase). In one embodiment, the genetically
engineered
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bacteria comprise one or more gene sequence(s) which encode CCLB1 from homo
sapiens). In one embodiment, the genetically engineered bacteria comprise one
or more
gene sequence(s) which encode CCLB2 (kynurenine--oxoglutarate transaminase 3)
In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode CCLB2 from homo sapiens.In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode cclbl and/or cc1b2
and/or
aadat and/or got2.In one embodiment, the genetically engineered bacteria
comprise one or
more gene sequence(s) which encode one or more of idol and/or tdo2 and/or
bna2, in
combination with one or more of afmid and/or bna3, and in combination with one
or more
of. cclbl and/or cc1b2 and/or aadat and/or got2.
[0425] In any of these embodiments, the genetically engineered bacteria which
produce kynurenic acid from tryptophan also optionally comprise one or more
gene
sequence(s) comprising one or more enzymes for tryptophan production, and gene
deletions/or mutations as depicted and described in the figures and the
examples and
described elsewhere herein. In some embodiments, the genetically engineered
bacteria
which produce kynurenic acid from tryptophan also optionally comprise one or
more gene
sequence(s) which encode one or more transporter(s) as described herein,
through which
tryptophan can be imported. Optionally, in some embodiments, the genetically
engineered
bacteria which produce kynurenic acid from tryptophan also optionally comprise
one or
more gene sequence(s) which encode an exporter as described herein, which can
export
tryptophan or any of its metabolites.
[0426] In some embodiments, the one or more genes for producing kynurenic acid
are modified and/or mutated, e.g., to enhance stability, increase kynurenic
acid production
under inducing conditions. In some embodiments, the engineered bacteria have
enhanced
uptake or import of tryptophan, e.g., comprise a transporter or other
mechanism for
increasing the uptake of tryptophan into the bacterial cell.
[0427] To improve acetate production, while maintaining high levels of
kynurenic
acid production, targeted one or more deletions can be introduced in competing
metabolic
arms of mixed acid fermentation to prevent the production of alternative
metabolic
fermentative byproducts (thereby increasing acetate production). Non-limiting
examples
of competing such competing metabolic arms are frdA (converts
phosphoenolpyruvate to
succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA
to
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Ethanol). Deletions which may be introduced therefore include deletion of
adhE, ldh, and
frd. Thus, in certain embodiments, the genetically engineered bacteria
comprise one or
more kynurenic acid production cassette(s) and further comprise mutations
and/or
deletions in one or more of frdA, ldhA, and adhE.
[0428] In some embodiments, the genetically engineered bacteria comprise one
or
more kynurenic acid production cassette(s) described herein and one or more
mutation(s)
and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA
gene and
the adhE gene.
[0429] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) encoding one or more enzymes described herein for the
production
of kynurenic acid and further comprise a mutation and/or deletion in one or
more
endogenous genes selected from in the ldhA gene, the frdA gene and the adhE
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
kynurenic acid and further comprise a mutation and/or deletion in the
endogenous ldhA
gene. In some embodiments, the genetically engineered bacteria comprise one or
more
gene sequence(s) encoding one or more enzymes described herein for the
production of
kynurenic acid and further comprise a mutation and/or deletion in the
endogenous adhE
gene. In some embodiments, the genetically engineered bacteria comprise one or
more
gene sequence(s) encoding one or more enzymes described herein for the
production of
kynurenic acid and further comprise a mutation and/or deletion in the
endogenous frdA
gene. In some embodiments, the genetically engineered bacteria comprise one or
more
gene sequence(s) encoding one or more enzymes described herein for the
production of
kynurenic acid and further comprise a mutation and/or deletion in the
endogenous ldhA
and rdA genes. In some embodiments, the genetically engineered bacteria
comprise one or
more gene sequence(s) encoding one or more enzymes described herein for the
production
of kynurenic acid and further comprise a mutation and/or deletion in the
endogenous ldhA
genes and adhE genes. In some embodiments, the genetically engineered bacteria
comprise one or more gene sequence(s) encoding one or more enzymes described
herein
for the production of kynurenic acid and further comprise a mutation and/or
deletion in the
endogenous frdA and adhE genes. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) encoding one or more enzymes
described
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herein for the production of kynurenic acid and further comprise a mutation
and/or
deletion in the endogenous ldhA, the frdA, and adhE genes. In some
embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s) encoding
one or
more enzymes described herein for the production of kynurenic acid and further
comprise
a mutation and/or deletion in one or more endogenous genes selected from in
the ldhA
gene, the frdA gene and the adhE genes.
[0430] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more acetate than unmodified bacteria of the same
bacterial subtype
under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the the genetically engineered bacteria
produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than
unmodified
bacteria of the same bacterial subtype under the same conditions.
[0431] In some embodiments, the genetically engineered bacteria produce 0% to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more kynurenic acid than unmodified bacteria of the same
bacterial
subtype under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more kynurenic acid than unmodified bacteria of the same bacterial
subtype under the
same conditions. In yet another embodiment, the the genetically engineered
bacteria
produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold,
nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more
kynurenic acid than
unmodified bacteria of the same bacterial subtype under the same conditions.
[0432] In certain situations, the need may arise to prevent and/or reduce
acetate
production by of an engineered or naturally occurring strain, e.g., E. coli
Nissle, while
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maintaining high levels of kynurenic acid production. Without wishing to be
bound by
theory, one or more mutations and/or deletions in one or more gene(s) encoding
in one or
more enzymes described herein which function in the acetate producing
metabolic arm of
fermentation should reduce and/or prevent production of acetate. A non-
limiting example
of such an enzyme is phosphate acetyltransferase (Pta), which is the first
enzyme in the
metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of
the Pta gene
or a gene encoding another enzyme in this metabolic arm may also allow for
more acetyl-
CoA to be used for kynurenic acid production. Additionally, one or more
mutations
preventing or reducing the flow through other metabolic arms of mixed acid
fermentaion,
such as those which produce succinate, lactate, and/or ethanol can increase
the production
of acetyl-CoA, which is available for kynurenic acid synthesis. Such mutations
and/or
deletions, include but are not limited to mutations and/or deletions in the
frdA, ldhA,
and/or adhE genes.
[0433] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) encoding one or more enzymes described herein for the
production
of kynurenic acid and further comprise a mutation and/or deletion in the
endogenous pta
gene. In some embodiments, the genetically engineered bacteria comprise one or
more
gene sequence(s) encoding one or more enzymes described herein for the
production of
kynurenic acid and further comprise a mutation and/or deletion in the
endogenous pta gene
and in one or more endogenous genes selected from in the ldhA gene, the frdA
gene and
the adhE gene. In some embodiments, the genetically engineered bacteria
comprise one or
more gene sequence(s) encoding one or more enzymes described herein for the
production
of kynurenic acid and further comprise a mutation in the endogenous pta and
adhE genes.
In some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
kynurenic acid and further comprise a mutation in the endogenous pta and ldhA
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
kynurenic acid and further comprise a mutation in the endogenous pta and frdA
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
kynurenic acid and further comprise a mutation and/or deletion in the
endogenous pta,
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ldhA and frdA genes. In some embodiments, the genetically engineered bacteria
comprise
one or more gene sequence(s) encoding one or more enzymes described herein for
the
production of kynurenic acid and further comprise a mutation in the endogenous
pta, ldhA,
and adhE genes. In some embodiments, the genetically engineered bacteria
comprise one
or more gene sequence(s) encoding one or more enzymes described herein for the
production of kynurenic acid and further comprise a mutation in the endogenous
pta, frdA
and adhE genes. In some embodiments, the genetically engineered bacteria
comprise one
or more gene sequence(s) encoding one or more enzyme(s) for the production of
kynurenic acid and further comprise a mutation and/or deletion in the
endogenous pta,
ldhA, frdA, and adhE genes.
