Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR THE PRODUCTION OF XYLITOL FROM
XYLOSE UTILIZING DYNAMIC METABOLIC CONTROL
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application
Numbers
63/004,740 filed April 3, 2020, and 63/056,085 filed July 24, 2020, both of
which is
incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to metabolically engineered microorganisms, such
as bacterial
strains, and bioprocesses utilizing such strains. These strains provide
dynamic control of
metabolic pathways resulting in the production of xylitol from xylose.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which has been
filed electronically in
ASCII format as 49196-465T25 created March 29, 2021 that is 17051 bytes in
size and is
hereby incorporated by reference in its entirety.
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0004] This invention was made with government support under Federal Grant No.
EE0007563
awarded by the Department of Energy; Federal Contract No. HR0011-14-C-0075
awarded by the
United States Department of Defense; Federal Grant No. ONR YIP 12043956
awarded by the
United States Department of Defense; DARPA# HR0011-14-C-0075; ONR YIP #N00014-
16-1-
2558; DOE EERE grant #EE0007563; N00014-16-1-2558 awarded by NAVY/ONR, and NIH
Biotechnology Training Grant (T32GM008555). The government has certain rights
in the
invention.
BACKGROUND OF THE INVENTION
[0005] Xylitol is an industrial sugar alcohol primarily used as a sweetener,
having a similar
sweetness but fewer calories than sucrose. Annual production of Xylitol is
¨125,000 tons and is
produced via the reduction of xylose. Xylose is the second most abundant
natural sugar (after
glucose), therefore it is an attractive feedstock. Many studies have
demonstrated the use of
xylose as a feedstock for the biosynthesis of numerous products ranging from
biofuels (ethanol)
to chemicals, including lactic acid, succinic acid, xylonate, 1,2,4-
butanetriol, and xylitol.
[0006] The industrial production of xylitol relies on traditional chemistry,
and the process has
remained relatively unchanged for decades. This conversion requires expensive
catalysts and
requires relatively pure xylose as a feedstock. Efforts have been made to
identify more
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economical ways to produce xylitol from lower cost, cellulosic sugar streams,
including the
development of biosynthetic processes. Biosynthetic production has the
potential to decrease
costs, utilize lower quality feedstocks, avoid the use of organic solvents,
eliminate the need for
expensive reduction catalysts. However, most previous biosynthetic studies
producing xylitol
from xylose rely on a bioconversion requiring an additional sugar (usually
glucose) as an
electron donor. Oxidation of glucose (producing the byproduct gluconic acid)
generates
NAD(P)H which is then used for xylose reduction. While these processes offer
high xylitol titers
and a good yield when just considering xylose, the requirement for glucose at
equimolar levels to
xylose is a significant inefficiency.
[0007] Perhaps the simplest conversion is xylose to xylitol, which requires
only a single enzyme,
a xylose reductase. Biosynthetic production of xylitol, over chemical
conversion, has the
potential to decrease costs, while avoiding the use of organic solvents,
eliminating the need for
expensive reduction catalysts, and improving product purity.
SUMMARY OF THE INVENTION
[0008] We rationally designed genetically modified microorganism strains to
optimize xylitol
production from xylose utilizing two stage dynamic metabolic control. As
illustrated in FIG 1,
this design included overexpression of xylose reductase and the dynamic
reduction in xylose
isomerase (xylA) activity to reduce xylose metabolism which competes with
xylitol production.
Toward this goal we constructed strains and plasmids to enable the dynamic
induction of xyrA,
and dynamic reduction in XylA activity upon phosphate depletion, or other
causative event,
either through gene silencing, proteolysis of XylA or a combination of both
functions. Provided
herein are microbial strains for scalable biofermentation processes the use
synthetic metabolic
valves (SMVs) to decouple growth from product formation. The described strains
provide
dynamic control of metabolic pathways, including pathways that, when altered,
have negative
effects on microorganism growth under certain inducible conditions.
[0009] We also fully describe improved NADPH flux coincident with xylitol
biosynthesis in
engineered E. coli. Xylitol is produced from xylose via an NADPH dependent
reductase. We
utilize two-stage dynamic metabolic control to compare two approaches to
optimize xylitol
biosynthesis, a stoichiometric approach, wherein competitive fluxes are
decreased, and a
regulatory approach wherein the levels of key regulatory metabolites are
reduced. The
stoichiometric and regulatory approaches lead to a 16 fold and 100 fold
improvement in xylitol
production, respectively. Strains with reduced levels of enoyl-ACP reductase
and glucose-6-
phosphate dehydrogenase, led to altered metabolite pools resulting in the
activation of the
membrane bound transhydrogenase and a new NADPH generation pathway, namely
pyruvate
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ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase,
leading to
increased NADPH fluxes, despite a reduction in NADPH pools. These strains
produced titers of
200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented
bioreactors. Dynamic
control over enoyl-ACP reductase and glucose-6-phosphate dehydrogenase will
broadly enable
improved NADPH dependent bioconversions.
[0010] Also provided herein are multi-stage bioprocesses for xylitol
production that use the
described genetically modified microorganism containing one or more synthetic
metabolic
valves that provide dynamic flux control and result in improved xylitol
production. In certain
embodiments, carbon feedstocks can include xylose, or a combination of xylose
and glucose,
arabinose, mannose, lactose, or alternatively carbon dioxide, carbon monoxide,
methane,
methanol, formaldehyde, or oils. Additional genetic modifications may be added
to a
microorganism to provide further conversion of xylitol to additional chemical
or fuel products.
[0011] Other methods, features and/or advantages is, or will become, apparent
upon examination
of the following Figures and detailed description. It is intended that all
such additional methods,
features, and advantages be included within this description and be protected
by the
accompanying claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The novel features of the invention are set forth with particularity in
the claims. A better
understanding of the features and advantages of the present invention will be
obtained by
reference to the following detailed description that sets forth illustrative
embodiments, in which
the principles of the invention are used, and the accompanying drawings of
which:
[0013] FIG 1 depicts the design of metabolic valves for the bioproduction of
xylitol. The
biosynthesis process of xylitol in E. coli by xylose reductase (XyrA) with
NADPH as cofactor
(bold arrow). The main competitive pathway for the consumption of xylose is to
xylose by
xylose isomerase (XylA, valve).
[0014] FIG 2A-C depicts Xylose Reductase Expression and Enzyme Kinetics.
FIG2A,
Expression of XyrA in BL21 using media combination of SM10++(for growth) and
SM10-No
phos(for expression). After the expression, the postproduction cells were
lysed by freeze-thawing
cycle. Next, the xyrA protein was extracted by N-N Resin because of the His-
tag on XyrA which
was design into plasmid sequence. FIG2B, Activity of xyrA with NADPH as co-
factor. Reaction
velocity is plotted as function of xylose concentration. In these assays,
NADPH was held at a
constant initial level of 50 uM. FIG 2C, Kinetic Parameters for XyrA from this
project and from
other research sources as comparison.
[0015] FIG 3 depicts the xylitol titer/OD (g/L-OD) were measured under
different xylA
silencing and xylA proteolysis combinations. The specific productivity of
different strains was
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significantly different with the control strain DLF-0025-EV. While all three
valve combinations
made statistically significant amount more than the DLF25-EV control, xylA
silencing or
proteolysis alone were better than the combination.
