Note: Descriptions are shown in the official language in which they were submitted.
1
MICROORGANISM AND METHOD FOR THE IMPROVED PRODUCTION OF SERINE
AND/OR CYSTEINE
Field of the invention
The present invention relates to a microorganism genetically modified for the
improved production of serine and/or cysteine and to a method for the improved
production
of serine and/or cysteine using said microorganism.
Background of the invention
Amino acids are used in many industrial fields, including the food, animal
feed,
cosmetics, pharmaceutical, and chemical industries and have an annual
worldwide market
growth rate of an estimated 5 to 7% (Leuchtenberger, et al., 2005). Among
these, serine
and derivatives thereof as cysteine are particularly important for use in
cosmetics and
medical industry, in particular in pharmaceutics. Indeed, serine has been
identified as one
the most interesting biochemicals due to its potential use as a building block
biochemical.
The biosynthesis of serine and cysteine which derives thereof, requires the
assembly
of an amine function in place of the ketonic function of the 3 phospho-
hydroxypyruvate to
give phospho-serine which will give serine after a dephosphorylation, which
can be
subsequently converted into cysteine.
Serine and cysteine may be produced via chemical synthesis, or microbial
fermentation. Due to the associated environmental advantages, replacement of
chemical
production by fermentation is considered as attractive and promising
approaches. In
addition, fermentation provides a useful way of using abundant, renewable,
and/or
inexpensive materials as the main source of carbon. In particular, E. coil is
a bacterial model
organism for metabolic engineering, which is successfully employed as a cell
factory for
production of a range of biochemicals. The E. coil bacterium has long been
used for the
production of proteinogenic amino acids such as serine and cysteine, for many
biotechnological applications.
Engineered strains which accumulates serine were constructed in the art, with
different strategies to improve serine production. Some of which were mainly
focusing on
decreasing degradation of serine in the production organism. For example,
genetically
engineered microorganisms deficient in serine degradation pathways catalyzed
by serine
deaminases activity and serine hydroxymethyltransferase activity were
disclosed in
W02016/120326. As a further example, others rather described stimulation of
enzymes
involved in the biosynthesis of serine as such, for example those leading to L-
serine
production from 3-phosphoglycerate as in U52019/233857.
Date Recue/Date Received 2023-12-29
2
However, it is difficult to obtain optimal productions of serine, and
consequently
cysteine, because their biosynthesis competes with energy production and cell
division for
carbon utilization.
In view of the ever-increasing demand for serine and cysteine in industrial
applications, there remains a need for further improvements in the production
of these
amino acids. In particular, there remains a need for improved microorganisms
that are able
to produce serine or cysteine with high levels of productivity, titer, and
yield, in particular
from an inexpensive and/or abundant carbon source such as glucose. There also
remains
a need for improved methods for the production of serine or cysteine on an
industrial scale,
ideally wherein the productivity, titer, and yield of serine or cysteine is at
least similar to that
obtained with current methods.
Brief description of the invention
The present invention addresses the above needs, providing a microorganism
genetically modified for the production of serine and/or cysteine and methods
for the
production of serine and/or cysteine using said microorganism.
The microorganism genetically modified for the production of serine and/or
cysteine
notably expresses a heterologous gapN gene coding an NADP-dependent
glyceraldehyde-
3-phosphate dehydrogenase and has attenuated expression of gapA gene coding
glyceraldehyde-3-phosphate dehydrogenase A and attenuated expression of sdaA
and/or
sdaB gene(s) coding L-serine deaminases (ie. L-serine dehydratases), as
compared to an
unmodified microorganism. Indeed, the inventors have found that by such a
microorganism
advantageously shows improved production of serine or cysteine as
productivity, titer and
yield are increased.
Preferably, the gapN gene codes an NADP-dependent glyceraldehyde-3-phosphate
.. dehydrogenase having at least 80% identity with GapN from Streptococcus
mutans.
Preferably, the gapA gene is deleted.
Preferably, the microorganism further comprises an attenuation of the
expression of
the gapB and/or gapC genes as compared to an unmodified microorganism,
preferably a
deletion of the gapB and gapC genes.
Preferably, the microorganism further comprises attenuation of the gpmA gene
and/or
glyA gene(s), as compared to an unmodified microorganism.
Preferably, the microorganism further comprises an overexpression of at least
one
gene selected in the group consisting of: serA, serB, serC and eamA,
preferably at least
serA, more preferably at least serA and serB, as compared to an unmodified
microorganism; where advantageously the gene serA is serA*.
Preferably, the microorganism further comprises an overexpression of the gdhA
gene
as compared to an unmodified microorganism.
Date Recue/Date Received 2023-12-29
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Preferably, the microorganism further comprises the attenuation of at least
one gene
selected in the group consisting of tdcG and tdcB as compared to an unmodified
microorganism.
Preferably, the microorganism further comprises: a) an attenuation of the
expression
of the genes ptsHIcrr and/or the ptsG gene, preferably a deletion of the genes
ptsHIcrr
and/or the ptsG gene,
b) an attenuation of the expression of the gpmM gene, preferably a deletion of
the
gpmM gene, and
c) an overexpression of the galP gene,
as compared to an unmodified microorganism.
Preferably, the microorganism further comprises:
a) an attenuation of the expression of the gpmM gene, preferably a deletion of
the
gpmM gene, and
b) an overexpression of the csc genes,
as compared to an unmodified microorganism.
Preferably, the microorganism further comprises:
a) an attenuation of the expression of the pykA and pykF genes, preferably a
deletion
of the pykA and pykF genes, and
b) an overexpression of the scr genes,
as compared to an unmodified microorganism.
Preferably, the microorganism is genetically modified for the production of
cysteine
and comprises the overexpression of at least one gene selected in the group
consisting of
cysE, cysK and cysM, preferably the overexpression of cysE and cysK or cysE
and cysM,
more preferably of cysE and cysK, as compared to an unmodified microorganism;
where
advantageously the gene cysE is cysE*.
Preferably, in the microorganism of the invention, the expression of at least
one gene
selected from the group consisting of udhA, mgsA, ackA, ptA, pflAB, frdABCD,
IdhA, adhE,
zwf, edd, eda, gnd is attenuated, preferably the expression of the genes udhA,
mgsA, zwf,
edd, eda and gnd is attenuated, more preferably the expression of the genes
udhA, mgsA,
edd and eda is attenuated, as compared to an unmodified microorganism.
Preferably, the microorganism belongs to the Escherichia genus, more
preferably
wherein the microorganism is Escherichia coli, the Corynebacterium genus, more
preferably
wherein the microorganism is Corynebacterium glutamicum, or the Streptococcus
genus,
more preferably wherein the microorganism is chosen among Streptococcus
thermophilus
and Streptococcus salivarius, most preferably wherein the microorganism is
Escherichia
coil.
Date Recue/Date Received 2023-12-29
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The invention further relates to a method for the production of serine and/or
cysteine
comprising the steps of:
a) culturing a microorganism genetically modified for the production of
serine and/or
cysteine as provided herein in an appropriate culture medium comprising a
source of carbon, and
b) recovering serine and/or cysteine from the culture medium.
Preferably, the source of carbon is glucose, fructose, galactose, lactose,
sucrose or
any combination thereof.
Preferably step b) of the method comprises a step of purification of serine
and/or
cysteine.
Detailed Description
Before describing the present invention in detail, it is to be understood that
the
invention is not limited to particularly exemplified microorganism and/or
methods and may,
of course, vary. Indeed, various modifications, substitutions, omissions, and
changes may
be made without departing from the scope of the invention. It shall also be
understood that
the terminology used herein is for the purpose of describing particular
embodiments of the
invention only, and is not intended to be limiting.
All publications, patents and patent applications cited herein, whether supra
or infra,
are hereby incorporated by reference in their entirety. Furthermore, the
practice of the
present invention employs, unless otherwise indicated, conventional
microbiological and
molecular biological techniques that are within the skill of the art. Such
techniques are well-
known to the skilled person, and are fully explained in the literature.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as are commonly understood by one of ordinary skill in the art to
which this
invention belongs. Although any materials and methods similar or equivalent to
those
described herein can be used to practice or test the present invention,
preferred material
and methods are provided.
It must be noted that as used herein and in the appended claims, the singular
forms
"a," "an," and "the," include plural reference unless the context clearly
dictates otherwise.
Thus, for example, a reference to "a microorganism" includes a plurality of
such
microorganisms, and so forth.
The terms "comprise," "contain," "include," and variations thereof such as
"comprising"
are used herein in an inclusive sense, i.e., to specify the presence of the
stated features
but not to preclude the presence or addition of further features in various
embodiments of
the invention.
A first aspect of the invention relates to a microorganism genetically
modified for the
production of serine and/or cysteine. The term "microorganism," as used
herein, refers to
Date Recue/Date Received 2023-12-29
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a living microscopic organism, which may be a single cell or a multicellular
organism and
which can generally be found in nature. The microorganism provided herein is
preferably a
bacterium. Preferably, the microorganism is selected within the
Enterobacteriaceae,
Streptococcaceae, or Corynebacteriaceae family. More preferably, the
microorganism is a
species of the Escherichia, Streptococcus, or Corynebacterium genus. Even more
preferably, said Enterobacteriaceae bacterium is Escherichia coil, said
Streptococcaceae
bacterium is Streptococcus thermophilus or Streptococcus saliva rius, and said
Corynebacteriaceae bacterium is Corynebacterium glutamicum. Most preferably,
the
microorganism is Escherichia coil.
The terms "recombinant microorganism," "genetically modified microorganism,"
or
"microorganism genetically modified" are used interchangeably herein and refer
to a
microorganism or a strain of microorganism that has been genetically modified
or
genetically engineered. This means, according to the usual meaning of these
terms, that
the microorganism of the invention is not found in nature and is genetically
modified when
compared to the "parental" microorganism from which it is derived. The
"parental"
microorganism may occur in nature (i.e., a wild-type microorganism) or may
have been
previously modified. The recombinant microorganism of the invention may
notably be
modified by the introduction, deletion, and/or modification of genetic
elements. Such
modifications can be performed, e.g., by genetic engineering or by adaptation,
wherein a
microorganism is cultured in conditions that apply a specific stress on the
microorganism
and induce mutagenesis and/or by forcing the development and evolution of
metabolic
pathways by combining directed mutagenesis and evolution under specific
selection
pressure.
A microorganism genetically modified for the increased production of serine
and/or
cysteine means that said microorganism is a recombinant microorganism that has
increased production of serine and/or cysteine as compared to a parent
microorganism
which does not comprise the genetic modification. In other words, said
microorganism has
been genetically modified for increased production of serine and/or cysteine
as compared
to a corresponding unmodified microorganism.
A microorganism may notably be modified to modulate the expression level of an
endogenous gene or the level of production of the corresponding protein or the
activity of
the corresponding enzyme. The term "endogenous gene" means that the gene was
present
in the microorganism before any genetic modification. Endogenous genes may be
overexpressed by introducing heterologous sequences in addition to, or to
replace,
endogenous regulatory elements. Endogenous genes may also be overexpressed by
introducing one or more supplementary copies of the gene into the chromosome
or on a
plasmid. In this case, the endogenous gene initially present in the
microorganism may be
Date Recue/Date Received 2023-12-29
6
deleted. Endogenous gene expression levels, protein production levels, or the
activity of the
encoded protein, can also be increased or attenuated by introducing mutations
into the
coding sequence of a gene or into non-coding sequences. These mutations may be
synonymous, when no modification in the corresponding amino acid occurs, or
non-
synonymous, when the corresponding amino acid is altered. Synonymous mutations
do not
have any impact on the function of translated proteins, but may have an impact
on the
regulation of the corresponding genes or even of other genes, if the mutated
sequence is
located in a binding site for a regulator factor. Non-synonymous mutations may
have an
impact on the function or activity of the translated protein as well as on
regulation,
depending the nature of the mutated sequence.