[0434] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% less acetate than unmodified bacteria of the same
bacterial subtype
under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold less acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the genetically engineered bacteria
produce three-
fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-
fold, fifteen-fold,
twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than
unmodified bacteria of
the same bacterial subtype under the same conditions.
[0435] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more kynurenic acid than unmodified bacteria of the same
bacterial
subtype under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more kynurenic acid than unmodified bacteria of the same bacterial
subtype under the
same conditions. In yet another embodiment, the genetically engineered
bacteria produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
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fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more kynurenic acid
than
unmodified bacteria of the same bacterial subtype under the same conditions.
[0436] In some embodiments, the genetically engineered bacteria are capable of
producing kynurenic acid under inducing conditions, e.g., under a condition(s)
associated
with inflammation. In some embodiments, the genetically engineered bacteria
are capable
of producing kynurenic acid in low-oxygen conditions, in the presence of
certain
molecules or metabolites, in the presence of molecules or metabolites
associated with
inflammation or an inflammatory response, or in the presence of some other
metabolite
that may or may not be present in the gut, such as arabinose.
[0437] . In some embodiments, the gene sequences(s) are controlled by an
inducible promoter. In some embodiments, the gene sequences(s) are controlled
by a
constitutive promoter. In some embodiments, the gene sequences(s) are
controlled by an
inducible and/or constritutive promoter, and are expressed during bacterial
culture in vitro,
e.g., for bacterial expansion, production and/or manufacture, as described
herein.
[0438] In some embodiments, the genetically engineered bacteria are capable of
expressing any one or more of the described circuits in low-oxygen conditions,
in the
presence of disease or tissue specific molecules or metabolites, in the
presence of
molecules or metabolites associated with inflammation or an inflammatory
response or
immune suppression or in the presence of some other metabolite that may or may
not be
present in the gut, such as arabinose and others described herein. In some
embodiments,
any one or more of the described circuits are present on one or more plasmids
(e.g., high
copy or low copy) or are integrated into one or more sites in the bacterial
chromosome.
Also, in some embodiments, the genetically engineered bacteria are further
capable of
expressing any one or more of the described circuits and further comprise one
or more of
the following: (1) one or more auxotrophies, such as any auxotrophies known in
the art
and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch
circuits, such as
any of the kill-switches described herein or otherwise known in the art, (3)
one or more
antibiotic resistance circuits, (4) one or more transporters for importing
biological
molecules or substrates, such any of the transporters described herein or
otherwise known
in the art, (5) one or more secretion circuits, such as any of the secretion
circuits described
herein and otherwise known in the art, and (6) combinations of one or more of
such
additional circuits.
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Producing Indole Tryptophan Metabolites and Tryptamine
[0439] In some embodiments, the genetically engineered bacteria comprise
genetic
circuits for the production of indole metabolites and/or tryptamine. Exemplary
circuits for
the production of indole metabolites/derivatives are shown in the figures.
[0440] In some embodiments, the genetically engineered bacteria comprise
genetic
circuitry for converting tryptophan to tryptamine. In some embodiments, the
engineered
bacteria comprise gene sequence encoding Tryptophan decarboxylase, e.g., from
Catharanthus roseus. In some embodiments, the engineered bacteria comprise
genetic
circuitry for producing indole-3-acetaldehyde and FICZ from tryptophan. In
some
embodiments, the genetically engineered bacteria comprise gene sequence
encoding one
or more of the following: aro9 ( L-tryptophan aminotransferase, e.g., from S.
cerevisae),
aspC (aspartate aminotransferase, e.g., from E. coli, taal (L-tryptophan-
pyruvate
aminotransferase, e.g., from Arabidopsis thaliana), sta0 (L-tryptophan
oxidase, e.g., from
streptomyces sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from Nostoc
punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from
Enterobacter cloacae). In some embodiments, the genetically engineered
bacteria comprise
gene sequence encoding one or more of the following: tdc (Tryptophan
decarboxylase,
e.g., from Catharanthus roseus and/or Clostridium sporogenes), and tynA
(Monoamine
oxidase, e.g., from E. coli). In some embodiments, the engineered bacteria
comprise
genetic circuitry for producing indole-3-acetonitrile from tryptophan. In some
embodiments, the genetically engineered bacteria comprise gene sequence
encoding one
or more of the following: cyp79B2, (tryptophan N-monooxygenase, e.g., from
Arabidopsis thaliana), cyp79B3 (tryptophan N-monooxygenase, e.g., from
Arabidopsis
thaliana). In some embodiments, the engineered bacteria comprise genetic
circuitry for
producing kynurenine from tryptophan. In some embodiments, the genetically
engineered
bacteria comprise gene sequence encoding one or more of the following:
ID01(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TD02 (tryptophan
2,3-
dioxygenase, e.g., from homo sapiens), BNA2 (indoleamine 2,3-dioxygenase,
e.g., from
S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse), BNA3
(kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae). In some
embodiments,
the engineered bacteria comprise genetic circuitry for producing kynureninic
acid from
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tryptophan. In some embodiments, the genetically engineered bacteria comprise
gene
sequence encoding one or more of the following: ID01(indoleamine 2,3-
dioxygenase,
e.g., from homo sapiens or TD02 (tryptophan 2,3-dioxygenase, e.g., from homo
sapiens), BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and
Afmid:
Kynurenine formamidase, e.g., from mouse), BNA3 (kynurenine--oxoglutarate
transaminase, e.g., from S. cerevisae) and GOT2 (Aspartate aminotransferase,
mitochondrial, e.g. ,from homo sapiens or AADAT (Kynurenine/alpha-aminoadipate
aminotransferase, mitochondrial, e.g., from homo sapiens), or CCLB1
(Kynurenine--
oxoglutarate transaminase 1, e.g., from homo sapiens) or CCLB2 (kynurenine--
oxoglutarate transaminase 3, e.g., from homo sapiens. In some embodiments, the
engineered bacteria comprise genetic circuitry for producing indole from
tryptophan. In
some embodiments, the genetically engineered bacteria comprise gene sequence
encoding
one or more of the following: tnaA (tryptophanase, e.g., from E. coli). In
some
embodiments, the engineered bacteria comprise genetic circuitry for producing
indole-3-
carbinol, indole-3-aldehyde, 3,3' diindolylmethane (DIM), indolo(3,2-b)
carbazole (ICZ)
from indole glucosinolate (taken up through the diet). The genetically
engineered bacteria
comprise a gene sequence encoding pne2 (myrosinase, e.g., from Arabidopsis
thaliana).