[0016] FIG 4 depicts xylitol Production in E. coli utilizing 2-stage Dynamic
Control. Strain
metabolic network design. The main metabolic pathways include: Fatty Acid
Biosynthesis, the
Citric Acid Cycle (TCA), NADPH supply, the Pentose Phosphate Pathway
Transhydrogenase
and Glycolysis. The valves which may be 'switched off in the metabolic system
include xylose
isomerase (xy1A-X), the soluble transhydrogenase (udhA-U) enoyl-ACP reductase
(fabI-F),
citrate synthase (g1tA-G) and glucose-6-phosphate dehydrogenase (zwf-Z). These
valves are all
highlighted by red valves. Xylose reductase (xyrA) may be dynamically
'switched on' for xylitol
production with NADPH as cofactor.
[0017] FIG 5A-B depicts (5A) Rank order plot for average xylitol titer of all
valve strains
examined in 2-stage micro fermentation, as well as with standard deviation.
Xylitol production in
the control strain was colored in red. A post hoc Dunnett test shows
combinations that differ
from the DLF025-Empty vector control significantly at p <0.05, which are
indicated as darkened
(instead of gray bar, meaning non-significant) in the sorted titer per unit OD
plot. (5B) Heatmap
of xylitol titer in 2-stage production in response to different proteolysis
and silencing
combinations, from 0 g/L (white) to 12 g/L (darker). The x-axis stands for
different proteolysis
valves while the y-axis represents the different pCASCADE silencing. The DLF
25 empty valve
control is in the red circle. The gray dots indicate combinations that are not
assayed or have no
proper cell growth for all replicates. According to the heatmap result, for
the combinations which
the titer/OD >3, 6 replications were performed to avoid the false positive
results.
[0018] FIG 6 depicts p-value map of micro-fermentation results of FIGS.
[0019] FIG 7A-B depicts plots of instrumented fermentation of (7A) an
exemplary production
strain Z-FZ (Silencing of zwf("Z"), proteolysis of fabI and zwf ("FZ")) and
(7B) the control
strain (DLF-0025-EV) to 1L bioreactors The Blue lines indicate the 0D600
values and orange
lines represents the xylitol titer at various time points. The Z-FZ
combination resulted in a titer
of 104+/- 11.31g/L after 160 hours of production, while the control strain
(DLF 0025-EV) only
produced -3 g/L at the same production time. We replicated the Z-FZ tank
fermentation using the
same seed and fermentation conditions, the results here are the average of
these two replicated
tanks and standard deviation was noted in the plot sample points.
[0020] FIG 8 depicts a conceptual model of two-stage NADPH production in our
engineered
system. Glucose-6-phosphate dehydrogenase (encoded by the zwf gene) is
normally responsible
for the biosynthesis of a majority of NADPH. This irreversible reaction drives
an NADPH set
point, in which the SoxRS oxidative stress response is OFF (gray area).
Dynamic reduction in
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Zwf levels reduces NADPH pools activating the SoxRS response, which in turn
activates
expression of Pyruvate ferredoxin oxidoreductase (Pfo, encoded by the ydbK
gene) and NADPH
dependent ferredoxin reductase (Fpr). Together Pfo and Fpr (operating in
reverse) constitute a
new pathway to generate NADPH as well as allow for continued pyruvate
oxidation and
generation of acetyl-CoA for entry into the tricarboxylic acid cycle (TCA
cycle). NADPH flux is
further enhanced by reducing fatty acid biosynthesis whose products inhibit
the membrane
bound transhydrogenase (encoded by the pntAB genes). Activated PntAB uses the
proton motive
force to convert NADH from the TCA cycle to NADPH. NADPH can be used for
bioconversions such as for xylitol production.
[0021] FIG 9A-B depict enzyme levels of 9A) XylA and 9B) UdhA in response to
inducible
proteolysis and/or gene silencing in a phosphate depleted stationary phase. ev
-empty vector, x-
xylA promoter, u- udhA promoter.
[0022] FIG 10 A-C: Specific xylitol production in strains engineered for
dynamic control over
levels of 10A) xylose isomerase (XylA), 10B) soluble transhydrogenase (UdhA)
and 10C) the
combined control over xylose isomerase soluble transhydrogenase. ev -empty
vector, x- xylA
promoter, u- udhA promoter. All results were obtained from microfermentations.
[0023] FIG 11: XyrA expression and purification from BL21(DE3). Left: A time
course of
expression post phosphate depletion, whole cell lysates demonstrate expression
of XyrA.
Densitometry indicates an expression level of ¨ 20%. Middle: Purification of
XyrA (which
contains an N-terminal 6 X histidine tag) via IMAC. Right Kinetic analysis of
purified XyrA.
Initial velocity ( M/s) is plotted as a function of substrate (xylose)
concentration.
[0024] FIG 12 A-D: 12A) An overview of xylitol production and the location of
metabolic
valves in central metabolism. Xylitol is produced from xylose by a xylose
reductase (xyrA).
Valves comprise inducible proteolysis and/or silencing of 5 enzymes: citrate
synthase (g1tA) ,
xylose isomerase (xylA), glucose-6-phosphate dehydrogenase (zwf), enoyl-ACP
reductase (fabI)
and soluble transhydrogenase (udhA). The membrane bound transhydrogenase
(pntAB) is also
shown. 12B) Specific xylitol production (g/L-0D600nm) in microfermentations as
a function of
silencing and or proteolysis. 12C) P-values for the data in 12B, comparing
each strain to the no-
valve control using a Welchs t-test. 12D) a rank order plot of the data from
panel. Bars indicate a
p-value <0.05. Abbreviations: xylE : xylose permease, xylFGH : xylose ABC
transporter, PPP:
pentose phosphate pathway, PDH: pyruvate dehydrogenase multienzyme complex,
TCA:
tricarboxylic acid, G6P: glucose-6-phosphate, 6-PGL: 6-phosphogluconolactone,
6PG: 6-
phosphogluconate, GA3P: glyceraldehyde-3-phosphate , PEP: phosphoenolpyrvate,
OAA:
oxaloacetic acid, X5P: xylulose-5-phosphate, Fd: ferredoxin. Silencing: ev:
empty vector, g2:
gltAp2 promoter, z: zwf promoter, x: xylA promoter, u: udhA promoter.
Proteolysis: F: fabI-
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DAS+4, G: gltA-DAS+4, Z: zwf-DAS+4, U: udha DAS+4, X: xy1A-DAS+4. All results
were
obtained from microfermentations.
[0025] FIG 13 A-D: Agarose gel electrophoretic analysis of gRNA array
stability. Colony PCR
was used to amplify and size gRNA arrays from 8 clones after transformation
into host strains
engineered for dynamic metabolic control. "Guide" indicates PCR products are
taken from
sequence confirmed gRNA arrays with 0, 1 , or 2 gRNAs respectively. 13A)
Strain
DLF Z0025, white labels: pCASCADE-ev, yellow labels: pCASCADE-g2, 13B) Strain
DLF Z0025, white labels: pCASCADE-z, yellow labels: pCASCADE-fg2, 13C) White
labels:
Strain DLF Z0044, pCASCADE-fg2, yellow labels: DLF Z0025, pCASCADE-fg2, 13D)
White
labels: Strain DLF Z0046 , pCASCADE-g2, gray labels: DLF Z1002 pCASCADE-fg2.
[0026] FIG 14: Dynamic Control over FabI (enoyl-ACP reductase) levels due to
inducible
proteolysis with a DAS+4 degron tag. The chromosomal fabI gene was tagged with
a C-terminal
sfGFP. Protein levels were measured by ELISA, 24 hour post induction by
phosphate depletion
in microfermentations.