In particular, mutations in non-coding sequences may be located upstream of
the
coding sequence (i.e., in the promoter region, in an enhancer, silencer, or
insulator region,
in a specific transcription factor binding site) or downstream of the coding
sequence.
Mutations introduced in the promoter region may be in the core promoter,
proximal promoter
or distal promoter. Mutations may be introduced by site-directed mutagenesis
using, for
example, Polymerase Chain Reaction (PCR), by random mutagenesis techniques for
example via mutagenic agents (Ultra-Violet rays or chemical agents like
nitrosoguanidine
(NTG) or ethylmethanesulfonate (EMS)) or DNA shuffling or error-prone PCR or
using
culture conditions that apply a specific stress on the microorganism and
induce
mutagenesis. The insertion of one or more supplementary nucleotide(s) in the
region
located upstream of a gene can notably modulate gene expression.
A particular way of modulating endogenous gene expression is to exchange the
endogenous promoter of a gene (e.g., wild-type promoter) with a stronger or
weaker
promoter to upregulate or downregulate expression of the endogenous gene. The
promoter
may be endogenous (i.e., originating from the same species) or exogenous
(i.e., originating
from a different species). It is well within the ability of the person skilled
in the art to select
an appropriate promoter for modulating the expression of an endogenous gene.
Such a
promoter be, for example, a Ptrc, Ptac, Ptet, or Plac promoter, or a lambda PL
(PL) or
lambda PR (PR) promoter. The promoter may be "inducible" by a particular
compound or by
specific external conditions, such as temperature or light or a small
molecule, such as an
antibiotic.
A particular way of modulating endogenous protein activity is to introduce
nonsynonymous mutations in the coding sequence of the corresponding gene,
e.g.,
according to any of the methods described above. A non-synonymous amino acid
mutation
that is present in a transcription factor may notably alter binding affinity
of the transcription
factor toward a cis-element, alter ligand binding to the transcription factor,
etc.
Date Recue/Date Received 2023-12-29
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A microorganism may also be genetically modified to express one or more
exogenous
or heterologous genes so as to overexpress the corresponding gene product
(e.g., an
enzyme). An "exogenous" or "heterologous" gene as used herein refers to a gene
encoding
a protein or polypeptide that is introduced into a microorganism in which said
gene does
not naturally occur. The gapN and Scr genes are notably heterologous genes in
the context
of the present invention. In particular, a heterologous gene may be directly
integrated into
the chromosome of the microorganism, or be expressed extra-chromosomally
within the
microorganism by plasmids or vectors. For successful expression, the
heterologous gene(s)
must be introduced into the microorganism with all of the regulatory elements
necessary for
their expression or be introduced into a microorganism that already comprises
all of the
regulatory elements necessary for their expression. The genetic modification
or
transformation of microorganisms with one or more exogenous genes is a routine
task for
those skilled in the art.
One or more copies of a given heterologous gene can be introduced on a
chromosome by methods well-known in the art, such as by genetic recombination.
When a
gene is expressed extra-chromosomally, it can be carried by a plasmid or a
vector. Different
types of plasmid are notably available, which may differ in respect to their
origin of
replication and/or on their copy number in the cell. For example, a
microorganism
transformed by a plasmid can contain 1 to 5 copies of the plasmid, about 20
copies, or even
up to 500 copies, depending on the nature of the selected plasmid. A variety
of plasmids
having different origins of replication and/or copy numbers are well-known in
the art and
can be easily selected by the skilled practitioner for such purposes,
including, for example,
pTrc, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, or
pCL1920.
It should be understood that, in the context of the present invention, when a
heterologous gene encoding a protein of interest is expressed in a
microorganism, such as
E. colt, a synthetic version of this gene is preferably constructed by
replacing non-preferred
codons or less preferred codons with preferred codons of said microorganism
which encode
the same amino acid. Indeed, it is well-known in the art that codon usage
varies between
microorganism species, and that this may impact the recombinant production
level of a
protein of interest. To overcome this issue, codon optimization methods have
been
developed, and are extensively described by Graf et aL (2000), Deml etal.
(2001) and Davis
& Olsen (2011). Several software programs have notably been developed for
codon
optimization determination such as the GeneOptimizer software
(Lifetechnologies) or the
OptimumGeneTM software of (GenScript). In other words, the heterologous gene
encoding
a protein of interest is preferably codon-optimized for production in the
chosen
Date Recue/Date Received 2023-12-29
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microorganism. As a particular example, the heterologous gapN gene may be
codon
optimized for expression in a microorganism such as E. coil.
On the basis of a given amino acid sequence, the skilled person is furthermore
able
to identify an appropriate polynucleotide coding for said polypeptide (e.g.,
in the available
databases, such as Uniprot), or to synthesize the corresponding polypeptide or
a
polynucleotide coding for said polypeptide. De novo synthesis of a
polynucleotide can be
performed, for example, by initially synthesizing individual nucleic acid
oligonucleotides and
hybridizing these with oligonucleotides complementary thereto, such that they
form a
double-stranded DNA molecule, and then ligating the individual double-stranded
.. oligonucleotides such that the desired nucleic acid sequence is obtained.
The terms "production," "overproducting," or "overproduction" of a protein of
interest,
such as an enzyme, refer herein to an increase in the production level and/or
activity of said
protein in a microorganism, as compared to the corresponding parent
microorganism that
does not comprise the modification present in the genetically modified
microorganism (i.e.,
in the unmodified microorganism). A heterologous gene or protein can be
considered to be
respectively "expressed" or "overexpressed" and "produced" or "overproduced"
in a
genetically modified microorganism when compared with a corresponding parent
microorganism in which said heterologous gene or protein is absent. In
contrast, the terms
"attenuating" or "attenuation" of the synthesis of a protein of interest refer
to a decrease in
.. the production level and/or activity of said protein in a microorganism, as
compared to the
parent microorganism. Similarly, an "attenuation" of gene expression refers to
a decrease
in the level of gene expression as compared to the parent microorganism. An
attenuation
of expression can notably be due to either the exchange of the wild-type
promoter for a
weaker natural or synthetic promoter or the use of an agent reducing gene
expression, such
as antisense RNA or interfering RNA (RNAi), and more particularly small
interfering RNAs
(siRNAs) or short hairpin RNAs (shRNAs). Promoter exchange may notably be
achieved by
the technique of homologous recombination (Datsenko & Wanner, 2000). The
complete
attenuation of the production level and/or activity of a protein of interest
means that
production and/or activity is abolished; thus, the production level of said
protein is null. The
complete attenuation of the production level and/or activity of a protein of
interest may be
due to the complete suppression of the expression of a gene. This suppression
can be
either an inhibition of the expression of the gene, a deletion of all or part
of the promoter
region necessary for expression of the gene, or a deletion of all or part of
the coding region
of the gene. A deleted gene can notably be replaced by a selection marker gene
that
.. facilitates the identification, isolation and purification of the modified
microorganism. As a
non-limiting example, suppression of gene expression may be achieved by the
technique
Date Recue/Date Received 2023-12-29
9
of homologous recombination, which is well-known to the person skilled in the
art (Datsenko
& Wanner, 2000).
Modulating the production level of one or more proteins may thus occur by
altering
the expression of one or more endogenous genes that encode said protein within
the
microorganism as described above and/or by introducing one or more
heterologous genes
that encode said protein(s) into the microorganism.
The term "production level" as used herein, refers to the amount (e.g.,
relative amount,
concentration) of a protein of interest (or of the gene encoding said protein)
expressed in a
microorganism, which is measurable by methods well-known in the art. The level
of gene
expression can be measured by various known methods including Northern
blotting,
quantitative RT-PCR, and the like. Alternatively, the level of production of
the protein coded
by said gene may be measured, for example by SDS-PAGE, HPLC, LC/MS and other
quantitative proteomic techniques (Bantscheff et al., 2007), or, when
antibodies against said
protein are available, by Western Blot-Immunoblot (Burnette, 1981), Enzyme-
linked
immunosorbent assay (e.g., ELISA) (Engvall and Perlman, 1971), protein
immunoprecipitation, immunoelectrophoresis, and the like. The copy number of
an
expressed gene can be quantified, for example, by restricting chromosomal DNA
followed
by Southern blotting using a probe based on the gene sequence, fluorescence in
situ
hybridization (FISH), qPCR, and the like.
Overexpression of a given gene or overproduction of the corresponding protein
may
be verified by comparing the expression level of said gene or the level of
synthesis of said
protein in the genetically modified organism to the expression level of the
same gene or the
level of synthesis of the same protein, respectively, in a control
microorganism that does
not have the genetic modification (i.e., the parental strain or unmodified
microorganism).
The microorganism genetically modified for the production of serine and/or
cysteine
provided herein comprises
- a heterologous enzyme having NADP-dependent glyceraldehyde-3-phosphate
dehydrogenase activity, and
- an attenuation of the activity of glyceraldehyde-3-phosphate
dehydrogenase A
(GapA) and of the activity of L-serine deaminase(s) (SdaA and/or SdaB) as
compared to an
unmodified microorganism.
Indeed, the inventors have shown that the above genetic modifications
advantageously improve serine and cysteine titer, productivity, and yield, as
compared to a
microorganism that does not comprise these modifications.
The "activity" or "function" of an enzyme designates the reaction that is
catalyzed by
said enzyme for converting its corresponding substrate(s) into another
molecule(s) (i.e.,
product(s)). As is well-known in the art, the activity of an enzyme may be
assessed by
Date Recue/Date Received 2023-12-29
10
measuring its catalytic efficiency and/or Michaelis constant. Such an
assessment is
described for example in Segel, 1993, in particular on pages 44-54 and 100-
112,
incorporated herein by reference.
The enzyme having NADP-dependent glyceraldehyde-3-phosphate dehydrogenase
activity may be either a phosphorylating or a non-phosphorylating enzyme. It
is preferably
GapN. GapN may be of bacterial, archaeal, or eukaryotic origin. Preferably,
GapN is of
bacterial origin. GapN may notably be one of those described in Figure 4 of
Iddar et al.,
2005, incorporated herein by reference. In particular, the GapN enzyme may be
from a
species of the Streptococcus genus (e.g., from S. mutans, S. pyogenes), a
species of the
Bacillus genus (e.g., B. cereus, B. licheniformis, B. thuringiensis), a
species of the
Clostridium genus (e.g., C. acetobutylicum), or from Pisum savitum.
Preferably, the GapN
enzyme is from S. mutans, S. pyo genes, C. acetobutylicum, B. cereus, or P.
sativum, more
preferably from S. mutans. GapN preferably has at least 80%, 90%, 95%, or 100%
sequence similarity or sequence identity with the GapN enzyme having the
sequence of
SEQ ID NO: 23, 107, 109, 111, or 113. More preferably, GapN has the sequence
of SEQ
ID NO: 23. GapN may be a functional variant or functional fragment of one of
the GapN
enzymes described herein. The corresponding gapN gene, which codes GapN,
preferably
has at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 22, 106,
108,
110, or 112, more preferably SEQ ID NO: 22.
A "functional fragment" of an enzyme, as used herein, refers to parts of the
amino
acid sequence of an enzyme comprising at least all the regions essential for
exhibiting the
biological activity of said enzyme. These parts of sequences can be of various
lengths,
provided that the biological activity of the amino acid sequence of the enzyme
of reference
is retained by said parts. In other words, a functional fragment of an enzyme
as provided
herein is enzymatically active.
A "functional variant" as used herein refers to a protein that is structurally
different
from the amino acid sequence of a reference protein but that generally retains
all the
essential functional characteristics of said reference protein. A variant of a
protein may be
a naturally-occurring variant or a non-naturally occurring variant. Such non-
naturally
occurring variants of the reference protein can be made, for example, by
mutagenesis
techniques on the coding nucleic acids or genes, for example by random
mutagenesis or
site-directed mutagenesis.