In some embodiments, the engineered bacteria comprise genetic circuitry for
producing
indole-3-acetic acid from tryptophan. In some embodiments, the genetically
engineered
bacteria comprise gene sequence encoding one or more of the following: aro9 (
L-
tryptophan aminotransferase, e.g., from S. cerevisae), aspC (aspartate
aminotransferase,
e.g., from E. coli, taal (L-tryptophan-pyruvate aminotransferase, e.g., from
Arabidopsis
thaliana), sta0 (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274),
trpDH
(Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108), ipdC
(Indole-3-
pyruvate decarboxylase, e.g., from Enterobacter cloacae), iadl ( Indole-3-
acetaldehyde
dehydrogenase, e.g., from Ustilago maydis), AA01 (Indole-3-acetaldehyde
oxidase, e.g.,
from Arabidopsis thaliana). In some embodiments, the genetically engineered
bacteria
comprise gene sequence encoding one or more of the following: tdc (Tryptophan
decarboxylase, e.g.,from Catharanthus roseus and/or Clostridium sporogenes),
tynA
(Monoamine oxidase, e.g., from E. coli), iadl (Indole-3-acetaldehyde
dehydrogenase,
e.g., from Ustilago maydis), AA01 (Indole-3-acetaldehyde oxidase, e.g., from
Arabidopsis thaliana). In some embodiments, the genetically engineered
bacteria
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comprise gene sequence encoding one or more of the following: aro9 ( L-
tryptophan
aminotransferase, e.g., from S. cerevisae), aspC (aspartate aminotransferase,
e.g., from E.
coli, taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis
thaliana), sta0
(L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274), trpDH
(Tryptophan
dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and yuc2 ( indole-3-
pyruvate
monoxygenase, e.g., from Arabidopsis thaliana). In some embodiments, the
genetically
engineered bacteria comprise gene sequence encoding one or more of the
following: IaaM
(Tryptophan 2-monooxygenase e.g., from Pseudomonas savastanoi), iaaH
(Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi). In some
embodiments,
the genetically engineered bacteria comprise gene sequence encoding one or
more of the
following: cyp79B2 (tryptophan N-monooxygenase, e.g., from Arabidopsis
thaliana),
cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana, cyp71a13
(indoleacetaldoxime dehydratase, e.g., from Arabidopis thaliana), nitl
(Nitrilase, e.g.,
from Arabidopsis thaliana), iaaH (Indoleacetamide hydrolase, e.g., from
Pseudomonas
savastanoi). In some embodiments, the genetically engineered bacteria
comprises trpDH
(Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108), ipdC
(Indole-3-
pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together
produce indole-3-
acetaldehyde and FICZ though an (indo1-3y1)pyruvate intermediate, and iadl
(Indole-3-
acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-
3-
acetaldehyde into indole-3-acetat
[0441] In some embodiments, the genetically engineered bacteria comprise
genetic
circuits for the production of tryptophan, tryptamine, indole acetic acid, and
indole
propionic acid. In some embodiments, the engineered bacteria produces
tryptamine.
Tryptophan is optionally produced from chorismate precursor, and the bacteria
optionally
comprises circuits as depicted and/or described in FIG. 40A and/or FIG. 40B
and/or FIG.
40C and/or FIG. 40D. Additionally, the bacteria comprises tdc (tryptophan
decarboxylase,
e.g., from Catharanthus roseus and/or Clostridium sporogenes), which converts
tryptophan into tryptamine.
[0442] In some embodiments, the engineered bacteria comprise genetic circuits
for
the production of indole-3-acetate. Tryptophan is optionally produced from
chorismate
precursor, and the strain optionally comprises circuits as depicted and/or
described in FIG.
40A and/or FIG. 40B and/or FIG. 40C and/or FIG. 40D. Additionally, the strain
comprises
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trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and
ipdC
(Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which
together
produce indole-3-acetaldehyde and FICZ though an (indo1-3y1)pyruvate
intermediate, and
iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which
converts
indole-3-acetaldehyde into indole-3-acetate.
[0443] In some embodiments, the engineered bacteria comprise genetic circuits
for
the production of indole-3-propionate. Tryptophan is optionally produced from
chorismate precursor, and the strain optionally comprises circuits as depicted
and/or
described in FIG. 40A and/or FIG. 40B and/or FIG. 40C and/or FIG. 40D.
Additionally,
the strain comprises a circuit as described in FIG. 48, comprising trpDH
(Tryptophan
dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indo1-
3y1)pyruvate from tryptophan), fldA (indole-3-propionyl-CoA:indole-3-lactate
CoA
transferase, e.g., from Clostridium sporogenes, which converts converts indole-
3-lactate
and indo1-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-
CoA), fldB
and fldC (indole-3-lactate dehydratase e.g., from Clostridium sporogenes,
which converts
indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or AcuI: (indole-3-
acrylyl-CoA
reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase,
e.g., from
Rhodobacter sphaeroides, which convert indole-3-acrylyl-CoA to indole-3-
propionyl-
CoA). The circuits further comprisefldH/ and/or fldH2 (indole-3-lactate
dehydrogenase 1
and/or 2, e.g., from Clostridium sporogenes), which converts (indo1-3-
yl)pyruvate into
indole-3-lactate).
[0444] In some embodiments, the engineered bacteria comprises genetic
circuitry
for the production of indole-3-propionic acid (IPA). In some embodiments, the
engineered bacteria comprises gene sequence encoding tryptophan ammonia lyase
and an
indole-3-acrylate reductase (e.g., Tryptophan ammonia lyase (WAL) (e.g., from
Rubrivivax benzoatilyticus) and indole-3-acrylate reductase (e.g., from
Clostridum
botulinum). Tryptophan ammonia lyase converts tryptophan to indole-3-acrylic
acid, and
indole-3-acrylate reductase converts indole-3-acrylic acid into IPA. Without
wishing to be
bound by theory, no oxygen is needed for this reaction, allowing it to proceed
under low
or no oxygen conditions, e.g., as those found in the mammalian gut. In some
embodiments, the genetically engineered bacteria further comprise one or more
circuits for
the production of tryptophan, e.g., as shown in FIG. 40 (A-D) and FIG. 44 and
as
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described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced
with
feedback resistant versions to improve tryptophan production in the
genetically engineered
bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a
tryptophanase
converting tryptophan into indole) are deleted to further increase levels of
tryptophan
produced.
[0445] In some embodiments, the engineered bacteria comprise genetic circuitry
for producing indole-3-propionic acid (IPA), indole acetic acid (IAA), and/or
tryptamine
synthesis(TrA) circuits. In some embodiments, the engineered bacteria comprise
gene
sequence encoding one or more of the following: TrpDH: tryptophan
dehydrogenase, e.g.,
from from Nostoc punctiforme NIES-2108; FldHl/F1dH2: indole-3-lactate
dehydrogenase, e.g., from Clostridium sporogenes; FldA: indole-3-propionyl-
CoA:indole-
3-lactate CoA transferase, e.g., from Clostridium sporogenes; FldBC: indole-3-
lactate
dehydratase, e.g., from Clostridium sporogenes; FldD: indole-3-acrylyl-CoA
reductase,
e.g., from Clostridium sporogenes; AcuI: acrylyl-CoA reductase, e.g., from
Rhodobacter
sphaeroides. 1pdC: Indole-3-pyruvate decarboxylase, e.g., from Enterobacter
cloacae;
ladl: Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis; Tdc:
Tryptophan
decarboxylase, e.g., from Catharanthus roseus or from Clostridium sporogenes.