[0027] FIG 15 A-D: Identification of pathways responsible for NADPH and
xylitol production
in the "FZ" valve strain 15A) the impact of deletions of ydbK and fpr on
specific xylitol
production, 15B) the impact of pntAB overexpression on xylitol production.
(15C-D) "FZ" valve
strains further modified for dynamic control over 15C) GltA levels and 15D)
UdhA levels. ev -
empty vector, z- zwf promoter, g2- gltAp2 promoter, u- udhA promoter. All
results were
obtained from microfermentations.
[0028] FIG 16 A-B: Stoichiometric flux models of 16A) cellular growth and 16B)
stationary
phase xylitol production in "FZ" valve strains. Pathway flux is relative to
xylose uptake rates.
During growth the majority of flux is through the pentose phosphate pathway
(PPP), pyruvate
dehydrogenase multienzyme complex (PDH) with minimal flux through the pentose
membrane
bound transhydrogenase. Upon dynamic control, a 4-fold increase in membrane
bound
transhydrogenase flux is accompanied by increased flux through Pfo (ydbK) and
FPr.
Abbreviations: G6P: glucose-6-phosphate, 6-PGL: 6-phosphogluconolactone, 6PG:
6-
phosphogluconate, GA3P: glyceraldehyde-3-phosphate , OAA: oxaloacetic acid.
[0029] FIG 17 A-B Modeled NADPH producing reactions and pathways for xylitol
production
in different production strains. 17A) Specific reactions fluxes during xylitol
production. 17B)
Pathway percentage fluxes for xylitol production.
[0030] FIG 18 A-C: Xylitol production in minimal media fed batch fermentations
in
instrumented bioreactors by 18A) the control strain expressing xylose
reductase (DLF Z0025,
pCASCADE-ev, pHCKan-xyrA), 18B) the "FZ" valve strain (DLF Z0025-fabI-DAS+4-
zwf-
DAS+4, pCASCADE-z, pHCKan-xyrA), 18C) the "FZ" valve strain also
overexpressing the
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membrane bound transhydrogenase pntAB (DLF Z0025-fabI-DAS+4-zwf-DAS+4,
pCASCADE-z, pHCKan-xyrA, pCDF-pntAB). Biomass (black) and xylitol (blue) are
given as a
function of time. For FIG 18B and 18C, x's and triangles represent the
measured values of two
duplicate runs.
[0031] FIG 19 depicts stationary phase NADPH pools in engineered strain. Pools
were
measured 24 hours post phosphate depletion.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention is related to various genetically modified
microorganisms that have
utility for production of xylitol or a related chemical products to methods of
making such
chemical products using these microorganisms.
Definitions
[0033] As used in the specification and the claims, the singular forms "a,"
"an," and "the"
include plural referents unless the context clearly dictates otherwise. Thus,
for example,
reference to an "expression vector" includes a single expression vector as
well as a plurality of
expression vectors, either the same (e.g., the same operon) or different;
reference to
"microorganism" includes a single microorganism as well as a plurality of
microorganisms; and
the like.
[0034] The term "heterologous DNA," "heterologous nucleic acid sequence," and
the like as
used herein refers to a nucleic acid sequence wherein at least one of the
following is true: (a) the
sequence of nucleic acids is foreign to (i.e., not naturally found in) a given
host microorganism;
(b) the sequence may be naturally found in a given host microorganism, but in
an unnatural (e.g.,
greater than expected) amount; or (c) the sequence of nucleic acids comprises
two or more
subsequences that are not found in the same relationship to each other in
nature. For example,
regarding instance (c), a heterologous nucleic acid sequence that is
recombinantly produced will
have two or more sequences from unrelated genes arranged to make a new
functional nucleic
acid, such as a nonnative promoter driving gene expression. The term
"heterologous" is
intended to include the term "exogenous" as the latter term is generally used
in the art. With
reference to the host microorganism's genome prior to the introduction of a
heterologous nucleic
acid sequence, the nucleic acid sequence that codes for the enzyme is
heterologous (whether or
not the heterologous nucleic acid sequence is introduced into that genome). As
used herein,
chromosomal, and native and endogenous refer to genetic material of the host
microorganism.
[0035] The term "synthetic metabolic valve," and the like as used herein
refers to either the use
of controlled proteolysis, gene silencing or the combination of both
proteolysis and gene
silencing to alter metabolic fluxes.
[0036] As used herein, the term "gene disruption," or grammatical equivalents
thereof (and
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including "to disrupt enzymatic function," "disruption of enzymatic function,"
and the like), is
intended to mean a genetic modification to a microorganism that renders the
encoded gene
product as having a reduced polypeptide activity compared with polypeptide
activity in or from a
microorganism cell not so modified. The genetic modification can be, for
example, deletion of
the entire gene, deletion or other modification of a regulatory sequence
required for transcription
or translation, deletion of a portion of the gene which results in a truncated
gene product (e.g.,
enzyme) or by any of various mutation strategies that reduces activity
(including to no detectable
activity level) the encoded gene product. A disruption may broadly include a
deletion of all or
part of the nucleic acid sequence encoding the enzyme, and also includes, but
is not limited to
other types of genetic modifications, e.g., introduction of stop codons, frame
shift mutations,
introduction or removal of portions of the gene, and introduction of a
degradation signal, those
genetic modifications affecting mRNA transcription levels and/or stability,
and altering the
promoter or repressor upstream of the gene encoding the enzyme.
[0037] Bio-production, Micro-fermentation (microfermentation) or Fermentation,
as used herein,
may be aerobic, microaerobic, or anaerobic.
[0038] When the genetic modification of a gene product, i.e., an enzyme, is
referred to herein,
including the claims, it is understood that the genetic modification is of a
nucleic acid sequence,
such as or including the gene, that normally encodes the stated gene product,
i.e., the enzyme.
[0039] As used herein, the term "metabolic flux" and the like refers to
changes in metabolism
that lead to changes in product and/or byproduct formation, including
production rates,
production titers and production yields from a given substrate.
[0040] Species and other phylogenic identifications are according to the
classification known to
a person skilled in the art of microbiology.
[0041] Enzymes are listed here within, with reference to a UniProt
identification number, which
would be well known to one skilled in the art. The UniProt database can be
accessed at
http://www.UniProt.org/. When the genetic modification of a gene product,
i.e., an enzyme, is
referred to herein, including the claims, it is understood that the genetic
modification is of a
nucleic acid sequence, such as or including the gene, that normally encodes
the stated gene
product, i.e., the enzyme.
[0042] Where methods and steps described herein indicate certain events
occurring in certain
order, those of ordinary skill in the art will recognize that the ordering of
certain steps may be
modified and that such modifications are in accordance with the variations of
the invention.
Additionally, certain steps may be performed concurrently in a parallel
process when possible, as
well as performed sequentially.