Structural differences may be limited in such a way that the amino acid
sequence of
reference protein and the amino acid sequence of the variant may be closely
similar overall,
and identical in many regions. Structural differences may result from
conservative or non-
conservative amino acid substitutions, deletions and/or additions between the
amino acid
sequence of the reference protein and the variant. The only proviso is that,
even if some
Date Recue/Date Received 2023-12-29
11
amino acids are substituted, deleted and/or added, the biological activity of
the amino acid
sequence of the reference protein is retained by the variant. As a non-
limiting example,
such a variant of GapN conserves its NADP-dependent glyceraldehyde-3-phosphate
dehydrogenase activity. The capacity of the variants to exhibit such activity
can be assessed
according to in vitro tests known to the person skilled in the art. It should
be noted that the
activity of said variants may differ in efficiency as compared to the activity
of the amino acid
sequences of the enzymes of reference provided herein (e.g., the genes/enzymes
provided
herein of a particular species of microorganism or having particular sequences
as provided
in the corresponding SEQ ID NO).
A "functional variant" of an enzyme as described herein includes, but is not
limited to,
enzymes having amino acid sequences which are at least 60% similar or
identical after
alignment to the amino acid sequence encoding an enzyme as provided herein.
According
to the present invention, such a variant preferably has at least 70%, 80%,
85%, 90%, 95%,
96%, 97%, 98%, 99%, or 100% amino acid sequence similarity or identity to the
protein
described herein. Said functional variant furthermore has the same enzymatic
function as
the enzyme provided herein. As a non-limiting example, a functional variant of
GapN of
SEQ ID NO: 23 has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100%
sequence identity to said sequence. As a non-limiting example, means of
determining
sequence identity are further provided below.
Preferably, the attenuation of GapA activity and SdaA and/or SdaB activity
results
from an inhibition of expression of the gapA gene and the sdaA and/or sdaB
gene(s) as
compared to an unmodified microorganism. The activity of the GapA enzyme
and/or the
activity of SdaA and/or SdaB enzyme(s) may be completely attenuated. Complete
attenuation is preferably due to a partial or complete deletion of the gene
coding for the
enzyme. Preferably, GapA has at least 80%, 90%, 95%, or 100% sequence
similarity or
sequence identity with the sequence of SEQ ID NO: 21. Preferably, the gapA
gene has at
least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO:
20.
Preferably, the gapA gene is deleted. Preferably, SdaA has at least 80%, 90%,
95%, or
100% sequence similarity or sequence identity with the sequence of SEQ ID NO:
10.
Preferably, the sdaA gene has at least 80%, 90%, 95%, or 100% sequence
identity with the
sequence of SEQ ID NO: 9. Preferably, SdaB has at least 80%, 90%, 95%, or 100%
sequence similarity or sequence identity with the sequence of SEQ ID NO: 12.
Preferably,
the sdaB gene has at least 80%, 90%, 95%, or 100% sequence identity with the
sequence
of SEQ ID NO: 11.
The microorganism genetically modified for the production of serine and/or
cysteine
microorganism of the present invention preferably comprises:
Date Recue/Date Received 2023-12-29
12
- the expression of a heterologous gapN gene coding an NADP-dependent
glyceraldehyde-3-phosphate dehydrogenase, and
- an attenuation of the expression of the gapA gene and of the expression
of the sdaA
and/or sdaB gene(s) as compared to an unmodified microorganism.
In addition to the modifications described above, the genetically modified
microorganism for production of serine and/or cysteine may comprise one or
more
additional modifications among those described below.
In particular, said microorganism may further comprise an attenuation of D-
erythrose-
4-phosphate dehydrogenase (GapB) activity. Preferably, production of GapB is
partially or
completely attenuated. Preferably, GapB has at least 80%, 90%, 95%, or 100%
sequence
similarity or sequence identity with the sequence of SEQ ID NO: 26.
Preferably, attenuation
of GapB activity results from an inhibition of the expression of the gapB gene
coding said
enzyme. Preferably, attenuation of expression results from a partial or
complete deletion of
the gapB gene. Preferably, the gapB gene has at least 80%, 90%, 95%, or 100%
sequence
identity with the sequence of SEQ ID NO: 25.
Said microorganism may further comprise an attenuation of glyceraldehyde-3-
phosphate dehydrogenase (GapC) activity. Preferably, production of GapC is
partially or
completely attenuated. Preferably, GapC has at least 80%, 90%, 95%, or 100%
sequence
similarity or sequence identity with the sequence of SEQ ID NO: 115, 117, or
119.
Preferably, said attenuation results from an inhibition of expression of the
gapC gene coding
said enzyme. Preferably, attenuation of expression results from a partial or
complete
deletion of the gapC gene. In some microorganisms, the gapC gene is a
pseudogene. Thus,
"gapC" as used herein may refer to a functional gene or to a pseudogene. The
gapC
pseudogene or functional gene, preferably has at least 80%, 90%, 95%, or 100%
sequence
identity with the sequence of SEQ ID NO: 27, 114, 116 or 118. In cases where
gapC is a
pseudogene, said pseudogene is advantageously deleted in order to avoid
reversion of
pseudogene into functional gene.
Preferably, the microorganism comprises an attenuation of the expression of
the gapB
gene and deletion of gapC pseudogene as compared to an unmodified
microorganism,
more preferably a deletion of the gapB and gapC genes.
The microorganism genetically modified for the production of serine and/or
cysteine
preferably further comprises attenuated activity of the phosphoglycerate
mutase GpmA, as
compared to an unmodified microorganism.
Preferably, GpmA has at least 80%, 90%, 95%, or 100% sequence similarity or
sequence identity with the sequence of SEQ ID NO: 29.
Preferably, the attenuation of this phosphoglycerate mutase results from an
attenuation of the gene coding said protein (i.e. the gene gpmA). Preferably,
the gpmA gene
Date Recue/Date Received 2023-12-29
13
has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ
ID NO:
28.
The microorganism genetically modified for the production of serine and/or
cysteine
preferably further comprises attenuated activity of the serine
hydroxymethyltransferase
GlyA, as compared to an unmodified microorganism.
Preferably, GlyA has at least 80%, 90%, 95%, or 100% sequence similarity or
sequence identity with the sequence of SEQ ID NO: 121.
Preferably, the attenuation of this serine hydroxymethyltransferase results
from an
attenuation of the gene coding said protein (i.e. the gene glyA). Preferably,
the glyA gene
has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ
ID NO:
120.
Preferably, the microorganism comprises attenuation of the gpmA and/or glyA
gene(s), as compared to an unmodified microorganism.
The microorganism for the production of serine may comprise an increased
activity of
at least one of the following L-serine deaminases: phosphoglycerate
dehydrogenase
(SerA), SerA*, phosphoserine phosphatase (SerB) and
phosphoserine/phosphohydroxythreonine aminotransferase (SerC), as compared to
an
unmodified microorganism. The microorganism for the production of serine may
also
comprise an increased activity of the cysteine/O-acetylserine exporter (EamA,
also known
as YdeD).
Preferably, the microorganism for the production of serine comprises an
overproduction of at least one of the following proteins: SerA, SerA*, SerB,
SerC and EamA.
SerA* is a feedback resistant (FBR) protein.
The term "feedback resistant protein" as used herein refers to a protein which
has
been modified such that feedback inhibition of the protein (i.e., the
reduction in enzyme
activity mediated by the binding of the product to the enzyme) is reduced or
even eliminated.
Preferably, SerA, SerB, SerC and EamA have at least 80%, 90%, 95%, or 100%
sequence similarity or sequence identity with the sequences of SEQ ID NOs: 14,
16, 18 and
123, respectively. When SerA* is overproduced rather than SerA, said protein
preferably
has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity
with the
sequence of SEQ ID NO: 14. SerA* comprises the substitution of asparagine
residue at
position 364 by an alanine residue when compared to SEQ ID NO: 14.
Preferably, the overproduction of said one or more proteins results from an
overexpression of at least one of the genes coding said protein (i.e., serA
(or serA*), serB,
serC and/or eamA genes). Preferably, the serA, serB, serC and eamA genes have
at least
80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NOs: 13,
15, 17
and 122, respectively. Preferably, the serA* gene has at least 80%, 90%, 95%,
or 100%
Date Recue/Date Received 2023-12-29
14
sequence identity with the sequence of SEQ ID NO: 13, wherein serA* codes for
a protein
having the substitution of asparagine residue at position 364 by an alanine
residue with
reference to the wild-type protein having the sequence SEQ ID NO: 14.
Preferably, the microorganism is genetically modified for the production of
serine and
.. comprises an overexpression of the following genes: serA, serB, serC and
eamA, and more
preferably serA*, serB, serC and eamA, as compared to an unmodified
microorganism.
Preferably, overexpression of serA, serA*, serB, serC occurs by replacing the
native
promoter with an artificial promoter, such as the Ptrc promoter.
Alternatively, a vector
comprising one or more genes under the control of a strong or inducible
promoter (e.g., the
pCL1920 vector) may be introduced into the microorganism and the gene(s)
overexpressed.
Preferably, eamA is overexpressed under the native promoter.
The microorganism for the production of serine may further comprise an
increased
activity of the glutamate dehydrogenase GdhA, as compared to an unmodified
microorganism. Preferably, the microorganism for the production of serine
comprises an
overproduction of the glutamate dehydrogenase GdhA.
Preferably, GdhA has at least 80%, 90%, 95%, or 100% sequence similarity or
sequence identity with the sequences of SEQ ID NO: 31.
Preferably, the overproduction of said protein results from an overexpression
of the
gene coding said protein (i.e., gdhA gene). Preferably, the gdhA gene has at
least 80%,
90%, 95%, or 100% sequence identity with the sequences of SEQ ID NO: 30.
Preferably, the microorganism is genetically modified for the production of
serine and
comprises an overexpression of the gdhA gene, as compared to an unmodified
microorganism.
The microorganism genetically modified for the production of serine and/or
cysteine
preferably further comprises attenuated activity of at least one of the
following L-serine
deaminases: TdcG and TdcB, as compared to an unmodified microorganism.
Preferably, TdcG and TdcB have at least 80%, 90%, 95%, or 100% sequence
similarity or sequence identity with the sequences of SEQ ID NOs: 36 and 38,
respectively.
Preferably, the attenuation of said one or more proteins results from an
attenuation of
the gene coding said protein (i.e., tdcG and/or tdcB genes). Preferably, the
tdcG and tdcB
genes have at least 80%, 90%, 95%, or 100% sequence identity with the
sequences of SEQ
ID NOs: 35 and 37, respectively.
Preferably, the microorganism is genetically modified for the production of
serine
and/or cysteine and comprises an attenuation of at least one of the following
genes: tdcG
and tdcB genes, and more preferably of tdcG and tdcB genes, as compared to an
unmodified microorganism.
Date Recue/Date Received 2023-12-29
15
In the following first specific embodiment, the microorganism genetically
modified for
the production of serine or cysteine as described herein is further modified
to be able to use
glucose as a carbon source.
Thus, according to this first embodiment, the microorganism of the invention
which is
genetically modified for the production of serine and/or cysteine as described
above, further
comprises:
a) an attenuation of phosphoenolpyruvate-dependent phosphotransferase system
(PTS) activity, preferably an inhibition of said activity,
b) an attenuation of the phosphoglycerate mutase GpmM and/or GpmB,
preferably
at least GpmM, and more preferably an inhibition of the GpmM activity,
c) an increased galactose-proton symporter (GalP) activity,
as compared to an unmodified microorganism.
Preferably, the microorganism for the production of serine and/or cysteine
comprises
an attenuation of the proteins PtsHICrr and/or PtsG. Preferably, PtsH, Ptsl,
and PtsG have
at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with
the
sequence of SEQ ID NO: 42, 44, and 77, respectively. Preferably, Crr has at
least 80%,
90%, 95%, or 100% sequence similarity or sequence identity with the sequence
of SEQ ID
NO: 46.