[0446] In some embodiments, the engineered bacteria comprise genetic circuitry
for producing (indo1-3-yl)pyruvate (IPyA). In some embodiments, the engineered
bacteria
comprise gene sequence encoing one or more of the following: tryptophan
dehydrogenase
(EC 1.4.1.19) (enzyme that catalyzes the reversible chemical reaction
converting L-
tryptophan, NAD(P) and water to (indo1-3-yl)pyruvate (IPyA), NH3, NAD(P)H and
H ));
Indole-3-lactate dehydrogenase ((EC 1.1.1.110, e.g., Clostridium sporogenes or
Lactobacillus casei) (converts (indo1-3y1)pyruvate (IpyA) and NADH and H+ to
indole-3-
lactate (ILA) and NAD+); Indole-3-propionyl-CoA:indole-3-lactate CoA
transferase
(F1dA ) (converts indole-3-lactate (ILA) and indo1-3-propionyl-CoA to indole-3-
propionic
acid (IPA) and indole-3-lactate-CoA); Indole-3-acrylyl-CoA reductase (F1dD )
and
acrylyl-CoA reductase (AcuI) (convert indole-3-acrylyl-CoA to indole-3-
propionyl-CoA);
Indole-3-lactate dehydratase (FldBC ) (converts indole-3-lactate-CoA to indole-
3-acrylyl-
CoA); Indole-3-pyruvate decarboxylase (1pdC:) (converts Indole-3-pyruvic acid
(IPyA)
into Indole-3-acetaldehyde (IAA1d)); ladl: Indole-3-acetaldehyde dehydrogenase
(coverts
Indole-3-acetaldehyde (IAA1d) into Indole-3-acetic acid (IAA)); Tdc:
Tryptophan
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decarboxylase (converts tryptophan (Trp) into tryptamine (TrA)). In some
embodiments,
the genetically engineered bacteria further comprise one or more circuits for
the
production of tryptophan, e.g., as shown in FIG. 40 (A-D) and FIG. 44 and as
described
elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with
feedback
resistant versions to improve tryptophan production in the genetically
engineered bacteria.
In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase
converting
tryptophan into indole) are deleted to further increase levels of tryptophan
produced.
[0447] In any of the described embodiments, any of the gene(s), gene
sequence(s)
and/or gene circuit(s) or cassette(s) are optionally expressed from an
inducible promoter.
In certain embodiments, the one or more cassettes are under the control of
constitutive
promoters. Exemplary inducible promoters which may control the expression of
the
gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen
level-
dependent promoters (e.g., FNR-inducible promoter), promoters induced by
inflammation
or an inflammatory response (RNS, ROS promoters), and promoters induced by a
metabolite that may or may not be naturally present (e.g., can be exogenously
added) in
the gut, e.g., arabinose and tetracycline. The bacteria may also include an
auxotrophy,
e.g., deletion of thyA (A thyA; thymidine dependence).
[0448] In some embodiments, the genetically engineered bacteria further
comprise
one or more circuits for the production of tryptophan, e.g., as shown in FIG.
40 (A-D) and
FIG. 44 and as described elsewhere herein. In some embodiments, AroG and/or
TrpE are
replaced with feedback resistant versions to improve tryptophan production in
the
genetically engineered bacteria. In some embodiments, trpR and/or the tnaA
gene
(encoding a tryptophanase converting tryptophan into indole) are deleted to
further
increase levels of tryptophan produced.
[0449] In in any of these embodiments the expression of the gene sequences for
the production of the indole and other tryptophan metabolites, including, but
not limited
to, tryptamine and/or indole-3 acetaladehyde, indole-3acetonitrile, indole,
indole acetic
acid FICZ, indole-3-propionic acid, is under the control of an inducible
promoter.
Exemplary inducible promoters which may control the expression of the
biosynthetic
cassettes include oxygen level-dependent promoters (e.g., FNR-inducible
promoter),
promoters induced by inflammation or an inflammatory response (RNS, ROS
promoters),
and promoters induced by a metabolite characteristic of a disorder described
herein, or that
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may or may not be naturally present (e.g., can be exogenously added) in the
gut, e.g.,
arabinose and tetracycline. In some embodiments, the gene sequences(s) are
controlled by
an inducible promoter. In some embodiments, the gene sequences(s) are
controlled by a
constitutive promoter. In some embodiments, the gene sequences(s) are
controlled by an
inducible and/or constritutive promoter, and are expressed during bacterial
culture in vitro,
e.g., for bacterial expansion, production and/or manufacture, as described
herein.
[0450] In some embodiments, the genetically engineered bacteria are capable of
expressing any one or more of the described circuits in low-oxygen conditions,
in the
presence of disease or tissue specific molecules or metabolites, in the
presence of
molecules or metabolites associated with inflammation or an inflammatory
response or
immune suppression, or in the presence of some other metabolite that may or
may not be
present in the gut, such as arabinose. In some embodiments, any one or more of
the
described circuits are present on one or more plasmids (e.g., high copy or low
copy) or are
integrated into one or more sites in the bacterial chromosome. Also, in some
embodiments, the genetically engineered bacteria are further capable of
expressing any
one or more of the described circuits and further comprise one or more of the
following:
(1) one or more auxotrophies, such as any auxotrophies known in the art and
provided
herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as
any of the kill-
switches described herein or otherwise known in the art, (3) one or more
antibiotic
resistance circuits, (4) one or more transporters for importing biological
molecules or
substrates, such any of the transporters described herein or otherwise known
in the art, (5)
one or more secretion circuits, such as any of the secretion circuits
described herein and
otherwise known in the art, and (6) combinations of one or more of such
additional
circuits.
Tryptamine
[0451] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) which encode one or more tryptophan catabolism enzymes,
which
produce tryptamine from tryptophan. The monoamine alkaloid, tryptamine, is
derived
from the direct decarboxylation of tryptophan. Tryptophan is converted to
indole-3-acetic
acid (IAA) via the enzymes tryptophan monooxygenase (IaaM) and indole-3-
acetamide
hydrolase (IaaH), which constitute the indole-3-acetamide (IAM) pathway, as
described in
the figures and examples.
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[0452] A non-limiting example of such as strain is shown in FIG. 41A. Another
non-limiting example of such as strain is shown in FIG. 43A. In one
embodiment, the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode one
or more Tryptophan decarboxylase(s), e.g., from Catharanthus roseus. In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode one or more Tryptophan decarboxylase(s), e.g., from Clostridium
sporgenenes. In one embodiment, the genetically engineered bacteria comprise
one or
more gene sequence(s) which encode one or more Tryptophan decarboxylase(s)
e.g., from
Ruminococcus Gnavus.
[0453] Table 15, Table 11A, and Table 12 lists exemplary sequences for
tryptamine production in genetically engineered bacteria.
[0454] In some embodiments, the genetically engineered bacteria which produce
tryptamine from tryptophan also optionally comprise one or more gene
sequence(s)
comprising one or more enzymes for tryptophan production, and gene
deletions/or
mutations as depicted and described in FIG. 40, FIG. 44A and/or FIG. 44B and
described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced
with
feedback resistant versions to improve tryptophan production in the
genetically engineered
bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a
tryptophanase
converting tryptophan into indole) are deleted to further increase levels of
tryptophan
produced. In some embodiments, the genetically engineered bacteria which
produce
tryptamine from tryptophan also optionally comprise one or more gene
sequence(s) which
encode one or more transporter(s) as described herein, through which
tryptophan can be
imported. Optionally, In some embodiments, the genetically engineered bacteria
which
produce tryptamine from tryptophan also optionally comprise one or more gene
sequence(s) which encode an exporter as described herein, which can export
tryptophan or
any of its metabolites.
[0455] To improve acetate production, while maintaining high levels of
tryptamine
production, targeted one or more deletions can be introduced in competing
metabolic arms
of mixed acid fermentation to prevent the production of alternative metabolic
fermentative
byproducts (thereby increasing acetate production). Non-limiting examples of
competing
such competing metabolic arms are frdA (converts phosphoenolpyruvate to
succinate),
ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol).
Deletions
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which may be introduced therefore include deletion of adhE, ldh, and frd.