[0043] The meaning of abbreviations is as follows: "C" means Celsius or
degrees Celsius, as is
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clear from its usage, DCW means dry cell weight, "s" means second(s), "min"
means minute(s),
"h," "hr," or "hrs" means hour(s), "psi" means pounds per square inch, "nm"
means nanometers,
"d" means day(s), "4" or "uL" or "ul" means microliter(s), "mL" means
milliliter(s), "L"
means liter(s), "mm" means millimeter(s), "nm" means nanometers, "mM" means
millimolar,
" M" or "uM" means micromolar, "M" means molar, "mmol" means millimole(s),
"[tmol" or
"uMol" means micromole(s)", "g" means gram(s), "jig" or "ug" means
microgram(s) and "ng"
means nanogram(s), "PCR" means polymerase chain reaction, "OD" means optical
density,
"OD600" means the optical density measured at a photon wavelength of 600 nm,
"kDa" means
kilodaltons, "g" means the gravitation constant, "bp" means base pair(s),
"kbp" means kilobase
pair(s), "% w/v" means weight/volume percent, "% v/v" means volume/volume
percent, "IPTG"
means isopropyl-D-thiogalactopyranoiside, "aTc" means anhydrotetracycline,
"RBS" means
ribosome binding site, "rpm" means revolutions per minute, "HPLC" means high
performance
liquid chromatography, and "GC" means gas chromatography.
I. Carbon Sources
[0044] Bio-production media, which is used in the present invention with
recombinant
microorganisms must contain suitable carbon sources or substrates for both
growth and
production stages. Suitable substrates may include but are not limited to
xylose or a combination
of xylose and glucose, sucrose, xylose, mannose, arabinose, oils, carbon
dioxide, carbon
monoxide, methane, methanol, formaldehyde, or glycerol. It is contemplated
that all of the above
mentioned carbon substrates and mixtures thereof are suitable in the present
invention as a
carbon source(s).
II. Microorganisms
[0045] Features as described and claimed herein may be provided in a
microorganism selected
from the listing herein, or another suitable microorganism, that also
comprises one or more
natural, introduced, or enhanced product bio-production pathways. Thus, in
some embodiments
the microorganism(s) comprise an endogenous product production pathway (which
may, in some
such embodiments, be enhanced), whereas in other embodiments the microorganism
does not
comprise an endogenous product production pathway.
[0046] More particularly, based on the various criteria described herein,
suitable microbial hosts
for the bio-production of a chemical product generally may include, but are
not limited to the
organisms described in the Methods Section.
[0047] The host microorganism or the source microorganism for any gene or
protein described
here may be selected from the following list of microorganisms: Citrobacter,
Enterobacter,
Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus,
Saccharomyces,
Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida,
Hansenula,
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Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella,
Bacillus,
Streptomyces, and Pseudomonas. In some aspects the host microorganism is an
E.coli
microorganism.
III. Media and Culture Conditions
[0048] In addition to an appropriate carbon source, such as selected from one
of the herein-
disclosed types, bio-production media must contain suitable minerals, salts,
cofactors, buffers
and other components, known to those skilled in the art, suitable for the
growth of the cultures
and promotion of chemical product bio-production under the present invention.
[0049] Another aspect of the invention regards media and culture conditions
that comprise
genetically modified microorganisms of the invention and optionally
supplements.
[0050] Typically cells are grown at a temperature in the range of about 25 C
to about 40 C in
an appropriate medium, as well as up to 70 C for thermophilic microorganisms.
Suitable growth
media are well characterized and known in the art. Suitable pH ranges for the
bio-production are
between pH 2.0 to pH 10.0, where pH 6.0 to pH 8.0 is a typical pH range for
the initial
condition. However, the actual culture conditions for a particular embodiment
are not meant to
be limited by these pH ranges. Bio-productions may be performed under aerobic,
microaerobic
or anaerobic conditions with or without agitation.
IV. Bio-production Reactors and Systems
[0051] Fermentation systems utilizing methods and/or compositions according to
the invention
are also within the scope of the invention. Any of the recombinant
microorganisms as described
and/or referred to herein may be introduced into an industrial bio-production
system where the
microorganisms convert a carbon source into a product in a commercially viable
operation. The
bio-production system includes the introduction of such a recombinant
microorganism into a
bioreactor vessel, with a carbon source substrate and bio-production media
suitable for growing
the recombinant microorganism, and maintaining the bio-production system
within a suitable
temperature range (and dissolved oxygen concentration range if the reaction is
aerobic or
microaerobic) for a suitable time to obtain a desired conversion of a portion
of the substrate
molecules to a selected chemical product. Bio-productions may be performed
under aerobic,
microaerobic, or anaerobic conditions, with or without agitation. Industrial
bio-production
systems and their operation are well-known to those skilled in the arts of
chemical engineering
and bioprocess engineering.
[0052] The amount of a product produced in a bio-production media generally
can be
determined using a number of methods known in the art, for example, high
performance liquid
chromatography (HPLC), gas chromatography (GC), or GC/Mass Spectroscopy (MS).
V. Genetic Modifications, Nucleotide Sequences, and Amino Acid Sequences
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[0053] Embodiments of the present invention may result from introduction of an
expression
vector into a host microorganism, wherein the expression vector contains a
nucleic acid sequence
coding for an enzyme that is, or is not, normally found in a host
microorganism.
[0054] The ability to genetically modify a host cell is essential for the
production of any
genetically modified (recombinant) microorganism. The mode of gene transfer
technology may
be by electroporation, conjugation, transduction, or natural transformation. A
broad range of host
conjugative plasmids and drug resistance markers are available. The cloning
vectors are tailored
to the host organisms based on the nature of antibiotic resistance markers
that can function in
that host. Also, as disclosed herein, a genetically modified (recombinant)
microorganism may
comprise modifications other than via plasmid introduction, including
modifications to its
genomic DNA.
[0055] More generally, nucleic acid constructs can be prepared comprising an
isolated
polynucleotide encoding a polypeptide having enzyme activity operably linked
to one or more
(several) control sequences that direct the expression of the coding sequence
in a microorganism,
such as E. coil, under conditions compatible with the control sequences. The
isolated
polynucleotide may be manipulated to provide for expression of the
polypeptide. Manipulation
of the polynucleotide's sequence prior to its insertion into a vector may be
desirable or necessary
depending on the expression vector. The techniques for modifying
polynucleotide sequences
utilizing recombinant DNA methods are well established in the art.
[0056] The control sequence may be an appropriate promoter sequence, a
nucleotide sequence
that is recognized by a host cell for expression of a polynucleotide encoding
a polypeptide of the
present invention. The promoter sequence may contain transcriptional control
sequences that
mediate the expression of the polypeptide. The promoter may be any nucleotide
sequence that
shows transcriptional activity in the host cell of choice including mutant,
truncated, and hybrid
promoters, and may be obtained from genes encoding extracellular or
intracellular polypeptides
either homologous or heterologous to the host cell. The techniques for
modifying and utilizing
recombinant DNA promoter sequences are well established in the art.
[0057] For various embodiments of the invention the genetic manipulations may
include a
manipulation directed to change regulation of, and therefore ultimate activity
of, an enzyme or
enzymatic activity of an enzyme identified in any of the respective pathways.
Such genetic
modifications may be directed to transcriptional, translational, and post-
translational
modifications that result in a change of enzyme activity and/or selectivity
under selected culture
conditions. Genetic manipulation of nucleic acid sequences may increase copy
number and/or
comprise use of mutants of an enzyme related to product production. Specific
methodologies and
approaches to achieve such genetic modification are well known to one skilled
in the art.