Preferably, the attenuation of said one or more proteins results from an
attenuation of
the gene coding said protein (i.e., ptsHIcrr and/or ptsG genes). Preferably,
the ptsH, pstl,
ptsG genes have at least 80%, 90%, 95%, or 100% sequence identity with the
sequences
of SEQ ID NOs: 41, 43, and 76, respectively. Preferably, the crr gene has at
least 80%,
90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 45.
Preferably, the
microorganism is genetically modified for the production of serine an/or
cysteine and
comprises an attenuation of the ptsHIcrr and ptsG genes, as compared to an
unmodified
microorganism.
Preferably, expression of PtsHICrr and/or PtsG is partially or completely
attenuated.
Preferably, attenuation of PtsHICrr and/or PtsG activity results from an
inhibition of
expression of the ptsHIcrr and/or ptsG genes coding said enzymes. Preferably,
attenuation
of expression results from a partial or complete deletion of the ptsHIcrr
and/or ptsG genes,
more preferably from a partial or complete deletion of the ptsHIcrr and ptsG
genes.
Preferably, the GpmM and GpmB proteins have at least 80%, 90%, 95%, or 100%
sequence similarity or sequence identity with the sequence of SEQ ID NO: 40
and 79,
respectively. Preferably, the microorganism for the production of serine
and/or cysteine
comprises an attenuation of the protein GpmM, as compared to an unmodified
microorganism.
Date Recue/Date Received 2023-12-29
16
Preferably, the attenuation of said one or more proteins results from an
attenuation of
the gene coding said protein (i.e., gpmM and/or gpmB genes). Preferably, the
gpmM and
gpmB genes have at least 80%, 90%, 95%, or 100% sequence identity with the
sequences
of SEQ ID NOs: 39 and 78, respectively. Preferably, the microorganism for the
production
of serine and/or cysteine comprises an attenuation of the gpmM gene, as
compared to an
unmodified microorganism.
Preferably, expression of GpmM and/or GpmB is partially or completely
attenuated.
Preferably, attenuation of GpmM and/or GpmB activity results from an
inhibition of
expression of the gpmM and/or gpmB genes coding said enzymes, most preferably
from an
inhibition of expression of the gpmM gene. Preferably, attenuation of
expression results
from a partial or complete deletion of the gpmM and/or gpmB genes, more
preferably from
a partial or complete deletion of the gpmM and/or gpmB genes, most preferably
from a
partial or complete deletion of the gpmM gene.
Preferably, the microorganism for the production of serine and/or cysteine
comprises
an overproduction of the galactose-proton symporter GalP.
Preferably, GalP has at least 80%, 90%, 95%, or 100% sequence similarity or
sequence identity with the sequences of SEQ ID NO: 48.
Preferably, the overproduction of said protein results from an overexpression
of the
gene coding said protein (i.e., galP gene), as compared to an unmodified
microorganism.
Preferably, the galP gene has at least 80%, 90%, 95%, or 100% sequence
identity with the
sequences of SEQ ID NO: 47.
Preferably in this first embodiment, the microorganism is genetically modified
for the
production of serine and/or cysteine as described above and further comprises:
a) an attenuation of the expression of the ptsl-lIcrr genes and/or the ptsG
gene,
preferably a deletion of the genes ptsl-lIcrr and/or the ptsG gene,
b) an attenuation of the expression of the gpmM gene and/or the gpmB gene,
preferably a deletion of the gpmM gene and/or the gpmB gene, and
c) an overexpression of the galP gene,
as compared to an unmodified microorganism.
Preferably, in the first specific embodiment as described herein, the
microorganism is
further modified to comprise attenuation of glyA and/or overexpression of
eamA, as
compared to an unmodified microorganism, more preferably to comprise deletion
of glyA
and/or overexpression of eamA, and most preferably to comprise deletion of
glyA and
overexpression of eamA, as compared to an unmodified microorganism.
In the two following other specific embodiments, the microorganism genetically
modified for the production of serine or cysteine as described herein is
further modified to
be able to use sucrose as a carbon source. Preferably, proteins involved in
the import and
Date Recue/Date Received 2023-12-29
17
metabolism of sucrose are overproduced. Preferably, the following proteins are
overproduced:
- CscB sucrose permease, CscA sucrose hydrolase, CscK fructokinase, and
CscR
csc-specific repressor, or
- ScrA Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase
system and, said ScrK gene encodes ATP-dependent fructokinase, said ScrB
sucrose 6-
phosphate hydrolase (invertase), said ScrY sucrose porine, ScrR sucrose operon
repressor.
Preferably, genes coding for said proteins are overexpressed according to one
of the
methods provided herein. Preferably, the microorganism overexpresses:
- the heterologous cscBKAR genes of E. coil EC3132, or
- the heterologous scrKYABR genes of Salmonella sp.
Thus according to the second specific embodiment, the microorganism of the
invention which is genetically modified for the production of serine and/or
cysteine as
described above, further comprises:
a) an attenuation of the phosphoglycerate mutase GpmM and/or GpmB, preferably
at
least GpmM, and more preferably an inhibition of the GpmM activity,
b) an increased activity of Csc proteins involved in the import and metabolism
of
sucrose,
as compared to an unmodified microorganism.
Preferably, the GpmM and GpmB proteins have at least 80%, 90%, 95%, or 100%
sequence similarity or sequence identity with the sequence of SEQ ID NO: 40
and 79,
respectively. Preferably, the microorganism for the production of serine
and/or cysteine
comprises an attenuation of the protein GpmM, as compared to an unmodified
microorganism.
Preferably, the attenuation of said one or more proteins results from an
attenuation of
the gene coding said protein (i.e., gpmM and/or gpmB genes). Preferably, the
gpmM and
gpmB genes have at least 80%, 90%, 95%, or 100% sequence identity with the
sequences
of SEQ ID NOs: 39 and 78, respectively. Preferably, the microorganism for the
production
of serine and/or cysteine comprises an attenuation of the gpmM gene, as
compared to an
unmodified microorganism.
Preferably, expression of GpmM and/or GpmB is partially or completely
attenuated.
Preferably, attenuation of GpmM and/or GpmB activity results from an
inhibition of
expression of the gpmM and/or gpmB genes coding said enzymes, most preferably
from an
inhibition of expression of the gpmM gene. Preferably, attenuation of
expression results
from a partial or complete deletion of the gpmM and/or gpmB genes, more
preferably from
Date Recue/Date Received 2023-12-29
18
a partial or complete deletion of the gpmM and/or gpmB genes, most preferably
from a
partial or complete deletion of the gpmM gene.
Preferably, the microorganism for the production of serine and/or cysteine
comprises
an overproduction of the proteins CscBKAR.
Preferably, the CscB sucrose permease, CscK fructokinase, CscA sucrose
hydrolase,
and CscR csc-specific repressor have at least 80%, 90%, 95%, or 100% sequence
similarity
or sequence identity with the sequences of SEQ ID NO: 51, 53, 55 and 57,
respectively.
Preferably, the overproduction of said proteins results from an overexpression
of the
gene coding said protein (i.e., cscBKAR genes), as compared to an unmodified
microorganism. Preferably, the cscBKAR genes have at least 80%, 90%, 95%, or
100%
sequence identity with the sequences of SEQ ID NO: 50, 52, 54 and 56,
respectively.
Preferably in this second embodiment, the microorganism is genetically
modified for
the production of serine and/or cysteine as described above and further
comprises:
a) an attenuation of the expression of the gpmM gene and/or the gpmB gene,
preferably a deletion of the gpmM gene and/or the gpmB gene, and
b) an overexpression of the csc genes,
as compared to an unmodified microorganism.
According to the third specific embodiment, the microorganism of the invention
which
is genetically modified for the production of serine and/or cysteine as
described above,
further comprises:
a) an attenuation of the pyruvate kinase activity, and more preferably an
inhibition of
the pyruvate kinase activity,
b) an increased activity of Scr proteins involved in the import and metabolism
of
sucrose,
as compared to an unmodified microorganism.
Preferably, the PykA and PykF proteins have at least 80%, 90%, 95%, or 100%
sequence similarity or sequence identity with the sequence of SEQ ID NO: 59
and 61,
respectively.
Preferably, the attenuation of said proteins results from an attenuation of
the gene
coding said protein (i.e., pykA and pykF genes). Preferably, the pykA and pykF
genes have
at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID
NOs:
58 and 60, respectively.
Preferably, expression of PykA and PykF is partially or completely attenuated.
Preferably, attenuation of PykA and PykF activity results from an inhibition
of expression of
the pykA and pykF genes coding said enzymes. Preferably, attenuation of
expression
results from a partial or complete deletion of the pykA and pykF genes, more
preferably
from a partial or complete deletion of the pykA and pykF genes.
Date Recue/Date Received 2023-12-29
19
Preferably, the microorganism for the production of serine and/or cysteine
comprises
an overproduction of the proteins ScrKYABR.
Preferably, the ScrK ATP-dependent fructokinase, the ScrY sucrose porine, the
ScrA
Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system, the
ScrB
sucrose 6-phosphate hydrolase (invertase), and the ScrR sucrose operon
repressor have
at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with
the
sequences of SEQ ID NO: 63, 65, 67, 69 and 71, respectively.
Preferably, the overproduction of said proteins results from an overexpression
of the
gene coding said protein (i.e., scrKYABR genes), as compared to an unmodified
microorganism. Preferably, the scrKYABR genes have at least 80%, 90%, 95%, or
100%
sequence identity with the sequences of SEQ ID NO: 62, 64, 66, 68 and 70,
respectively.
Preferably in this third embodiment, the microorganism is genetically modified
for the
production of serine and/or cysteine as described above and further comprises:
a) an attenuation of the expression of the pykA and pykF genes, preferably a
deletion
of the pykA and pykF genes, and
b) an overexpression of the scr genes,
as compared to an unmodified microorganism.
Still for this third specific embodiment, the microorganism is genetically
modified for
the production of serine and/or cysteine advantageously comprise a wild-type,
not modified
(not attenuated or deleted) GpmM activity or gpmM gene.
Preferably, in the third specific embodiment as described herein, the
microorganism
is further modified to comprise attenuation of glyA and/or overexpression of
eamA, as
compared to an unmodified microorganism, more preferably to comprise deletion
of glyA
and/or overexpression of eamA, and most preferably to comprise deletion of
glyA and
overexpression of eamA, as compared to an unmodified microorganism.
The microorganism for the production of cysteine may comprise an increased
activity
of at least one of the following enzymes: serine acetyltransferase (CysE),
cysteine synthase
(CysK), and cysteine synthase (CysM), as compared to an unmodified
microorganism.
Preferably, the microorganism for the production of serine comprises an
overproduction of
at least one of the following proteins: CysE, CysE*, CysK and CysM. CysE* is a
feedback
resistant (FBR) protein.
Preferably, CysE, CysK and CysM have at least 80%, 90%, 95%, or 100% sequence
similarity or sequence identity with the sequences of SEQ ID NOs: 73, 75 and
81,
respectively. When CysE* is overproduced rather than CysE, said protein
preferably has at
least 80%, 90%, 95%, or 99,9% sequence similarity or sequence identity with
the sequence
of SEQ ID NO: 73. CysE* comprises the substitution of threonine residue at
position 167 by
an alanine residue when compared to SEQ ID NO: 73.
Date Recue/Date Received 2023-12-29
20
Preferably, the overproduction of said one or more proteins results from an
overexpression of the gene coding said protein (i.e., cysE (or cysE *), cysK,
and/or cysM
genes). Preferably, the cysE, cysK, and cysM genes have at least 80%, 90%,
95%, or 100%
sequence identity with the sequences of SEQ ID NOs: 72, 74 and 80,
respectively.