Thus, in certain
embodiments, the genetically engineered bacteria comprise one or more
tryptamine
production cassette(s) and further comprise mutations and/or deletions in one
or more of
frdA, ldhA, and adhE.
[0456] In some embodiments, the genetically engineered bacteria comprise one
or
more tryptamine production cassette(s) described herein and one or more
mutation(s)
and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA
gene and
the adhE gene.
[0457] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) encoding one or more enzymes described herein for the
production
of tryptamine and further comprise a mutation and/or deletion in one or more
endogenous
genes selected from in the ldhA gene, the frdA gene and the adhE genes. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes described herein for the production of tryptamine
and
further comprise a mutation and/or deletion in the endogenous ldhA gene. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes described herein for the production of tryptamine
and
further comprise a mutation and/or deletion in the endogenous adhE gene. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes described herein for the production of tryptamine
and
further comprise a mutation and/or deletion in the endogenous frdA gene. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes described herein for the production of tryptamine
and
further comprise a mutation and/or deletion in the endogenous ldhA and rdA
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
tryptamine and further comprise a mutation and/or deletion in the endogenous
ldhA genes
and adhE genes. In some embodiments, the genetically engineered bacteria
comprise one
or more gene sequence(s) encoding one or more enzymes described herein for the
production of tryptamine and further comprise a mutation and/or deletion in
the
endogenous frdA and adhE genes. In some embodiments, the genetically
engineered
bacteria comprise one or more gene sequence(s) encoding one or more enzymes
described
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herein for the production of tryptamine and further comprise a mutation and/or
deletion in
the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the
genetically
engineered bacteria comprise one or more gene sequence(s) encoding one or more
enzymes described herein for the production of tryptamine and further comprise
a
mutation and/or deletion in one or more endogenous genes selected from in the
ldhA gene,
the frdA gene and the adhE genes.
[0458] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and
AtrpR and
AtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA
gene. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and
AtnaA,
and further comprise a mutation and/or deletion in the endogenous adhE gene.
In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and
further
comprise a mutation and/or deletion in the endogenous frdA gene. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
selected from
trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise
a
mutation and/or deletion in the endogenous ldhA and frdA genes. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
selected from
trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise
a
mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and
further
comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and
further
comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE
genes.
[0459] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more acetate than unmodified bacteria of the same
bacterial subtype
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under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the the genetically engineered bacteria
produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than
unmodified
bacteria of the same bacterial subtype under the same conditions.
[0460] In some embodiments, the genetically engineered bacteria produce 0% to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more tryptamine than unmodified bacteria of the same
bacterial
subtype under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more tryptamine than unmodified bacteria of the same bacterial subtype
under the
same conditions. In yet another embodiment, the the genetically engineered
bacteria
produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold,
nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more
tryptamine than
unmodified bacteria of the same bacterial subtype under the same conditions.
[0461] In certain situations, the need may arise to prevent and/or reduce
acetate
production by of an engineered or naturally occurring strain, e.g., E. coli
Nissle, while
maintaining high levels of tryptamine production. Without wishing to be bound
by theory,
one or more mutations and/or deletions in one or more gene(s) encoding in one
or more
enzymes described herein which function in the acetate producing metabolic arm
of
fermentation should reduce and/or prevent production of acetate. A non-
limiting example
of such an enzyme is phosphate acetyltransferase (Pta), which is the first
enzyme in the
metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of
the Pta gene
or a gene encoding another enzyme in this metabolic arm may also allow for
more acetyl-
CoA to be used for tryptamine production. Additionally, one or more mutations
preventing
or reducing the flow through other metabolic arms of mixed acid fermentaion,
such as
those which produce succinate, lactate, and/or ethanol can increase the
production of
acetyl-CoA, which is available for tryptamine synthesis. Such mutations and/or
deletions,
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include but are not limited to mutations and/or deletions in the frdA, ldhA,
and/or adhE
genes.
[0462] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) encoding one or more enzymes described herein for the
production
of tryptamine and further comprise a mutation and/or deletion in the
endogenous pta gene.
In some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
tryptamine and further comprise a mutation and/or deletion in the endogenous
pta gene
and in one or more endogenous genes selected from in the ldhA gene, the frdA
gene and
the adhE gene. In some embodiments, the genetically engineered bacteria
comprise one or
more gene sequence(s) encoding one or more enzymes described herein for the
production
of tryptamine and further comprise a mutation in the endogenous pta and adhE
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
tryptamine and further comprise a mutation in the endogenous pta and ldhA
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
tryptamine and further comprise a mutation in the endogenous pta and frdA
genes. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) encoding one or more enzymes described herein for the production
of
tryptamine and further comprise a mutation and/or deletion in the endogenous
pta, ldhA
and frdA genes. In some embodiments, the genetically engineered bacteria
comprise one
or more gene sequence(s) encoding one or more enzymes described herein for the
production of tryptamine and further comprise a mutation in the endogenous
pta, ldhA,
and adhE genes. In some embodiments, the genetically engineered bacteria
comprise one
or more gene sequence(s) encoding one or more enzymes described herein for the
production of tryptamine and further comprise a mutation in the endogenous
pta, frdA and
adhE genes. In some embodiments, the genetically engineered bacteria comprise
one or
more gene sequence(s) encoding one or more enzyme(s) for the production of
tryptamine
and further comprise a mutation and/or deletion in the endogenous pta, ldhA,
frdA, and
adhE genes.
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[0463] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and
AtrpR and
AtnaA, and further comprise a mutation and/or deletion in the endogenous pta
gene. In
some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and
AtnaA,
and further comprise a mutation and/or deletion in the endogenous pta gene and
in one or
more endogenous genes selected from in the ldhA gene, the frdA gene and the
adhE gene.
In some embodiments, the genetically engineered bacteria comprise one or more
gene
sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and
AtnaA,
and further comprise a mutation in the endogenous pta and adhE genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and
further
comprise a mutation in the endogenous pta and ldhA genes.
[0464] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and
AtrpR and
AtnaA, and further comprise a mutation in the endogenous pta and frdA genes.
In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and
further
comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA
genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and
further
comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some
embodiments,
the genetically engineered bacteria comprise one or more gene sequence(s)
selected from
trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise
a
mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s) selected
from
trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise
a
mutation in the endogenous pta, ldhA, frdA, and adhE genes.
[0465] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
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90%, or 90% to 100% less acetate than unmodified bacteria of the same
bacterial subtype
under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold less acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the genetically engineered bacteria
produce three-
fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-
fold, fifteen-fold,
twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than
unmodified bacteria of
the same bacterial subtype under the same conditions.
[0466] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more tryptamine than unmodified bacteria of the same
bacterial
subtype under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more tryptamine than unmodified bacteria of the same bacterial subtype
under the
same conditions. In yet another embodiment, the genetically engineered
bacteria produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptamine
than unmodified
bacteria of the same bacterial subtype under the same conditions.
[0467] In some embodiments, the genetically engineered bacteria are capable of
producing Tryptamine under inducing conditions, e.g., under a condition(s)
associated
with inflammation. In some embodiments, the genetically engineered bacteria
are capable
of producing kynurenine in low-oxygen conditions, in the presence of certain
molecules or
metabolites, in the presence of molecules or metabolites associated with
inflammation or
an inflammatory response, or in the presence of some other metabolite that may
or may
not be present in the gut, such as arabinose and others described herein.