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[0058] In various embodiments, to function more efficiently, a microorganism
may comprise
one or more gene deletions. For example, in E. colt, the genes encoding the
lactate
dehydrogenase (ldhA), phosphate acetyltransferase (pta), pyruvate oxidase
(poxB), pyruvate-
formate lyase (pflB), methylglyoxal synthase (mgsA), acetate kinase (ackA),
alcohol
dehydrogenase (adhE), the clpXP protease specificity enhancing factor (sspB),
the ATP-
dependent Lon protease (lon), the outer membrane protease (ompT), the arcA
transcriptional
dual regulator (arcA), and the ic1R transcriptional regulator (ic1R) may be
disrupted, including
deleted. Such gene disruptions, including deletions, are not meant to be
limiting, and may be
implemented in various combinations in various embodiments. Gene deletions may
be
accomplished by numerous strategies well known in the art, as are methods to
incorporate
foreign DNA into a host chromosome.
[0059] In various embodiments, to function more efficiently, a microorganism
may comprise
one or more synthetic metabolic valves, composed of enzymes targeted for
controlled
proteolysis, expression silencing or a combination of both controlled
proteolysis and expression
silencing. For example, one enzyme encoded by one gene or a combination of
numerous
enzymes encoded by numerous genes in E. colt may be designed as synthetic
metabolic valves to
alter metabolism and improve product formation. Representative genes in E.
colt may include
but are not limited to the following: fabl, zwf gltA, ppc, udhA, 1pd, sucD,
aceA, pfkA, ion, rpoS,
pykA, pykF, tktA or tktB. It is appreciated that it is well known to one
skilled in the art how to
identify homologues of these genes and or other genes in additional microbial
species.
[0060] For all nucleic acid and amino acid sequences provided herein, it is
appreciated that
conservatively modified variants of these sequences are included and are
within the scope of the
invention in its various embodiments. Functionally equivalent nucleic acid and
amino acid
sequences (functional variants), which may include conservatively modified
variants as well as
more extensively varied sequences, which are well within the skill of the
person of ordinary skill
in the art, and microorganisms comprising these, also are within the scope of
various
embodiments of the invention, as are methods and systems comprising such
sequences and/or
microorganisms.
[0061] Accordingly, as described in various sections above, some compositions,
methods and
systems of the present invention comprise providing a genetically modified
microorganism that
comprises both a production pathway to make a desired product from a central
intermediate in
combination with synthetic metabolic valves to redistribute flux.
[0062] Aspects of the invention also regard provision of multiple genetic
modifications to
improve microorganism overall effectiveness in converting a selected carbon
source into a
selected product. Particular combinations are shown, such as in the Examples,
to increase
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specific productivity, volumetric productivity, titer and yield substantially
over more basic
combinations of genetic modifications.
[0063] In addition to the above-described genetic modifications, in various
embodiments genetic
modifications, including synthetic metabolic valves also are provided to
increase the pool and
availability of the cofactor NADPH and/or NADH which may be consumed in the
production of
a product.
[0064] VI. Synthetic Metabolic Valves
[0065] Use of synthetic metabolic valves allows for simpler models of
metabolic fluxes and
physiological demands during a production phase, turning a growing cell into a
stationary phase
biocatalyst. These synthetic metabolic valves can be used to turn off
essential genes and redirect
carbon, electrons, and energy flux to product formation in a multi-stage
fermentation process.
One or more of the following provides the described synthetic valves: 1)
transcriptional gene
silencing or repression technologies in combination with 2) inducible and
selective enzyme
degradation and 3) nutrient limitation to induce a stationary or non-dividing
cellular state. SMVs
are generalizable to any pathway and microbial host. These synthetic metabolic
valves allow for
novel rapid metabolic engineering strategies useful for the production of
renewable chemicals
and fuels and any product that can be produced via whole cell catalysis.
[0066] In particular, the invention describes the construction of synthetic
metabolic valves
comprising one or more or a combination of the following: controlled gene
silencing and
controlled proteolysis. It is appreciated that one well skilled in the art is
aware of several
methodologies for gene silencing and controlled proteolysis.
VI.A Gene Silencing
[0067] In particular, the invention describes the use of controlled gene
silencing to provide the
control over metabolic fluxes in controlled multi-stage fermentation
processes. There are several
methodologies known in the art for controlled gene silencing, including but
not limited to
mRNA silencing or RNA interference, silencing via transcriptional repressors
and CRISPR
interference. Methodologies and mechanisms for RNA interference are taught by
Agrawal et al.
"RNA Interference: Biology, Mechanism, and Applications" Microbiology and
Molecular
Biology Reviews, December 2003; 67(4) p657-685. DOT: 10.1128/MMBR.67.657-
685.2003.
Methodologies and mechanisms for CRISRPR interference are taught by Qi et al.
"Repurposing
CRISPR as an RNA-guided platform for sequence-specific control of gene
expression" Cell
February 2013; 152(5) p1173-1183. DOT: 10.1016/j.ce11.2013.02.022. In
addition,
methodologies, and mechanisms for CRISRPR interference using the native E.
coil CASCADE
system are taught by Luo et al. "Repurposing endogenous type I CRISPR-Cas
systems for
programmable gene repression" NAR. October 2014; DOT: 10.1093. In additional
numerous
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transcriptional repressor systems are well known in the art and can be used to
turn off gene
expression.
VI.B Controlled Proteolysis
[0068] In particular, the invention describes the use of controlled protein
degradation or
proteolysis to provide the control over metabolic fluxes in controlled multi-
stage fermentation
processes. There are several methodologies known in the art for controlled
protein degradation,
including but not limited to targeted protein cleavage by a specific protease
and controlled
targeting of proteins for degradation by specific peptide tags. Systems for
the use of the E. coil
clpXP protease for controlled protein degradation are taught by McGinness et
al, "Engineering
controllable protein degradation", Mol Cell. June 2006; 22(5) p701-707. This
methodology relies
upon adding a specific C-terminal peptide tag such as a DAS4 (or DAS+4) tag.
Proteins with this
tag are not degraded by the clpXP protease until the specificity enhancing
chaperone sspB is
expressed. sspB induces degradation of DAS4 tagged proteins by the clpXP
protease. In
additional numerous site specific protease systems are well known in the art.
Proteins can be
engineered to contain a specific target site of a given protease and then
cleaved after the
controlled expression of the protease. In some embodiments, the cleavage can
be expected lead
to protein inactivation or degradation. For example Schmidt et al("ClpS is the
recognition
component for Escherichia coli substrates of the N-end rule degradation
pathway" Molecular
Microbiology March 2009. 72(2), 506-517. doi:10.1111), teaches that an N-
terminal sequence
can be added to a protein of interest in providing clpS dependent clpAP
degradation. In addition,
this sequence can further be masked by an additional N-terminal sequence,
which can be
controllable cleaved such as by a ULP hydrolase. This allows for controlled N-
rule degradation
dependent on hydrolase expression. It is therefore possible to tag proteins
for controlled
proteolysis either at the N-terminus or C-terminus. The preference of using an
N-terminal vs. C-
terminal tag will largely depend on whether either tag affects protein
function prior to the
controlled onset of degradation.
[0069] The invention describes the use of controlled protein degradation or
proteolysis to
provide the control over metabolic fluxes in controlled multi-stage
fermentation processes, in E.
coil. There are several methodologies known in the art for controlled protein
degradation in other
microbial hosts, including a wide range of gram-negative as well as gram-
positive bacteria, yeast
and even archaea. In particular, systems for controlled proteolysis can be
transferred from a
native microbial host and used in a non-native host. For example Grilly et al,
"A synthetic gene
network for tuning protein degradation in Saccharomyces cerevisiae" Molecular
Systems
Biology 3, Article 127. doi:10.1038, teaches the expression and use of the E.
coil clpXP protease
in the yeast Saccharomyces cerevisiae . Such approaches can be used to
transfer the
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methodology for synthetic metabolic valves to any genetically tractable host.