.. Preferably, the cysE* gene has at least 80%, 90%, 95%, or 99,9% sequence
identity with
the sequence of SEQ ID NO: 72, wherein cysE * codes for a protein having the
substitution
of threonine residue at position 167 by an alanine residue with reference to
the wild-type
protein having the sequence SEQ ID NO: 73.
Preferably, the microorganism is genetically modified for the production of
serine and
comprises an overexpression of the genes cysE and cysK or of the genes cysE
and cysM,
more preferably of the genes cysE and cysK, and more preferably comprises an
overexpression of the genes cysE* and cysK or of the genes cysE* and cysM,
more
preferably of the genes cysE* and cysK, as compared to an unmodified
microorganism.
Preferably, the microorganism is genetically modified for the production of
serine
.. and/or cysteine and further comprises:
- an attenuation activity of at least one of the following proteins: soluble
pyridine
nucleotide transhydrogenase (UdhA), methylglyoxal synthase (MgsA), acetyl-CoA
carboxylase (AckA), phosphate acetyltransferase (PtA), pyruvate formate lyase
(PflAB),
fumarate reductase enzyme complex (FrdABCD), lactate dehydrogenase (LdhA),
alcohol
.. dehydrogenase (AdhE), glucose-6-phosphate 1-dehydrogenase (Zwf),
phosphogluconate
dehydratase (Edd), KHG/KDPG aldolase (Eda), and 6-phosphogluconate
dehydrogenase
(Gnd), as compared to an unmodified microorganism.
Preferably, UdhA has at least 80%, 90%, 95%, or 100% sequence similarity or
sequence identity with the sequence of SEQ ID NO: 8. Preferably, MgsA has at
least 80%,
.. 90%, 95%, or 100% sequence similarity or sequence identity with the
sequence of SEQ ID
NO: 2. Preferably, AckA has at least 80%, 90%, 95%, or 100% sequence
similarity or
sequence identity with the sequence of SEQ ID NO: 83. Preferably, PtA has at
least 80%,
90%, 95%, or 100% sequence similarity or sequence identity with the sequence
of SEQ ID
NO: 85. Preferably, PflA and PfIB have at least 80%, 90%, 95%, or 100%
sequence
similarity or sequence identity with the sequences of SEQ ID NO: 87 and 89,
respectively.
Preferably, FrdA, FrdB, FrdC, and FrdD have at least 80%, 90%, 95%, or 100%
sequence
similarity or sequence identity with the sequences of SEQ ID NO: 91, 93, 95,
and 97,
respectively. Preferably, LdhA has at least 80%, 90%, 95%, or 100% sequence
similarity or
sequence identity with the sequence of SEQ ID NO: 99. Preferably, AdhE has at
least 80%,
90%, 95%, or 100% sequence similarity or sequence identity with the sequence
of SEQ ID
NO: 101. Preferably, Zwf has at least 80%, 90%, 95%, or 100% sequence
similarity or
sequence identity with the sequence of SEQ ID NO: 103. Preferably Edd has at
least 80%,
Date Recue/Date Received 2023-12-29
21
90%, 95%, or 100% sequence similarity or sequence identity with the sequence
of SEQ ID
NO: 4. Preferably Eda has at least 80%, 90%, 95%, or 100% sequence similarity
or
sequence identity with the sequence of SEQ ID NO: 6. Preferably Gnd has at
least 80%,
90%, 95%, or 100% sequence similarity or sequence identity with the sequence
of SEQ ID
NO: 105.
Preferably, attenuation of expression results from a partial or complete
deletion of the
gene encoding said protein (i.e., at least one of the udhA, mgsA, ackA, ptA,
pflAB, frdABCD,
IdhA, adhE, zwf, edd, eda and gnd genes).
Preferably, the udhA gene has at least 80%, 90%, 95%, or 100% sequence
identity
with the sequence of SEQ ID NO: 7. Preferably, the mgsA gene has at least 80%,
90%,
95%, or 100% sequence identity with the sequence of SEQ ID NO: 1. Preferably,
the ackA
gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence
of SEQ
ID NO: 82. Preferably, the ptA gene has at least 80%, 90%, 95%, or 100%
sequence identity
with the sequence of SEQ ID NO: 84. Preferably, the pflAB genes have at least
80%, 90%,
95%, or 100% sequence identity with the sequences of SEQ ID NOs: 86 and 88,
respectively. Preferably, the frdABCD genes have at least 80%, 90%, 95%, or
100%
sequence identity with the sequences of SEQ ID NOs: 90, 92, 94, and 96,
respectively.
Preferably, the IdhA gene has at least 80%, 90%, 95%, or 100% sequence
identity with the
sequence of SEQ ID NO: 98. Preferably, the adhE gene has at least 80%, 90%,
95%, or
100% sequence identity with the sequence of SEQ ID NO: 100. Preferably, the
zwf gene
has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ
ID NO:
102. Preferably, the edd gene has at least 80%, 90%, 95%, or 100% sequence
identity with
the sequence of SEQ ID NO: 3. Preferably, the eda gene has at least 80%, 90%,
95%, or
100% sequence identity with the sequence of SEQ ID NO: 5. Preferably, the gnd
gene has
at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID
NO: 104.
Preferably, at least one gene selected from among udhA, mgsA, ackA, ptA,
pflAB,
frdABCD, IdhA, adhE, zwf, edd, eda and gnd is deleted. Preferably, the genes
udhA, mgsA,
zwf, edd, eda and gnd are attenuated as compared to an unmodified
microorganism, more
preferably deleted. More preferably, the genes udhA, mgsA, edd and eda are
attenuated
as compared to an unmodified microorganism, and most preferably deleted.
Genes and proteins are identified herein using the denominations of the
corresponding genes in E. colt (e.g., E. coil K12 MG1655 having the Genbank
accession
number U00096.3) unless otherwise specified. However, in some cases use of
these
denominations has a more general meaning according to the invention and covers
all of the
corresponding genes and proteins in microorganisms. This is notably the case
for the genes
and proteins described herein that are not present in the microorganism (i.e.,
that are
heterologous) such as GapN or Scr, etc. Reference provided herein to any
protein (e.g.,
Date Recue/Date Received 2023-12-29
22
enzyme) or gene further comprises functional fragments, mutants, and
functional variants
thereof. As provided herein, said functional fragments, mutants, and
functional variants
preferably have at least 90% similarity to said protein or gene, or
alternatively, at least 80%,
90%, 95%, or even 100%, identity to said protein or gene.
A degree of sequence identity between proteins is a function of the number of
identical
amino acid residues or nucleotides at positions shared by the sequences of
said proteins.
The term "sequence identity" or "identity" as used herein in the context of
two nucleotide or
amino acid sequences more particularly refers to the residues in the two
sequences that
are identical when aligned for maximum correspondence. When percentage of
sequence
identity is used in reference to amino acid sequences, it is recognized that
positions at which
amino acids are not identical often differ by conservative amino acid
substitutions, where
amino acid residues are substituted for other amino acid residues having
similar chemical
properties (e.g., charge or hydrophobicity). When sequences differ due to
conservative
substitutions, percent identity between sequences may be adjusted upwards to
correct for
the conservative nature of the substitution. Sequences that differ by such
conservative
substitutions are said to have "sequence similarity" or "similarity". Thus,
the degree of
sequence similarity between polypeptides is a function of the number of
similar amino acid
residues at positions shared by the sequences of said proteins. The means of
identifying
similar sequences and their percent similarity or their percent identities are
well-known to
those skilled in the art, and include in particular the BLAST programs, which
can be used
from the website http://www.ncbi.nlm.nih.gov/BLAST/ with the default
parameters indicated
on that website. The sequences obtained can then be exploited (e.g., aligned)
using, for
example, the programs CLUSTALW (http://www.ebi.ac.uk/clustalw/) or MULTALIN
(http://prodes.toulouse.inra.fr/multalin/cgi-bin/multalin.p1), with the
default parameters
indicated on those websites.
Using the references given in GenBank for known genes, the person skilled in
the art
is able to determine the equivalent genes in other organisms, bacterial
strains, yeasts, fungi,
mammals, plants, etc. This routine work is advantageously done using consensus
sequences that can be determined by carrying out sequence alignments with
genes derived
from other microorganisms, and designing degenerate probes to clone the
corresponding
gene in another organism. These routine methods of molecular biology are well-
known to
those skilled in the art.
Specifically, sequence similarity and sequence identity between amino acid
sequences can be determined by comparing a position in each of the sequences
which may
be aligned for the purposes of comparison. When a position in the compared
sequences is
occupied by a similar amino acid or by the same amino acid then the sequences
are,
respectively, similar or identical at that position.
Date Recue/Date Received 2023-12-29
23
Sequence similarity may notably be expressed as the percent similarity of a
given
amino acid sequence to that of another amino acid sequence. This refers to the
similarity
between sequences on the basis of a "similarity score" that is obtained using
a particular
amino acid substitution matrix. Such matrices and their use in quantifying the
similarity
between two sequences are well-known in the art and described, for example in
Dayhoff et
al., 1978, and in Henikoff and Henikoff, 1992. Sequence similarity may be
calculated from
the alignment of two sequences, and is based on a substitution score matrix
and a gap
penalty function. As a non-limiting example, the similarity score is
determined using the
BLOSUM62 matrix, a gap existence penalty of 10, and a gap extension penalty of
0.1 or
the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension
penalty of 1.
Preferably, no compositional adjustments are made to compensate for the amino
acid
compositions of the sequences being compared and no filters or masks (e.g., to
mask off
segments of the sequence having low compositional complexity) are applied when
determining sequence similarity using web-based programs, such as BLAST. The
maximum similarity score obtainable for a given amino acid sequence is that
obtained when
comparing a sequence with itself. The skilled person is able to determine such
maximum
similarity scores on the basis of the above-described parameters for any amino
acid
sequence. A statistically relevant similarity can furthermore be indicated by
a "bit score" as
described, for example, in Durbin et al., Biological Sequence Analysis,
Cambridge
University Press (1998).
To determine if a given amino acid sequence has at least 80% similarity with a
protein
provided herein, said amino acid sequence can be optimally aligned as provided
above,
preferably using the BLOSUM62 matrix, a gap existence penalty of 10, and a gap
extension
penalty of 0.1. Two sequences are "optimally aligned" when they are aligned
for similarity
scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), gap
existence
penalty and gap extension penalty so as to arrive at the highest score
possible for that pair
of sequences. The skilled person is able to determine 80% similarity with a
maximum score
determined on the basis of the above-described parameters for any amino acid
sequence.
Percent similarity or percent identities as referred to herein are determined
after
optimal alignment of the sequences to be compared, which may therefore
comprise one or
more insertions, deletions, truncations and/or substitutions. This percent
identity may be
calculated by any sequence analysis method well-known to the person skilled in
the art.
The percent similarity or percent identity may be determined after global
alignment of the
sequences to be compared of the sequences taken in their entirety over their
entire length.
In addition to manual comparison, it is possible to determine global alignment
using the
algorithm of Needleman and Wunsch (1970). Optimal alignment of sequences may
preferably be conducted by the global alignment algorithm of Needleman and
Wunsch
Date Recue/Date Received 2023-12-29
24
(1970), by computerized implementations of this algorithm (such as CLUSTAL W)
or by
visual inspection.
For nucleotide sequences, the sequence comparison may be performed using any
software well-known to a person skilled in the art, such as the Needle
software. The
parameters used may notably be the following: "Gap open" equal to 10.0, "Gap
extend"
equal to 0.5, and the EDNAFULL matrix (NCBI EMBOSS Version NUC4.4).
For amino acid sequences, the sequence comparison may be performed using any
software well-known to a person skilled in the art, such as the Needle
software. The
parameters used may notably be the following: "Gap open" equal to 10, "Gap
extend" equal
to 0.1, and the BLOSUM62 matrix.
Preferably, the percent similarity or identity as defined herein is determined
via the
global alignment of sequences compared over their entire length.