Indole-3-acetaldehyde and FICZ
[0468] In one embodiment, the genetically engineered bacteria comprise one or
more gene sequence(s) which encode one or more tryptophan catabolism enzymes,
which
produce indole-3-acetaldehyde and FICZ from tryptophan. Exemplary gene
cassettes for
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the production of produce indole-3-acetaldehyde and FICZ from tryptophan are
shown in
FIG. 41B.
[0469] In one embodiment, the genetically engineered bacteria comprise one or
more gene sequence(s) which encode aro9 ( L-tryptophan aminotransferase). In
one
embodiment, the (L-tryptophan aminotransferase is from S. cerevisiae. In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter
cloacae). In
one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode aro9 and ipdC. In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode aspC (aspartate
aminotransferase. In one embodiment, the genetically engineered bacteria
comprise one or
more gene sequence(s) which encode aspC from E. coli. In one embodiment, the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
aspC and ipdC. In one embodiment, the genetically engineered bacteria comprise
one or
more gene sequence(s) which encode taal (L-tryptophan-pyruvate
aminotransferase, In
one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode taal from Arabidopsis thaliana. In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode taal
and ipdC. In one embodiment, the genetically engineered bacteria comprise one
or more
gene sequence(s) which encode sta0 (L-tryptophan oxidase). In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode sta0
from streptomyces sp. TP-A0274. In one embodiment, the genetically engineered
bacteria
comprise one or more gene sequence(s) which encode sta0 and ipdC. In one
embodiment,
the genetically engineered bacteria comprise one or more gene sequence(s)
which encode
trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc
punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria
comprise one or more gene sequence(s) which encode trpDH and ipdC. In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode one or more of aro9 or aspC or taal or sta0 or trpDH. In one
embodiment,
the genetically engineered bacteria comprise one or more gene sequence(s)
which encode
one or more of aro9 or aspC or taal or sta0 or trpDH and ipdC.
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[0470] Further exemplary gene cassettes for the production of produce indole-3-
acetaldehyde and FICZ from tryptophan are shown in FIG. 41C. In one
embodiment, the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode tdc
(Tryptophan decarboxylase). In one embodiment, the genetically engineered
bacteria
comprise one or more gene sequence(s) which encode tdc from Catharanthus
roseus. In
one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode tynA (Monoamine oxidase). In one embodiment, the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
tynA from E. coli. In one embodiment, the genetically engineered bacteria
comprise one
or more gene sequence(s) which encode tdc and tynA.
[0471] In any of these embodiments, the genetically engineered bacteria which
produce produce indole-3-acetaldehyde and FICZ also optionally comprise one or
more
gene sequence(s) comprising one or more enzymes for tryptophan production, and
gene
deletions/or mutations as depicted and described in FIG. 40, FIG. 44A and/or
FIG. 44B
and described elsewhere herein. In some embodiments, AroG and/or TrpE are
replaced
with feedback resistant versions to improve tryptophan production in the
genetically
engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding
a
tryptophanase converting tryptophan into indole) are deleted to further
increase levels of
tryptophan produced. In some embodiments, the genetically engineered bacteria
which
produce indole-3-acetaldehyde and FICZ also optionally comprise one or more
gene
sequence(s) which encode one or more transporter(s) as described herein,
through which
tryptophan can be imported. Optionally, in some embodiments, the genetically
engineered
bacteria which produce indole-3-acetaldehyde and FICZ also optionally comprise
one or
more gene sequence(s) which encode an exporter as described herein, which can
export
tryptophan or any of its metabolites.
[0472] To improve acetate production, while maintaining high levels of Indole-
3-
acetaldehyde and/or FICZ production, targeted one or more deletions can be
introduced in
competing metabolic arms of mixed acid fermentation to prevent the production
of
alternative metabolic fermentative byproducts (thereby increasing acetate
production).
Non-limiting examples of competing such competing metabolic arms are frdA
(converts
phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and
adhE (converts
Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include
deletion of
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adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered
bacteria
comprise one or more Indole-3-acetaldehyde and/or FICZ production cassette(s)
and
further comprise mutations and/or deletions in one or more of frdA, ldhA, and
adhE.
[0473] In some embodiments, the genetically engineered bacteria comprise one
or
more Indole-3-acetaldehyde and/or FICZ production cassette(s) described herein
and one
or more mutation(s) and/or deletion(s) in one or more genes selected from the
ldhA gene,
the frdA gene and the adhE gene.
[0474] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) encoding one or more enzymes described herein for the
production
of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or
deletion in
one or more endogenous genes selected from in the ldhA gene, the frdA gene and
the adhE
genes. In some embodiments, the genetically engineered bacteria comprise one
or more
gene sequence(s) encoding one or more enzymes described herein for the
production of
Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or
deletion in the
endogenous ldhA gene. In some embodiments, the genetically engineered bacteria
comprise one or more gene sequence(s) encoding one or more enzymes described
herein
for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a
mutation
and/or deletion in the endogenous adhE gene. In some embodiments, the
genetically
engineered bacteria comprise one or more gene sequence(s) encoding one or more
enzymes described herein for the production of Indole-3-acetaldehyde and/or
FICZ and
further comprise a mutation and/or deletion in the endogenous frdA gene. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes described herein for the production of Indole-3-
acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in
the
endogenous ldhA and rdA genes. In some embodiments, the genetically engineered
bacteria comprise one or more gene sequence(s) encoding one or more enzymes
described
herein for the production of Indole-3-acetaldehyde and/or FICZ and further
comprise a
mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes described herein for the production of Indole-3-
acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in
the
endogenous frdA and adhE genes. In some embodiments, the genetically
engineered
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bacteria comprise one or more gene sequence(s) encoding one or more enzymes
described
herein for the production of Indole-3-acetaldehyde and/or FICZ and further
comprise a
mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In
some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes described herein for the production of Indole-3-
acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in
one or more
endogenous genes selected from in the ldhA gene, the frdA gene and the adhE
genes.
[0475] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more acetate than unmodified bacteria of the same
bacterial subtype
under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold more acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the the genetically engineered bacteria
produce
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-
fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than
unmodified
bacteria of the same bacterial subtype under the same conditions.
[0476] In some embodiments, the genetically engineered bacteria produce 0% to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more Indole-3-acetaldehyde and/or FICZ than unmodified
bacteria
of the same bacterial subtype under the same conditions. In yet another
embodiment, the
genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-
fold, 1.6-1.8-
fold, 1.8-2-fold, or two-fold more Indole-3-acetaldehyde and/or FICZ than
unmodified
bacteria of the same bacterial subtype under the same conditions. In yet
another
embodiment, the the genetically engineered bacteria produce three-fold, four-
fold, five-
fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold,
twenty-fold, thirty-
fold, forty-fold, or fifty-fold, more Indole-3-acetaldehyde and/or FICZ than
unmodified
bacteria of the same bacterial subtype under the same conditions.
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[0477] In certain situations, the need may arise to prevent and/or reduce
acetate
production by of an engineered or naturally occurring strain, e.g., E. coli
Nissle, while
maintaining high levels of Indole-3-acetaldehyde and/or FICZ production.
Without
wishing to be bound by theory, one or more mutations and/or deletions in one
or more
gene(s) encoding in one or more enzymes described herein which function in the
acetate
producing metabolic arm of fermentation should reduce and/or prevent
production of
acetate. A non-limiting example of such an enzyme is phosphate
acetyltransferase (Pta),
which is the first enzyme in the metabolic arm converting acetyl-CoA to
acetate. Deletion
and/or mutation of the Pta gene or a gene encoding another enzyme in this
metabolic arm
may also allow for more acetyl-CoA to be used for Indole-3-acetaldehyde and/or
FICZ
production. Additionally, one or more mutations preventing or reducing the
flow through
other metabolic arms of mixed acid fermentaion, such as those which produce
succinate,
lactate, and/or ethanol can increase the production of acetyl-CoA, which is
available for
Indole-3-acetaldehyde and/or FICZ synthesis. Such mutations and/or deletions,
include but
are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE
genes.