VI.0 Synthetic Metabolic Valve Control
[0070] In particular the invention describes the use of synthetic metabolic
valves to control
metabolic fluxes in multi-stage fermentation processes. There are numerous
methodologies
known in the art to induce expression that can be used at the transition
between stages in multi-
stage fermentations. These include but are not limited to artificial chemical
inducers including:
tetracycline, anhydrotetracycline, lactose, IPTG (isopropyl-beta-D-1-
thiogalactopyranoside),
arabinose, raffinose, tryptophan and numerous others. Systems linking the use
of these well-
known inducers to the control of gene expression silencing and/or controlled
proteolysis can be
integrated into genetically modified microbial systems to control the
transition between growth
and production phases in multi-stage fermentation processes.
[0071] In addition, it may be desirable to control the transition between
growth and production
in multi-stage fermentations by the depletion of one or more limiting
nutrients that are consumed
during growth. Limiting nutrients can include but are not limited to:
phosphate, nitrogen, sulfur,
and magnesium. Natural gene expression systems that respond to these nutrient
limitations can
be used to operably link the control of gene expression silencing and/or
controlled proteolysis to
the transition between growth and production phases in multi-stage
fermentation processes.
[0072] Within the scope of the invention are genetically modified
microorganism, wherein the
microorganism is capable of producing xylitol at a specific rate selected from
the rates of greater
than 0.05 g/gDCW-hr, 0.08g/gDCW-hr, greater than 0.1g/gDCW-hr, greater than
0.13g/gDCW-
hr, greater than 0.15g/gDCW-hr, greater than 0.175g/gDCW-hr, greater than
0.2g/gDCW-hr,
greater than 0.25g/gDCW-hr, greater than 0.3g/gDCW-hr, greater than 0.35g/gDCW-
hr, greater
than 0.4g/gDCW-hr, greater than 0.45g/gDCW-hr, or greater than 0.5g/gDCW-hr.
[0073] Within the scope of the invention are genetically modified
microorganism, wherein the
microorganism is capable of producing xylitol from xylose or another sugar
source at a yield
greater than 0.5 g product /g xylose, greater than 0.6 g product /g xyloseõ
greater than 0.7 g
product /g xyloseõ greater than 0.8 g product /g xyloseõ greater than 0.9 g
product /g xyloseõ
greater than 0.95 g product /g xyloseõ or greater than 0.98 g product /g
xylose.
[0074] In various embodiments, the invention includes a culture system
comprising a carbon
source in an aqueous medium and a genetically modified microorganism according
to any one of
claims herein, wherein said genetically modified organism is present in an
amount selected from
greater than 0.05 gDCW/L, 0.1 gDCW/L, greater than 1 gDCW/L, greater than 5
gDCW/L,
greater than 10 gDCW/L, greater than 15 gDCW/L or greater than 20 gDCW/L, such
as when
the volume of the aqueous medium is selected from greater than 5 mL, greater
than 100 mL,
greater than 0.5L, greater than 1L, greater than 2 L, greater than 10 L,
greater than 250 L, greater
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than 1000L, greater than 10,000L, greater than 50,000 L, greater than 100,000
L or greater than
200,000 L, and such as when the volume of the aqueous medium is greater than
250 L and
contained within a steel vessel.
Overview of Invention Aspects
[0075] In one aspect, a genetically modified microorganism for producing
xylitol comprising is
provided. The genetically modified microorganism characterized by inducible
modification of
expression of xylose reductase (xyrA) and an inducible synthetic metabolic
valve. The synthetic
metabolic valve characterized by a gene expression-silencing synthetic
metabolic valve
characterized by silencing gene expression of one or more genes encoding one
or more enzymes;
or an enzymatic degradation synthetic metabolic valve characterized by
inducing enzymatic
degradation of one or more enzymes, or a combination thereof
[0076] In one aspect the xylose reductase of the genetically modified
microorganism is an
NADPH dependent xylose reductase or the xylose reductase maybe the xyrA gene
of A. niger. .
[0077] In one aspect, the genetically modified microorganism produces xylitol
from a xylose
feedstock. Of course the genetically modified microorganism may use a
feedstock comprising
xylose and a second sugar blending in any ratio.
[0078] In one aspect the gene-silencing synthetic metabolic valve or the
enzyme degradation
synthetic metabolic valve of the genetically modified microorganism maybe
directed to control
of the gene encoding xylose isomerase or the xylose isomerase enzyme; or the
gene encoding
glucose-6-phosphate dehydrogenase (zwf) or the glucose-6-phosphate
dehydrogenase (zwf)
enzyme.
[0079] In one aspect the gene-silencing synthetic metabolic valve or the
enzyme degradation
synthetic metabolic valve of the genetically modified microorganism maybe
directed to control
more than one gene, for example a gene encoding glucose-6-phosphate
dehydrogenase (zwf) or
the glucose-6-phosphate dehydrogenase (zwf) enzyme; and a gene encoding xylose
isomerase or
the xylose isomerase enzyme.
[0080] In yet another aspect, In one aspect the gene-silencing synthetic
metabolic valve or the
enzyme degradation synthetic metabolic valve of the genetically modified
microorganism maybe
directed to control more than one gene, for example a gene encoding glucose-6-
phosphate
dehydrogenase (zwf) or the glucose-6-phosphate dehydrogenase (zwf) enzyme; and
a gene
encoding enoyl-ACP reductase (fabl) or the enoyl-ACP reductase (fabl) enzyme.
[0081] In yet another aspect, In one aspect the gene-silencing synthetic
metabolic valve or the
enzyme degradation synthetic metabolic valve of the genetically modified
microorganism maybe
directed to control silencing of a gene encoding glucose-6-phosphate
dehydrogenase (zwf) and
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enzyme degradation of glucose-6-phosphate dehydrogenase (zwf) enzyme; and
enoyl-ACP
reductase (fab I) enzyme.
[0082] In another aspect, expression of xylose reductase, gene expression-
silencing synthetic
metabolic valve, and the enzymatic degradation synthetic metabolic valve are
induced under
conditions of a transition phrase of a multi-stage biofermentation process.
The induction may
occur via nutrient depletion or via phosphate depletion.
[0083] In one aspect, the genetically modified microorganism may further
comprise a
chromosomal deletion.
[0084] In one aspect, the silencing of gene expression comprises CRISPR
interference and the
genetically modified microorganism also expresses a CASCADE guide array, the
array
comprising two or more genes encoding small guide RNAs each specific for
targeting a different
gene for simultaneous silencing of multiple genes.
[0085] In one aspect, the genetically modified microorganism produces a
xylitol product titer of
greater than 0.08 g/L at twenty four in a biofermentation process.
[0086] In one aspect, the invention provides for a multi-stage fermentation
bioprocess for
producing xylitol from a genetically modified microorganism, including the
steps of (a) providing
a genetically modified microorganism. The genetically modified microorganism
characterized by
a modification of expression of xylose reductase and a synthetic metabolic
valve comprising: a
gene expression-silencing synthetic metabolic valve characterized by silencing
gene expression of
one or more genes encoding one or more enzymes; or an enzymatic degradation
synthetic
metabolic valve characterized by inducing enzymatic degradation of one or more
enzymes, or a
combination thereof The one or more enzymes of each synthetic metabolic valve
are the same or
different. The method further includes the steps of growing the genetically
modified
microorganism in a media with a xylose feedstock and transitioning from a
growth phase to a
xylitol. The transition step includes inducing the synthetic metabolic
valve(s) to slow or stop the
growth of the microorganism; and inducing expression of xylose reductase,
thereby producing
xylitol.