As a particular example, to determine the percentage of similarity or identity
between
two amino acid sequences, the sequences are aligned for optimal comparison.
For
example, gaps can be introduced in the sequence of a first amino acid sequence
for optimal
alignment with the second amino acid sequence. The amino acid residues at
corresponding
amino acid positions are then compared. When a position in the first sequence
is occupied
by a different but conserved amino acid residue, the molecules are similar at
that position,
and accorded a particular score (e.g., as provided in a given amino acid
substitution matrix,
discussed previously). When a position in the first sequence is occupied by
the same amino
acid residue as the corresponding position in the second sequence, the
molecules are
identical at that position.
The percentage of identity between the two sequences is a function of the
number
of identical positions shared by the sequences. Hence % identity = number of
identical
positions / total number of overlapping positions x 100.
In other words, the percentage of sequence identity is calculated by comparing
two
optimally aligned sequences, determining the number of positions at which the
identical
amino acid occurs in both sequences to yield the number of matched positions,
dividing the
number of matched positions by the total number of positions and multiplying
the result by
100 to yield the percentage of sequence identity.
PFAM (protein family database of alignments and hidden Markov models;
http://www.sanger.ac.uk/Software/Pfam/) represents a large collection of
protein sequence
alignments which may also be consulted by the skilled person. Each PFAM makes
it
possible to visualize multiple alignments, see protein domains, evaluate
distribution among
organisms, gain access to other databases, and visualize known protein
structures.
Finally, COGs (clusters of orthologous groups of
proteins;
http://www.ncbi.nlm.nih.gov/COG/) may be obtained by comparing protein
sequences from
Date Recue/Date Received 2023-12-29
25
43 fully sequenced genomes representing 30 major phylogenic lines. Each COG is
defined
from at least three lines, which permits the identification of former
conserved domains.
The above definitions and preferred embodiments related to the functional
fragments
and functional variants of proteins apply mutatis mutandis to nucleotide
sequences, such
as genes, encoding said proteins.
According to a further aspect, the present invention relates to a method for
the
production of serine and/or cysteine using the microorganism described herein.
Said
method comprises the steps of:
a) culturing a microorganism genetically modified for the production of serine
and/or
cysteine as provided herein in an appropriate culture medium comprising a
source
of carbon, and
b) recovering serine and/or cysteine from the culture medium.
According to the invention, the terms "fermentative process," "fermentative
production," "fermentation," or "culture" are used interchangeably to denote
the growth of
microorganism. This growth is generally conducted in fermenters with an
appropriate growth
medium adapted to the microorganism being used.
An "appropriate culture medium" or a "culture medium" refers to a culture
medium
optimized for the growth of the microorganism and the synthesis of serine or
cysteine by
the cells. The culture medium (e.g., a sterile, liquid media) comprises
nutrients essential or
beneficial to the maintenance and/or growth of the microorganism such as
carbon sources
or carbon substrates, nitrogen sources; phosphorus sources, for example,
monopotassium
phosphate or dipotassium phosphate; trace elements (e.g., metal salts, for
example
magnesium salts, cobalt salts and/or manganese salts); as well as growth
factors such as
amino acids and vitamins. The fermentation process is generally conducted in
reactors with
a synthetic, particularly inorganic, culture medium of known defined
composition adapted
to the microorganism, e.g., E. coll. In particular, the inorganic culture
medium can be of
identical or similar composition to an M9 medium (Anderson, 1946), an M63
medium or a
medium such as defined by Schaefer et al. (1999). "Synthetic medium" refers to
a culture
medium comprising a chemically defined composition on which organisms are
grown.
The term "source of carbon," "carbon source," or "carbon substrate" according
to the
present invention refers to any carbon source capable of being metabolized by
a
microorganism wherein the substrate contains at least one carbon atom.
According to the
present invention, said source of carbon is preferably at least one
carbohydrate, and in
some cases a mixture of at least two carbohydrates. The term "carbohydrate"
refers to any
carbon source capable of being metabolized by a microorganism and containing
at least
three carbon atoms, two atoms of hydrogen. The one or more carbohydrates may
be
selected from among the group consisting of: monosaccharides such as glucose,
fructose,
Date Recue/Date Received 2023-12-29
26
mannose, galactose, and the like, disaccharides such as sucrose, cellobiose,
maltose,
lactose, and the like, oligosaccharides such as raffinose, stacchyose,
maltodextrins, and
the like, polysaccharides such as cellulose, starch, or glycerol. Preferred
carbon sources
are fructose, galactose, glucose, lactose, maltose, sucrose, or any
combination thereof,
more preferably glucose, fructose, galactose, lactose, and/or sucrose, most
preferably
glucose. In the present disclosure, the terms "glucose" and "dextrose" are
used
interchangeably since being synonymous and equivalent.
Preferably, the method for the production of serine and/or cysteine comprises
culturing the microorganism genetically modified as the first specific
embodiment described
above when the appropriate culture medium comprises glucose as a source of
carbon. In
this first specific embodiment, the microorganism of the invention which is
genetically
modified for the production of serine and/or cysteine as described above,
further comprises:
a) an attenuation of phosphoenolpyruvate-dependent phosphotransferase system
(PTS) activity, preferably an inhibition of said activity,
b) an attenuation of the phosphoglycerate mutase GpmM and/or GpmB, preferably
at
least GpmM, and more preferably an inhibition of the GpmM activity,
c) an increased galactose-proton symporter (GalP) activity,
as compared to an unmodified microorganism.
Preferably, the method for the production of serine and/or cysteine comprises
culturing the microorganism genetically modified as the second and third
specific
embodiments described above when the appropriate culture medium comprises
sucrose as
a source of carbon. In the second specific embodiment, the microorganism of
the invention
which is genetically modified for the production of serine and/or cysteine as
described
above, further comprises:
a) an attenuation of the phosphoglycerate mutase GpmM and/or GpmB, preferably
at
least GpmM, and more preferably an inhibition of the GpmM activity,
b) an increased activity of Csc proteins involved in the import and metabolism
of
sucrose,
as compared to an unmodified microorganism.
In this third specific embodiment, the microorganism of the invention which is
genetically modified for the production of serine and/or cysteine as described
above, further
comprises:
a) an attenuation of the pyruvate kinase activity, and more preferably an
inhibition of the
pyruvate kinase activity,
b) an increased activity of Scr proteins involved in the import and metabolism
of sucrose,
as compared to an unmodified microorganism.
Date Recue/Date Received 2023-12-29
27
The culture medium preferably comprises a nitrogen source capable of being
used by
the microorganism. Said source of nitrogen may be inorganic (e.g., (NH4)2504)
or organic
(e.g., urea or glutamate). Preferably, said source of nitrogen is in the form
of ammonium or
ammoniac. Preferably, said source of nitrogen is either an ammonium salt, such
as
ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium hydroxide and
ammonium phosphate, or ammoniac gas, corn steep liquor, peptone (e.g., BactoTM
peptone), yeast extract, meat extract, malt extract, urea, or glutamate, or
any combination
of two or more thereof. In some cases, the nitrogen source may be derived from
renewable
biomass of microbial origin (such as beer yeast autolysate, waste yeast
autolysate, bakers
yeast, hydrolyzed waste cells, algae biomass), vegetal origin (such as cotton
seed meal,
soy peptone, soybean peptide, soy flour, soybean flour, soy molasses, rapeseed
meal,
peanut meal, wheat bran hydro lysate, rice bran and defatted rice bran, malt
sprout, red
lentil flour, black gram, bengal gram, green gram, bean flour, flour of pigeon
pea,
protamylasse) or animal origin (such as fish waste hydrolysate, fish protein
hydrolysate,
chicken feather; feather hydrolysate, meat and bone meal, silk worm larvae,
silk fibroin
powder, shrimp wastes, beef extract), or any other nitrogen containing waste.
More
preferably, said source of nitrogen is peptone and/or yeast extract.
The person skilled in the art is able to define the culture conditions for the
microorganisms according to the invention. In particular the bacteria are
fermented at a
temperature between 20 C and 55 C, preferably between 25 C and 40 C, more
preferably
between about 30 C to 39 C, even more preferably about 37 C. In cases, where a
thermo-
inducible promoter is comprised in the microorganism provided herein, said
microorganism
is preferably fermented at about 39 C.
This process can be carried out either in a batch process, in a fed-batch
process or
in a continuous process. It can be carried out under aerobic, micro-aerobic or
anaerobic
conditions, or a combination thereof (for example, aerobic conditions followed
by anaerobic
conditions).
"Under aerobic conditions" means that oxygen is provided to the culture by
dissolving
the gas into the liquid phase. This could be obtained by (1) sparging oxygen
containing gas
(e.g., air) into the liquid phase or (2) shaking the vessel containing the
culture medium in
order to transfer the oxygen contained in the head space into the liquid
phase. The main
advantage of the fermentation under aerobic conditions is that the presence of
oxygen as
an electron acceptor improves the capacity of the strain to produce more
energy under the
form of ATP for cellular processes. Therefore, the strain has its general
metabolism
improved.
Micro-aerobic conditions are defined as culture conditions wherein low
percentages
of oxygen (e.g., using a mixture of gas containing between 0.1 and 15% of
oxygen,
Date Recue/Date Received 2023-12-29
28
completed to 100% with inert gas such as nitrogen, helium or argon, etc.), is
dissolved into
the liquid phase.
Anaerobic conditions are defined as culture conditions wherein no oxygen is
provided
to the culture medium. Strictly anaerobic conditions are obtained by sparging
an inert gas
like nitrogen into the culture medium to remove traces of other gas. Nitrate
can be used as
an electron acceptor to improve ATP production by the strain and improve its
metabolism.
The term "recovering" as used herein designates the process of separating or
isolating
the produced serine or cysteine using conventional laboratory techniques known
to the
person skilled in the art.
Preferably, step b) of the method comprises a step of filtration, ion
exchange,
crystallization, and/or distillation, more preferably a step of
crystallization. Serine or cysteine
may be recovered from the culture medium and/or from the microorganism itself.
Preferably,
serine or cysteine is recovered from at least the culture medium.
For example, serine may be purified by filtration or centrifugation for
removing cells,
by ion exchange and crystallization (at isoelectric point) (in particular as
described in
U53843441). Cysteine may also be purified by filtration or centrifugation for
removing cells,
by ion exchange, concentration and crystallization (in particular as described
in
U58088949).
EXAMPLES
The present invention is further defined in the following examples. It should
be
understood that these examples, while indicating preferred embodiments of the
invention,
are given by way of illustration only. The person skilled in the art will
readily understand that
these examples are not !imitative and that various modifications,
substitutions, omissions,
and changes may be made without departing from the scope of the invention.
Methods
The protocols used in the following examples are:
Protocol 1: (Chromosomal modifications by homologous recombination, selection
of
recombinants and antibiotic cassette excision flanked by FRT sequences) and
protocol 2
(Transduction of phage P1) used in this invention have been fully described in
patent
application W02013/001055 (see in particular the "Examples Protocols" section
and
Examples 1 to 8, incorporated herein by reference).
Protocol 3: Construction of recombinant plasmids.
Recombinant DNA technology is well described and known to the person skilled
in the art.
Briefly, DNA fragments were PCR amplified using oligonucleotides (that the
person skilled
Date Recue/Date Received 2023-12-29
29
in the art will be able to define) and E. coil MG1655 genomic DNA or an
adequate
synthetically synthesized fragment was used as a matrix. The DNA fragments and
chosen
plasmid were digested with compatible restriction enzymes (that the person
skilled in the
art is able to define), then ligated and transformed into competent cells.
Transformants were
analysed and recombinant plasmids of interest were verified by DNA sequencing.
Protocol 4: Evaluation of L-Serine fermentation performance.