[0478] In some embodiments, the genetically engineered bacteria comprise one
or
more gene sequence(s) encoding one or more enzymes described herein for the
production
of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or
deletion in
the endogenous pta gene. In some embodiments, the genetically engineered
bacteria
comprise one or more gene sequence(s) encoding one or more enzymes described
herein
for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a
mutation
and/or deletion in the endogenous pta gene and in one or more endogenous genes
selected
from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments,
the
genetically engineered bacteria comprise one or more gene sequence(s) encoding
one or
more enzymes described herein for the production of Indole-3-acetaldehyde
and/or FICZ
and further comprise a mutation in the endogenous pta and adhE genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes described herein for the production of Indole-3-
acetaldehyde and/or FICZ and further comprise a mutation in the endogenous pta
and
ldhA genes. In some embodiments, the genetically engineered bacteria comprise
one or
more gene sequence(s) encoding one or more enzymes described herein for the
production
of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation in the
endogenous
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pta and frdA genes. In some embodiments, the genetically engineered bacteria
comprise
one or more gene sequence(s) encoding one or more enzymes described herein for
the
production of Indole-3-acetaldehyde and/or FICZ and further comprise a
mutation and/or
deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the
genetically engineered bacteria comprise one or more gene sequence(s) encoding
one or
more enzymes described herein for the production of Indole-3-acetaldehyde
and/or FICZ
and further comprise a mutation in the endogenous pta, ldhA, and adhE genes.
In some
embodiments, the genetically engineered bacteria comprise one or more gene
sequence(s)
encoding one or more enzymes described herein for the production of Indole-3-
acetaldehyde and/or FICZ and further comprise a mutation in the endogenous
pta, frdA
and adhE genes. In some embodiments, the genetically engineered bacteria
comprise one
or more gene sequence(s) encoding one or more enzyme(s) for the production of
Indole-3-
acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in
the
endogenous pta, ldhA, frdA, and adhE genes.
[0479] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% less acetate than unmodified bacteria of the same
bacterial subtype
under the same conditions. In yet another embodiment, the genetically
engineered
bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-
fold, or two-
fold less acetate than unmodified bacteria of the same bacterial subtype under
the same
conditions. In yet another embodiment, the genetically engineered bacteria
produce three-
fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-
fold, fifteen-fold,
twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than
unmodified bacteria of
the same bacterial subtype under the same conditions.
[0480] In some embodiments, the genetically engineered bacteria produce 0% to
to
2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to
45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to
90%, or 90% to 100% more Indole-3-acetaldehyde and/or FICZ than unmodified
bacteria
of the same bacterial subtype under the same conditions. In yet another
embodiment, the
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genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-
fold, 1.6-1.8-
fold, 1.8-2-fold, or two-fold more Indole-3-acetaldehyde and/or FICZ than
unmodified
bacteria of the same bacterial subtype under the same conditions. In yet
another
embodiment, the genetically engineered bacteria produce three-fold, four-fold,
five-fold,
six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-
fold, thirty-fold,
forty-fold, or fifty-fold, more Indole-3-acetaldehyde and/or FICZ than
unmodified bacteria
of the same bacterial subtype under the same conditions.
[0481] In some embodiments, the genetically engineered bacteria are capable of
producing Indole-3-aldehyde under inducing conditions, e.g., under a
condition(s)
associated with inflammation. In some embodiments, the genetically engineered
bacteria
are capable of producing kynurenine in low-oxygen conditions, in the presence
of certain
molecules or metabolites, in the presence of molecules or metabolites
associated with
inflammation or an inflammatory response, or in the presence of some other
metabolite
that may or may not be present in the gut, such as arabinose.
[0482] In some embodiments, the gene sequences(s) are controlled by an
inducible
promoter. In some embodiments, the gene sequences(s) are controlled by a
constitutive
promoter. In some embodiments, the gene sequences(s) are controlled by an
inducible
and/or constritutive promoter, and are expressed during bacterial culture in
vitro, e.g., for
bacterial expansion, production and/or manufacture, as described herein.
Indole-3-acetic acid
[0483] In some embodiments, the genetically engineered bacteria comprise one
or
more gene cassettes which convert tryptophan to Indole-3-aldehyde and Indole
Acetic
Acid, e.g., via a tryptophan aminotransferase cassette. A non-limiting example
of such a
tryptophan aminotransferase expressed by the genetically engineered bacteria
is in the
tables. In some embodiments, the genetically engineered bacteria take up
tryptophan
through an endogenous or exogenous transporter, and further produce Indole-3-
aldehyde
and Indole Acetic Acid from tryptophan. In some embodiments, the genetically
engineered
bacteria optionally comprise a tryptophan and/or indole metabolite exporter.
[0484] The genetically engineered bacteria may comprise any suitable gene for
producing Indole-3-aldehyde and/or Indole Acetic Acid and/or Tryptamine. In
some
embodiments, the gene for producing kynurenine is modified and/or mutated,
e.g., to
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enhance stability, increase Indole-3-aldehyde and/or Indole Acetic Acid and/or
Tryptamine production, and/or increase anti-inflammatory potency under
inducing
conditions. In some embodiments, the engineered bacteria also have enhanced
uptake or
import of tryptophan, e.g., comprise a transporter or other mechanism for
increasing the
uptake of tryptophan into the bacterial cell, as discussed in detail above. In
some
embodiments, the engineered bacteria also have enhanced export of a indole
tryptophan
metabolite, e.g., comprise an exporter or other mechanism for increasing the
uptake of
tryptophan into the bacterial cell, as discussed in detail above. In some
embodiments, the
genetically engineered bacteria are capable of producing Indole-3-aldehyde
and/or Indole
Acetic Acid and/or Tryptamine under inducing conditions, e.g., under a
condition(s)
associated with inflammation. In some embodiments, the genetically engineered
bacteria
are capable of producing kynurenine in low-oxygen conditions, in the presence
of certain
molecules or metabolites, in the presence of molecules or metabolites
associated with
inflammation or an inflammatory response, or in the presence of some other
metabolite
that may or may not be present in the gut, such as arabinose.
[0485] In one embodiment, the genetically engineered bacteria comprise one or
more gene sequence(s) which encode one or more tryptophan catabolism enzymes,
which
produce indole-3-acetic acid.
[0486] Non-limiting example of such gene sequence(s) are shown in FIG. 42A,
FIG. 42B, FIG. 42C, FIG. 42D, and FIG. 42E, and FIG. 43B and FIG. 43E.
[0487] In one embodiment, the genetically engineered bacteria comprise one or
more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In
one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode aro9 from S. cerevisae). In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode aspC (aspartate
aminotransferase), In one embodiment, the genetically engineered bacteria
comprise one
or more gene sequence(s) which encode aspC from E. coli. In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode taal
(L-tryptophan-pyruvate aminotransferase. In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode taal from
Arabidopsis
thaliana). In one embodiment, the genetically engineered bacteria comprise one
or more
gene sequence(s) which encode sta0 (L-tryptophan oxidase). In one embodiment,
the
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genetically engineered bacteria comprise one or more gene sequence(s) which
encode sta0
from streptomyces sp. TP-A0274). In one embodiment, the genetically engineered
bacteria
comprise one or more gene sequence(s) which encode trpDH (Tryptophan
dehydrogenase). In one embodiment, the genetically engineered bacteria
comprise one or
more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108).