[0087] In some aspects, the multi-stage fermentation bioprocess may use a
genetically modified
microorganism characterized by the gene-silencing synthetic metabolic valve or
the enzyme
degradation synthetic metabolic valve of the genetically modified
microorganism are directed to
control of at least two genes, including a gene encoding glucose-6-phosphate
dehydrogenase
(zwf) or the glucose-6-phosphate dehydrogenase (zwf) enzyme; and a gene
encoding enoyl-ACP
reductase (fab I) or the enoyl-ACP reductase (fabl) enzyme.
[0088] In some aspects, the multi-stage fermentation bioprocess will produce a
xylitol product
titer of greater than 0.08 g/L at twenty four in a biofermentation process.
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[0089] In some aspects, the transition phase of the multi-stage fermentation
bioprocess occurs
via phosphate depletion of the growth media. In some aspects, the genetically
modified
microorganism of the multi-stage fermentation bioprocess is further
characterized by a
chromosomal deletion.
[0090] In one aspect the genetically modified microorganism for producing
xylitol, the
microorganism comprises: inducible reduction of xylose isomerase; inducible
reduction of
glucose-6-phosphate dehydrogenase activity so that the microorganism produces
xylitol from the
feedstock xylose upon induction. In another aspect the microorganism is an
E.coli
microorganism. In one aspect, the induction of the microorganism occurs by via
nutrient
depletion. In one aspect, the induction of the microorganism occurs via
phosphate depletion.
[0091] In one aspect, the invention provides a multi-stage fermentation
bioprocess for producing
xylitol from a genetically modified microorganism including inducible
reduction of xylose
isomerase and inducible reduction of glucose-6-phosphate dehydrogenase
activity. The
bioprocess includes the steps of (a) providing a genetically modified
microorganism, (b) growing
the genetically modified microorganism in a media with a xylose feedstock; (c)
transitioning
from a growth phase to a xylitol producing stage by inducing the synthetic
metabolic valve(s) to
slow or stop the growth of the microorganism; and inducing expression of
xylose isomerase,
thereby (d) producing xylitol.
[0092] In one aspect the genetically modified microorganism for producing
xylitol, the
microorganism comprises: inducible reduction of xylose reductase; inducible
reduction of
glucose-6-phosphate dehydrogenase activity; inducible reduction of enoyl-ACP
reductase;
wherein the strain produces xylitol from the feedstock xylose upon induction.
In one aspect, the
microorganism is an E.coli microorganism. In some aspect, induction of the
microorganism
occurs by via nutrient depletion or phosphate depletion.
[0093] In one aspect, the invention provides a multi-stage fermentation
bioprocess for producing
xylitol from a genetically modified microorganism including inducible
reduction of xylose
reductase; inducible reduction of glucose-6-phosphate dehydrogenase activity;
inducible
reduction of enoyl-ACP reductase. The bioprocess includes the steps of (a)
providing a
genetically modified microorganism; (b) growing the genetically modified
microorganism in a
media with a xylose feedstock; (c) transitioning from a growth phase to a
xylitol producing stage
by inducing the synthetic metabolic valve(s) to slow or stop the growth of the
microorganism;
and inducing expression of xylose reductase, thereby (d) producing xylitol.
[0094] In one aspect the genetically modified microorganism for producing
xylitol, the
microorganism comprises: activity of a membrane bound transhydrogenase
activity is increased;
activity of a pyruvate ferredoxin oxidoreductase is increased; activity of a
NADPH dependent
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ferredoxin reductase is increased; and wherein the microorganism produces at
least one chemical
product whose biosynthesis requires NADPH.
Disclosed Embodiments Are Non-Limiting
[0095] While various embodiments of the present invention have been shown and
described
herein, it is emphasized that such embodiments are provided by way of example
only. Numerous
variations, changes and substitutions may be made without departing from the
invention herein
in its various embodiments. Specifically, and for whatever reason, for any
grouping of
compounds, nucleic acid sequences, polypeptides including specific proteins
including
functional enzymes, metabolic pathway enzymes or intermediates, elements, or
other
compositions, or concentrations stated or otherwise presented herein in a
list, table, or other
grouping (such as metabolic pathway enzymes shown in a FIGs 1 and 4), unless
clearly stated
otherwise, it is intended that each such grouping provides the basis for and
serves to identify
various subset embodiments, the subset embodiments in their broadest scope
comprising every
subset of such grouping by exclusion of one or more members (or subsets) of
the respective
stated grouping. Moreover, when any range is described herein, unless clearly
stated otherwise,
that range includes all values therein and all sub-ranges therein.
[0096] Also, and more generally, in accordance with disclosures, discussions,
examples and
embodiments herein, there may be employed conventional molecular biology,
cellular biology,
microbiology, and recombinant DNA techniques within the skill of the art. Such
techniques are
explained fully in the literature. See, e.g., Sambrook and Russell, "Molecular
Cloning: A
Laboratory Manual," Third Edition 2001 (volumes 1 - 3), Cold Spring Harbor
Laboratory Press,
Cold Spring Harbor, N.Y.; Animal Cell Culture, R. I. Freshney, ed., 1986.
These published
resources are incorporated by reference herein.
[0097] The following published resources are incorporated by reference herein
for description
useful in conjunction with the invention described herein, for example,
methods of industrial
bio-production of chemical product(s) from sugar sources, and also industrial
systems that may
be used to achieve such conversion (Biochemical Engineering Fundamentals, 2nd
Ed. J. E. Bailey
and D. F. 011is, McGraw Hill, New York, 1986, e.g. Chapter 9, pages 533-657
for biological
reactor design; Unit Operations of Chemical Engineering, 5th Ed., W. L. McCabe
et al., McGraw
Hill, New York 1993, e.g., for process and separation technologies analyses;
Equilibrium Staged
Separations, P. C. Wankat, Prentice Hall, Englewood Cliffs, NJ USA, 1988,
e.g., for separation
technologies teachings).
[0098] All publications, patents, and patent applications mentioned in this
specification are
entirely incorporated by reference herein, including U.S. Provisional
Application No.s
62/010,574, filed June 11, 2014, and 62/461,436, filed February 21, 2017, and
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PCT/US2015/035306 filed June 11,2015 and PCT/US2018/019040, filed February 21,
2018.
EXAMPLES
[0099] The examples herein provide some examples, not meant to be limiting.
All reagents,
unless otherwise indicated, are obtained commercially. Species and other
phylogenic
identifications are according to the classification known to a person skilled
in the art of
microbiology, molecular biology, and biochemistry.
[00100] Common Methods
[00101] Reagents and Media
[00102] All reagents and chemicals were obtained from Sigma Aldrich (St.
Louis, MO)
unless otherwise noted. MOPS (3-(N-morpholino)propanesulfonic acid) was
obtained from
BioBasic, Inc. (Amherst, NY). Crystalline xylose was obtained from Profood
International
(Naperville, IL). All media: SM10++, SM10 No Phosphate, and FGM25 were
prepared as
previously reported (Menacho-Melgar, R. et al. Scalable, two-stage,
autoinduction of
recombinant protein expression in E. coli utilizing phosphate depletion.