Production strains were evaluated in 500 mL Erlenmeyer baffled flasks using
medium MMD
(Table 1) for serine fermentation on dextrose adjusted to pH 6.8 or using
medium MMS
(Table 2) for serine fermentation on Sucrose. A 10 mL preculture was grown at
37 C for 40
hours in a rich medium LB medium (10 g/L bactopeptone, 5 g/L yeast extract, 5
g/L NaCI).
It was used to inoculate a 50 mL culture to an 0D600 of 0.2. When necessary,
antibiotics
were added to the medium (spectinomycin at a final concentration of 50 mg.L-
1). The
temperature of the cultures was 37 C. When the culture had reached an
absorbance at 600
nm of 2 to 5 u0D/mL, extracellular amino acids were quantified by HPLC after
OPA/Fmoc
(Agilent Technologies) derivatization and other relevant metabolites were
analysed using
HPLC with refractometric detection (organic acids and glucose).
Table 1: Composition of MMD- medium
Compound Concentration (g.L-1)
Citric acid. H20 1.00
MgSO4. 7H20 1.00
CaCl2. 2H20 0.04
CoCl2. 6H20 0.0080
MnSO4. H20 0.0200
CuC12. 2H20 0.0020
H3B03 0.0010
Na2Mo04. 2H20 0.0004
ZnSO4. 7H20 0.0040
Na2HPO4 2.00
K2HPO4 2.00
(NH4)2HPO4 8.00
(NH4)2504 5.00
NH4CI 0.13
FeSO4. 7H20 0.01
Thiamin 0.0116
Date Recue/Date Received 2023-12-29
30
MOPS 40
Dextrose 10
In these cultures, Serine yield (Y
x . serine/dextrose) was expressed as followed:
g) serine produced(g)
Y serinel dextrose ( =
g dextrose consumed(g)
In these cultures, Serine productivity (P 1was expressed as followed:
x= serine,
g IL)= __________________
final serine concentration(g IL)
Pserine ( h ¨
time from inoculation(h)
Table 2: Composition of MMS- medium
Compound Concentration (g.L-1)
Citric acid. H20 1.00
MgSO4. 7H20 1.00
CaCl2. 2H20 0.04
CoCl2. 6H20 0.0080
MnSO4. H20 0.0200
CuC12. 2H20 0.0020
H3B03 0.0010
Na2Mo04. 2H20 0.0004
ZnSO4. 7H20 0.0040
Na2HPO4 2.00
K2HPO4 2.00
(NH4)2HPO4 8.00
(NH4)2504 5.00
NH4CI 0.13
FeSO4. 7H20 0.01
Thiamin 0.0116
MOPS 40
Sucrose 10
In these cultures, Serine yield (Y
x = serine/sucrose) was expressed as followed:
g) serine produced(g)
Y serine I sucrose ( =
g sucrose consumed(g)
In these cultures, Serine productivity (P 1was expressed as followed:
x= serine,
Date Recue/Date Received 2023-12-29
31
(g11, final serine
concentration(g I L)
Pserine
h )= ___________________________________________________
time from inoculation(h)
Protocol 5: Evaluation of L-Cysteine fermentation performance.
Production strains were evaluated in 500 mL Erlenmeyer baffled flasks using
medium MMD
(Table 1) for cysteine fermentation on dextrose adjusted to pH 6.8 or using
medium MMS
(Table 2) for cysteine fermentation on Sucrose. A 10 mL preculture was grown
at 37 C for
40 hours in a rich medium LB medium (10 g/L bactopeptone, 5 g/L yeast extract,
5 g/L
NaCI). It was used to inoculate a 50 mL culture to an 0D600 of 0.2. When
necessary,
antibiotics were added to the medium (spectinomycin at a final concentration
of 50 mg.L-1).
The temperature of the cultures was 37 C. When the culture had reached an
absorbance
at 600 nm of 2 to 5 u0D/mL, extracellular amino acids were quantified by HPLC
after
OPA/Fmoc (Agilent Technologies) derivatization and other relevant metabolites
were
analysed using HPLC with refractometric detection (organic acids and sucrose).
In the dextrose cultures, Cysteine yield (Y
k cysteme/dextrose) was expressed as followed:
g cysteine produced(g)
Ycysteineldextrose(
g ) dextrose consumed(g)
and Cysteine productivity (Pcysteine) was expressed as followed:
g final cysteine
concentration(g IL)
Pcysteine
h ) time from inoculation(h)
In the sucrose cultures, Cysteine yield (Y
, = cysteme/sucrose) was expressed as followed:
cysteine produced(g)
Y cysteine I sucrose (g) = _____________________________
g sucrose consumed(g)
and Cysteine productivity (Pcysteme) was expressed as followed:
g = final cysteine
concentration(g L)
Pcysteine
h ) time from inoculation(h)
Protocol 6: Biomass estimation.
Biomass quantity variation is monitored using a spectrophotometer (Nicolet
Evolution 100
UV-Vis, THERMOS). The biomass production increases the turbidity of the growth
medium.
It is assayed by measuring the absorbance at a 600 nm wavelength. Each unit of
absorbance corresponds to 2.2 x 109 +/- 2 x 108 cells/mL.
Date Recue/Date Received 2023-12-29
32
EXAMPLE 1: Strain constructions.
Serine producing strains: Strains 1 to 14.
Strain 1:
According to protocols 1, 2 and 3, strain 1 was obtained by sequentially
modifying the E.
coil MG1655 strain by knocking out:
- the mgsA gene encoding the methylglyoxal synthase MgsA protein (SEQ ID
NO:
1 and 2, respectively),
- the edd and eda operon (SEQ ID NO: 3 and 5, respectively) encoding the
phosphogluconate dehydratase Edd and the KHG/KDPG aldolase Eda proteins (SEQ
ID
NO: 4 and 6, respectively),
- the udhA gene encoding the soluble pyridine nucleotide transhydrogenase
UdhA
protein (SEQ ID NO: 7 and 8, respectively),
-the two genes sdaA and sdaB (SEQ ID NO: 9 and 11, respectively) encoding the
main serine deaminases SdaA and SdaB proteins respectively (SEQ ID NO: 10 and
12,
respectively);
And by overexpressing into the pCL1920 vector (Lerner & Inouye, 1990)
- a serA* gene encoding a phosphoglycerate dehydrogenase SerA* protein
havingthe substitution of asparagine residue at position 364 by an alanine
residue with
reference to the wild-type protein having the sequence SEQ ID NO: 14, and the
serB gene
encoding the phosphoserine phosphatase SerB protein (SEQ ID NO: 15 and 16,
respectively) under their native promoter.- the serC gene encoding the
phosphoserine /
phosphohydroxythreonine aminotransferase SerC protein (SEQ ID NO: 17 and 18,
respectively) under the trc promoter of SEQ ID NO: 19, leading to a construct
Ptrc30/RBS01-serC.
Strain 2:
According to protocols 1, 2 and 3, strain 2 was obtained by sequentially
modifying strain 1
by knocking out the gapA gene encoding the glyceraldehyde-3-phosphate
dehydrogenase
A GapA protein (SEQ ID NO: 20 and 21, respectively) and by overexpressing into
the
plasmid of strain 1 the heterologous gapN gene of Streptococcus mutans (SEQ ID
NO: 22)
encoding the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase GapN
protein
(SEQ ID NO: 23, Uniprot Q59931) under the IPTG inducible trc promoter of SEQ
ID NO:
24, thus leading to a construct Ptrc01/0P01/RBS09-gapNsm.
Strain 3:
Date Recue/Date Received 2023-12-29
33
According to protocols 1, 2 and 3, strain 3 was obtained by sequentially
modifying strain 2
by knocking out the gapB gene encoding the D-erythrose-4-phosphate
dehydrogenase
GapB protein (SEQ ID NO: 25 and 26, respectively) and the gapC pseudogene (SEQ
ID
NO: 27) encoding the glyceraldehyde-3-phosphate dehydrogenase when this gene
is intact,
Strain 4:
According to protocols 1, 2 and 3, strain 4 was obtained by modifying strain 3
by knocking
out the gpmA gene encoding the 2,3-bisphosphoglycerate dependent
phosphoglycerate
mutase GpmA protein (SEQ ID NO: 28 and 29, respectively)
Strain 5:
According to protocols 1, 2 and 3, strain 5 was obtained by modifying strain 4
by
overexpressing into the plasmid of strain 2 the gdhA gene encoding the
glutamate
dehydrogenase GdhA protein (SEQ ID NO: 30 and 31, respectively) under the the
PR
promoter of SEQ ID NO: 32, thus leading to a construct PR-gdhA, with the c1857
allele from
lambda phage (SEQ ID NO: 33 encoding the thermosensitive repressor protein of
SEQ ID
NO: 34) (amplified from the pFC1 vector, Mermet-Bouvier & Chauvat, 1994).
Strain 6:
According to protocols 1, 2 and 3, strain 6 was obtained by modifying strain 5
by knocking
out the tdcG gene encoding the L-serine deaminase III TdcG protein (SEQ ID NO:
35 and
36, respectively).
Strain 7:
According to protocols 1, 2 and 3, strain 7 was obtained by modifying strain 5
by knocking
out the tdcB gene encoding the catabolic threonine dehydratase TdcB protein
(SEQ ID NO:
37 and 38, respectively)
Strain 8:
According to protocols 1, 2 and 3, strain 8 was obtained by modifying strain 6
by knocking
out the gpmM gene encoding the 2,3-bisphosphoglycerate-independent
phosphoglycerate
mutase GpmM protein (SEQ ID NO: 39 and 40, respectively) and the ptsH-ptsl-crr
operon
(SEQ ID NO: 41, 43 and 45, respectively) encoding the phosphocarrier protein
HPr PtsH,
the PTS enzyme I Ptsl and the Enzyme 11A Glucose Crr proteins (SEQ ID NO: 42,
44 and
46, respectively), and by overexpressing the galP gene encoding the
galactose:H+
symporter GalP protein (SEQ ID NO: 47 and 48, respectively) by replacing the
native
Date Recue/Date Received 2023-12-29
34
promoter by the trc promoter of SEQ ID NO: 49, thus leading to a construct
Ptrc01/RBS01-
galP.
Strain 9:
According to protocols 1, 2 and 3, strain 9 was obtained by modifying strain 7
by knocking
out the gpmM gene encoding the 2,3-bisphosphoglycerate-independent
phosphoglycerate
mutase GpmM protein (SEQ ID NO: 39 and 40, respectively) and the ptsH-ptsl-crr
operon
(SEQ ID NO: 41, 43 and 45, respectively) encoding the phosphocarrier protein
HPr PtsH,
the PTS enzyme I Ptsl and the Enzyme IIA Glucose Crr proteins (SEQ ID NO: 42,
44 and
46, respectively), and by overexpressing the galP gene encoding the
galactose:H+
symporter GalP protein (SEQ ID NO: 47 and 48, respectively) by replacing the
native
promoter by the trc promoter of SEQ ID NO: 49, thus leading to a construct
Ptrc01/RBS01-
galP.
.. Strain 10:
According to protocols 1, 2 and 3, strain 10 was obtained by modifying strain
1 by
overexpressing into the plasmid of strain 1 the heterologous cscBKAR genes of
E. coil
EC3132 (SEQ ID NO: 50, 52, 54 and 56, respectively) encoding the sucrose
permease
CscB, sucrose fructokinase CscK, hydrolase CscA, and csc-specific repressor
CscR (SEQ
ID NO: 51, 53, 55 and 57, respectively) under the native promoter.
Strain 11:
According to protocols 1, 2 and 3, strain 11 was obtained by modifying strain
6 by knocking
out the gpmM gene as described for strain 8 and by overexpressing into the
plasmid of
strain 5 the heterologous cscBKAR genes as described for strain 10.
Strain 12:
According to protocols 1, 2 and 3, strain 12 was obtained by modifying strain
7 by knocking
out the gpmM gene as described for strain 8 and by overexpressing into the
plasmid of
.. strain 5 the heterologous cscBKAR genes as described for strain 10.