In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode iadl (Indole-3-acetaldehyde dehydrogenase). In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode iadl
from Ustilago maydis. In one embodiment, the genetically engineered bacteria
comprise
one or more gene sequence(s) which encode AA01 (Indole-3-acetaldehyde
oxidase). In
one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode AA01 from Arabidopsis thaliana. In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In
one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter
cloacae) in
combination with one or more sequences encoding enzymes selected from aro9
and/or
aspC and/or taal and/or sta0 and/or trpDH. In one embodiment, the genetically
engineered bacteria comprise one or more gene sequence(s) which encode ipdC
(Indole-3-
pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with
one or more
sequences encoding enzymes selected from iadl and/or aaol.In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in
combination
with one or more sequences encoding enzymes selected from aro9 and/or aspC
and/or taal
and/or sta0 and in combination with one or more sequences encoding enzymes
selected
from iadl and/or aaol (see, e.g., FIG. 42A).
[0488] Another non-limiting example of gene sequence(s) for the production of
indole-3-acetic acid are shown in FIG. 42B. In one embodiment, the genetically
engineered bacteria comprise one or more gene sequence(s) which encode tdc
(Tryptophan
decarboxylase). In one embodiment, the genetically engineered bacteria
comprise one or
more gene sequence(s) which encode tdc from Catharanthus roseus). In one
embodiment,
the genetically engineered bacteria comprise one or more gene sequence(s)
which encode
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tynA (Monoamine oxidase). In one embodiment, the genetically engineered
bacteria
comprise one or more gene sequence(s) which encode tynA from E. coli). In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode iadl (Indole-3-acetaldehyde dehydrogenase). In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode iadl
from Ustilago maydis). In one embodiment, the genetically engineered bacteria
comprise
one or more gene sequence(s) which encode AA01 (Indole-3-acetaldehyde
oxidase). In
one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode AA01 from Arabidopsis thaliana). In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode tdc
and tynA. In one embodiment, the genetically engineered bacteria comprise one
or more
gene sequence(s) which encode tdc and one or more sequence(s) selected from
iadl and/or
aaol. In one embodiment, the genetically engineered bacteria comprise one or
more gene
sequence(s) which encode tynA and one or more sequence(s) selected from iadl
and/or
aaol. In one embodiment, the genetically engineered bacteria comprise one or
more gene
sequence(s) which encode tdc and tynA and one or more sequence(s) selected
from iadl
and/or aaol.
[0489] Another non-limiting example of gene sequence(s) for the production of
indole-3-acetic acid are shown in FIG. 42C. In one embodiment, the genetically
engineered bacteria comprise one or more gene sequence(s) which encode yuc2
(indole-3-
pyruvate monooxygenase). In one embodiment, the genetically engineered
bacteria
comprise one or more gene sequence(s) which encode yuc2 from Enterobacter
cloacae. In
one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one
embodiment, the
(L-tryptophan aminotransferase is from S. cerevisiae. In one embodiment, the
genetically
engineered bacteria comprise one or more gene sequence(s) which encode aro9
and yuc2.
In one embodiment, the genetically engineered bacteria comprise one or more
gene
sequence(s) which encode aspC (aspartate aminotransferase. In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
aspC from E. coli. In one embodiment, the genetically engineered bacteria
comprise one
or more gene sequence(s) which encode aspC and yuc2. In one embodiment, the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode taal
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(L-tryptophan-pyruvate aminotransferase, In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode taal from
Arabidopsis
thaliana. In one embodiment, the genetically engineered bacteria comprise one
or more
gene sequence(s) which encode taal and yuc2.In one embodiment, the genetically
engineered bacteria comprise one or more gene sequence(s) which encode sta0 (L-
tryptophan oxidase). In one embodiment, the genetically engineered bacteria
comprise one
or more gene sequence(s) which encode sta0 from streptomyces sp. TP-A0274. In
one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode sta0 and yuc2. In one embodiment, the genetically engineered
bacteria
comprise one or more gene sequence(s) which encode trpDH (Tryptophan
dehydrogenase). In one embodiment, the genetically engineered bacteria
comprise one or
more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In
one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode trpDH and yuc2.. In one embodiment, the genetically engineered
bacteria
comprise one or more gene sequence(s) which encode one or more of aro9 or aspC
or taal
or sta0 or trpDH. In one embodiment, the genetically engineered bacteria
comprise one or
more gene sequence(s) which encode one or more of aro9 or aspC or taal or sta0
or
trpDH and yuc2.
[0490] Another non-limiting example of gene sequence(s) for the production of
acetic acid are shown in FIG. 42D. In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode IaaM (Tryptophan 2-
monooxygenase). In one embodiment, the genetically engineered bacteria
comprise one or
more gene sequence(s) which encode IaaM from Pseudomonas savastanoi). In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode iaaH (Indoleacetamide hydrolase). In one embodiment, the
genetically
engineered bacteria comprise one or more gene sequence(s) which encode iaaH
from
Pseudomonas savastanoi). In one embodiment, the genetically engineered
bacteria
comprise one or more gene sequence(s) which encode IaaM and iaaH.
[0491] Another non-limiting example of gene sequence(s) for the production of
acetic acid are shown in FIG. 42E. In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode cyp71a13
(indoleacetaldoxime dehydratase). In one embodiment, the genetically
engineered bacteria
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comprise one or more gene sequence(s) which encode cyp71a13 from Arabidopis
thaliana.
In one embodiment, the genetically engineered bacteria comprise one or more
gene
sequence(s) which encode nitl (Nitrilase). In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode nitl from
Arabidopsis
thaliana. In one embodiment, the genetically engineered bacteria comprise one
or more
gene sequence(s) which encode iaaH (Indoleacetamide hydrolase). In one
embodiment,
the genetically engineered bacteria comprise one or more gene sequence(s)
which encode
iaaH from Pseudomonas savastanoi),In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode cyp79B2
(tryptophan N-
monooxygenase). In one embodiment, the genetically engineered bacteria
comprise one or
more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s)
which encode cyp79B2 and cyp71a13. In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode cyp79B2 from
Arabidopsis
thaliana. In one embodiment, the genetically engineered bacteria comprise one
or more
gene sequence(s) which encode cyp79B2 and nitl and/or iaaH. In one embodiment,
the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
cyp79B3 (tryptophan N-monooxygenase). In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode cyp79B3 from
Arabidopsis
thaliana. In one embodiment, the genetically engineered bacteria comprise one
or more
gene sequence(s) which encode cyp79B3 and cyp71a13. In one embodiment, the
genetically engineered bacteria comprise one or more gene sequence(s) which
encode
cyp79B3 and cyp71a13 and nitl and/or iaaH. In one embodiment, the genetically
engineered bacteria comprise one or more gene sequence(s) which encode
cyp79B3,
cyp79B2 and cyp71a13. In one embodiment, the genetically engineered bacteria
comprise
one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13, and
nitl
and/or iaaH. In one embodiment, the genetically engineered bacteria comprise
one or more
gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one
embodiment,
the genetically engineered bacteria comprise one or more gene sequence(s)
which encode
cyp79B3 and cyp71a13 and nitl and iaaH. In one embodiment, the genetically
engineered
bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2
and
cyp71a13 and nitl and iaaH.
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