Strain 13:
According to protocols 1, 2 and 3, strain 13 was obtained by modifying strain
6 by knocking
out the pykA gene encoding pyruvate kinase 2 PykA protein (SEQ ID NO: 58 and
59,
respectively) and the pykF gene encoding pyruvate kinase 1 PykF protein (SEQ
ID NO: 60
and 61, respectively), and by overexpressing into the plasmid of strain 5 the
heterologous
scrKYABR genes of Salmonella sp. (SEQ ID NO: 62, 64, 66, 68 and 70,
respectively)
Date Recue/Date Received 2023-12-29
35
encoding the ATP-dependent fructokinase ScrK, the sucrose porine ScrY, the
sucrose
Enzyme II ScrA, the sucrose 6-phosphate hydrolase ScrB, the sucrose operon
repressor
ScrR proteins (SEQ ID NO: 63, 65, 67, 69 and 71, respectively) under the
native promoter.
Strain 14:
According to protocols 1, 2 and 3, strain 14 was obtained by modifying strain
7 by knocking
out the pykA and pykF genes as described for strain 13 and by overexpressing
into the
plasmid of strain 5 the heterologous scrKYABR genes as described for strain
13.
Cysteine producing strains: Strains 15 to 28.
Strains 15 to 28:
According to protocols 1, 2 and 3, strains 15 to 28 were obtained by
sequentially modifying
respectively strains 1 to 14.
- by knocking out the serB gene (SEQ ID NO: 15) encoding the phosphoserine
phosphatase SerB protein (SEQ ID NO: 16)
- by overexpressing in operon a cysE* gene encoding a serine acetyl
transferase
CysE* protein having the substitution of threonine residue at position 167 by
an
alanine residue with reference to the wild-type protein having the sequence
SEQ ID
NO: 73, and the cysK gene (SEQ ID NO: 74) encoding the cysteine synthase A
CysK protein (SEQ ID NO: 75) on the pCL1920 vector of each strain, under the
control of the PR promoter of SEQ ID NO: 32, thus leading to a construct PR-
cysE*+cysK.
EXAMPLE 2: Serine strain performance on dextrose as sole carbone source
Table 3: Biomass production, serine titer, productivity and yield for the
different strains
grown on the medium MMD described in Table 1.
Strain Biomass Final serine Serine productivity Ysenne
(gig)
production titer (g/L) (g/L.h)
(g/g)
Strain 1 Reference Reference Reference
Reference
Strain 2 = + + +
Strain 3 = + + +
Strain 4 - + + ++
Strain 5 - ++ ++ ++
Strain 6 - ++ ++ ++
Strain 7 - ++ ++ ++
Date Recue/Date Received 2023-12-29
36
Strain 8 -- +++ ++ +++
Strain 9 -- +++ ++ +++
The symbol "+" indicates an increase of a factor up to 2, the symbol "++" an
increase by a
factor between 2 and 3, and "+++" an increase by a factor greater than 4, as
compared to
the values of reference strain 1. The symbol "2 indicates a decrease of a
factor up to 2, the
symbol "--" a decrease by a factor between 2 and 3, as compared to the values
of reference
strain 1.
As can be seen in Table 3, the functional replacement of gapA by a gene
encoding an
enzyme reducing NADP (gapN) in strains 2 and 3 leads to an improvement in
strain
performance.
Results obtained using strain 4 (gpmA deletion) show that limitation of carbon
flux into the
3 last steps of the glycolysis pathway affect biomass production but
advantageously
improve serine yield.
The overexpression of gdhA gene in the strains 5 to 7 leads to an increase of
both, final
serine titer and productivity. It confirms the positive impact of increasing
the glutamate
availability into the bacterial cell.
Strains 8 and 9 (deletion of gpmM, ptsHl+crr, tdcG or tdcB and overexpression
of galP)
exhibits a further improvement in serine production ¨ specifically in the
final serine titer and
yield. Advantageously, the suppression of genes coding enzymes consuming
serine or
serine precursors combined increases serine production.
EXAMPLE 3: Serine strain performance on sucrose as sole Carbone source
Table 4: Biomass production, serine titer, productivity and yield for the
different strains
grown on the medium M MS described in Table 2.
Strain Biomass Final serine Serine productivity Ysenne
(gig)
production titer (g/L) (g/L.h)
(g/g)
Strain 10 Reference Reference Reference Reference
Strain 11 -- +++ ++ +++
Strain 12 -- +++ ++ +++
Strain 13 - +++ +++ ++
Strain 14 - +++ +++ ++
Date Recue/Date Received 2023-12-29
37
The symbol "+" indicates an increase of a factor up to 2, the symbol "++" an
increase by a
factor between 2 and 3, and "+++" an increase by a factor greater than 4, as
compared to
the values of reference strain 7. The symbol "2 indicates a decrease of a
factor up to 2, the
symbol "--" a decrease by a factor between 2 and 3, as compared to the values
of reference
strain 7.
The only difference when comparing respectively strains 1, 8 and 9 to strains
10, 11 and 12
deals with the overexpression of the cscBKAR genes. They are coding for
proteins
conferring the ability of bacteria to passively import sucrose from outside of
the cell. Strain
10 exhibits the same ability to grow and to produce serine, using sucrose as
sole carbon
source than the strain 1 when using dextrose.
The results obtained using strains 11 and 12 on sucrose are the same as those
obtained
using strain 8 and 9 on dextrose. These results demonstrate that changing the
capacity to
use different carbon source does not affect the serine production performances
Strains 13 and 14 contains the same metabolic pathway for the serine
production except
the absence of gpmM deletion but including the deletion of pyruvate kinase
coding genes
(pykA and pykF).
In this context, the passive sucrose transporter has been replaced by the
active scrKYABR
system. Results obtained with these strains shown on table 4 demonstrate a
significant
improvement of the productivity compare to strains 11 and 12 due to a better
growth of
bacterial culture. Nevertheless, the Yield is negatively affected for the same
reason.
EXAMPLE 4: Cysteine strain performance on dextrose as sole carbon source
Table 5: Biomass production, cysteine titer, productivity and yield for the
different strains
grown on the medium MMD described in Table 1.
Strain Biomass Final cysteine Cysteine productivity Ycysteme
(gig)
production titer (g/L) (g/L.h)
(g/g)
Strain 15 Reference Reference Reference Reference
Strain 16 = + + +
Strain 17 = + + +
Strain 18 - + + ++
Strain 19 - ++ ++ ++
Strain 20 - ++ ++ ++
Strain 21 - ++ ++ ++
Date Recue/Date Received 2023-12-29
38
Strain 22 -- +++ ++ +++
Strain 23 -- +++ ++ +++
The symbol "+" indicates an increase of a factor up to 2, the symbol "++" an
increase by a
factor between 2 and 3, and "+++" an increase by a factor greater than 4, as
compared to
the values of reference strain 1. The symbol "2 indicates a decrease of a
factor up to 2, the
symbol "--" a decrease by a factor between 2 and 3 as compared to the values
of reference
strain 1.
As can be seen in Table 5, the functional replacement of gapA by a gene
encoding an
enzyme reducing NADP (gapN) in strains 16 and 17 leads to an improvement in
strain
performance.
Results obtained using strain 18 (gpmA deletion) show that limitation of
carbon flux into the
3 last steps of the glycolysis pathway affect biomass production but
advantageously
improve cysteine yield.
The overexpression of gdhA gene in the strains 19 to 21 leads to an increase
of both, final
serine titer and productivity. It confirms the positive impact of increasing
the glutamate
availability into the bacterial cell.
Strains 22 and 23 (deletion of gpmM, ptsHl+crr, tdcG or tdcB and
overexpression of galP)
exhibits a further improvement in cysteine production ¨ specifically in the
final cysteine titer
and yield. Advantageously, the suppression of genes coding enzymes consuming
serine or
serine precursors combined increases cysteine production.
EXAMPLE 5: Cysteine strain performance on sucrose as sole Carbone source
Table 6: Biomass production, cysteine titer, productivity and yield for the
different strains
grown on the medium M MS described in Table 2.
Strain Biomass Final cysteine Cysteine
productivity Ycycsteine (gig)
production titer (g/L) (g/L.h)
(g/g)
Strain 24 Reference Reference Reference Reference
Strain 25 -- +++ ++ +++
Strain 26 -- +++ ++ +++
Strain 27 - +++ +++ ++
Strain 28 - +++ +++ ++
Date Recue/Date Received 2023-12-29
39
The symbol "+" indicates an increase of a factor up to 2, the symbol "++" an
increase by a
factor between 2 and 3 and "+++" an increase by a factor greater than 4, as
compared to
the values of reference strain 7. The symbol "2 indicates a decrease of a
factor up to 2, the
symbol "--" a decrease by a factor between 2 and 3, as compared to the values
of reference
strain 7.
The only difference when comparing respectively strains 15, 22 and 23 to
strains 24, 25
and 26 deals with the overexpression of the cscBKAR genes. They are coding for
proteins
conferring the ability of bacteria to passively import sucrose from outside of
the cell. Strain
24 exhibits the same ability to grow and to produce cysteine, using sucrose as
sole carbon
source than the strain 15 when using dextrose.
The results obtained using strains 25 and 26 on sucrose are the same as those
obtained
using strain 22 and 23 on dextrose. These results demonstrate that changing
the capacity
to use different carbon source does not affect the cysteine production
performances
Strains 27 and 28 contains the same metabolic pathway for the cysteine
production except
the absence of gpmM deletion but including the deletion of pyruvate kinase
coding genes
(pykA and pykF).
In this context, the passive sucrose transporter has been replaced by the
active scrKYABR
system. Results obtained with these strains shown on table 5 demonstrate a
significant
improvement of the productivity compare to strains 25 and 26 due to a better
growth of
bacterial culture. Nevertheless, the Yield is negatively affected for the same
reason.
EXEMPLE 6: Serine production improvement by modifying glyA and/or eamA
expression
According to protocols 1, 2 and 3, strains 29 to 34 were obtained by knocking
out the glyA
gene encoding the serine hydroxymethyltransferase GlyA protein (SEQ ID NOs:
120 and
121, respectively) and/or by overexpressing into the pCL1920-serA*-serB-serC
vector
described in example 1, the eamA gene encoding the cysteine/O-acetylserine
exporter
EamA protein (SEQ ID NOs: 122 and 123, respectively) under the native
promoter.
Table 7: strains constructed.
Strains eamA glyA deletion glyA deletion and eamA
overexpression overexpression
Strain 13 Strain 29 Strain 31 Strain 33
Strain 14 Strain 30 Strain 32 Strain 34
Date Recue/Date Received 2023-12-29
40
Table 8: Biomass production, serine titer, productivity and yield for the
different strains
grown on the medium M MS described in Table 2.
Strain Biomass Final serine Serine productivity Ysenne
(gig)
production titer (g/L) (g/L.h)
(g/g)
Strain 13 Reference Reference Reference Reference
Strain 14
Strain 29
Strain 30
Strain 31 ++ ++ ++
Strain 32 ++ ++ ++
Strain 33 = +++ ++ +++
Strain 34 = +++ ++ +++
The symbol "+" indicates an increase of a factor up to 2, the symbol "++" an
increase by a
factor between 2 and 3 and "+++" an increase by a factor greater than3, as
compared to
the values of reference strain 13. The symbol "2 indicates a decrease of a
factor up to 2,
the symbol "=" a decrease by a factor between 2 and 3, as compared to the
values of
reference strain 13.
Results obtained with these strains shown on table 7 demonstrate a significant
improvement
of the strain performances compare to strains 13 and 14 due to a better export
of serine
with the overexpression of eamA gene and a better stability of the strains
with the deletion
of glyA.
Similar results were obtained with dextrose as carbon source.
These modifications have little impact on the performance of the cysteine
producing strains
as described above in Example 5.
Date Recue/Date Received 2023-12-29
41
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Date Recue/Date Received 2023-12-29