Biotechnol. Bioeng. 26,
44 (2020)) except that xylose was substituted for glucose (1 gram xylose for 1
gram glucose) in
all media formulations. LB, Lennox formulation, was used for routine strain
propagation.
Working antibiotic concentrations were as follows: kanamycin: 35 ng/mL,
chloramphenicol: 35
ng/mL, gentamicin:10 ng/mL, 10 zeocin: 100 ng/mL, blasticidin: 100 ng/mL,
spectinomycin:
25 ng/mL, tetracycline: 5 ng/mL.
[00103] Strains & Plasmids Construction
[00104] Refer to Supplemental Table 51 for a list of strains and plasmids
used in this
study. Sequences of synthetic DNA used in this study are given in Supplemental
Table S2.
Chromosomal modifications were constructed using standard recombineering
methodologies
(Liochev, S. let al Proc. Natl. Acad. Sci. U S. A. 91, 1328-1331 (1994)). The
recombineering
plasmid pSIM5 was a kind gift from Donald Court (NCI,
https://redrecombineering.ncifcrfigov/court-lab.html). 53,54 C-terminal DAS+4
tags were added
by direct integration and selected through integration of antibiotic
resistance cassettes 3' of the
gene as previously described.24 All strains were confirmed by PCR, agarose gel
electrophoresis
and confirmed by sequencing. Refer to Table S3 for oligos used for strain
confirmation and
sequencing.
[00105] The xyrA gene from Aspergillus niger was codon optimized for
expression in E.
coli and the plasmid, pHCKan-xyrA (Addgene #58613), enabling the low phosphate
induction of
xylose reductase, was constructed by TWIST Biosciences (San Francisco, CA).
pCDF-pntAB
(Addgene # 158609) was constructed using PCR and Gibson Assembly from pCDF-ev
30 to
drive expression of the pntAB operon from the low phosphate inducible ugpBp
promoter
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(Moreb, E. A. et al. Media Robustness and scalability of phosphate regulated
promoters useful
for two-stage autoinduction in E. coli. ACS Synthetic Biology (2020)
doi:10.1021/acssynbio.0c00182). pCASCADE guide RNA array plasmids were
prepared by the
combination of PCR and Gibson assembly as previously described. Refer to Table
S4 for oligos
used for pCASCADE plasmid construction.
[00106] Table 1: Plasmids used in these Examples:
Addgene Source
Plasmids Promoter On Res
Number
pSMART-HC-Kan None colE1 Kan NA Lucigen
pHC-Kan-yibDp-xyrA yibDp colE1 Kan TBD
Previous Lab
Work
pCASCADE-EV ugpBp p15A Cm TBD
Previous Lab
Work
pCASCADE-gltAl ugpBp p15A Cm TBD
Previous Lab
Work
pCASCADE-gltA2 ugpBp p15A Cm 65817
Previous Lab
Work
pCASCADE-zwf ugpBp p15A Cm 65825
Previous Lab
Work
pCACADE-udhA ugpBp p15A Cm 65818
Previous Lab
Work
pCASCADE-xylA ugpBp p15A Cm TBD
Previous Lab
Work
pCASCADE-gltAl-zwf ugpBp p15A Cm TBD
Previous Lab
Work
pCASCADE-gltA2-zwf ugpBp p15A Cm TBD
Previous Lab
Work
pCASCADE-gltAl-udhA ugpBp p15A Cm TBD
Previous Lab
Work
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pCACADE-gltA2-udhA ugpBp p15A Cm 65819 Previous
Lab
Work
pCASCADE-gltAl-gltA2 ugpBp p15A Cm TBD Previous
Lab
Work
pCASCADE-gltAl-gltA2- ugpBp p15A Cm TBD Previous
Lab
zwf Work
pCASCADE-gltAl-gltA2- ugpBp p15A Cm TBD Previous
Lab
udhA Work
pCASCADE-zwf-xylA ugpBp p15A Cm TBD This
study
pCASCADE-udhA-xylA ugpBp p15A Cm TBD This
study
pCASCADE-gltAl-xylA ugpBp p15A Cm TBD This
study
pCASCADE-gltA2-xylA ugpBp p15A Cm TBD This
study
pCASCADE-gltAl -zwf- ugpBp p15A Cm TBD This
study
xylA
pCASCADE-gltA2-zwf- ugpBp p15A Cm TBD This
study
xylA
pCAS CADE-gltA 1 -udhA- ugpBp p15A Cm TBD This
study
xylA
pCACADE-gltA2-udhA- ugpBp p15A Cm TBD This
study
xylA
pCASCADE-gltAl-gltA2- ugpBp p15A Cm TBD This
study
xylA
pCASCADE-gltAl-gltA2- ugpBp p15A Cm TBD This
study
zwf-xylA
pCASCADE-gltAl-gltA2- ugpBp p15A Cm TBD This
study
udhA-xylA
[00107] Table Si: Additional plasmids used in the Examples:
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Plasmid Insert promot On Res Addge Source
er ne
pSMART-HC- None None colE1 Kan NA Lucigen
Kan
pCDF-ev None None c1oDF1 Sm 89596 Previous
3 Lab
Work
i-ittps://p
Mred<11,
OZSI 3
k3 NT
pHCKan-xyrA xyrA yibDp2 colE1 Kan 158610 This
study
pCDF-pntAB pntAB ugpBp2 c1oDF1 Sm 158609 This
3 study
pCASCADE-ev none ugpBp2 p15 Cm 65821 Previous
Lab
Work
pCASCADE-g2 gltAp2 gRNA ugpBp2 p15 Cm 65817 Previous
Lab
Work
pCASCADE-f fabIp gRNA ugpBp2 p15 Cm 66635 This
study
pCASCADE-z zwfp gRNA ugpBp2 p15 Cm 65825 Previous
Lab
Work
pCASCADE-u udhAp gRNA ugpBp2 p15 Cm 65818 This
study
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pCASCADE-x xylAp gRNA ugpBp2 p15 Cm 158611
This
study
pCASCADE- gltAp2, zwfp gRNA array ugpBp2
p15 Cm 71338 Previous
g2z Lab
Work
pCASCADE- gltAp2 udhAp gRNA array ugpBp2
p15 Cm 65819 This
g2u study
pCASCADE- gltAp2, xylAp gRNA array
ugpBp2 p15 Cm 158613 This
g2x study
pCASCADE-zx zwfp xylAp gRNA array ugpBp2 p15 Cm 158614
This
study
pCASCADE-ux udhAp , xylAp gRNA array ugpBp2 p15 Cm 158612
This
study
pCASCADE- fabIp, gltAp2 gRNA array
ugpBp2 p15 Cm 71341 This
fg2 study
pCASCADE-fz fabIp, zwf gRNA array ugpBp2 p15 Cm 71335
This
study
[00108] Table 2: Host strains used in
these Examples:
Strain Proteolytic
Genotype Source
Abbreviation
DLF 0025 Control/None F-, A(araD-araB)567, Previous
Lab
lacZ4787(del)(:=B-3) , rph-1, Work
A(rhaD-rhaB)568, hsdR514, AackA-
pta, ApoxB, ApflB, AldhA, AadhE,
AsspB, Aic1R, AarcA, Acas3::tm-
ugpb-sspB-pro [casA*]
DLF 0025-X X DLF 0025, xy1A-DAS+4-ampR This
Study
DLF 0025-F F DLF 0025, fabI-DAS+4-gentR Previous
Lab
Work
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DLF 0025-G G DLF 0025, gltA-DAS+4-zeoR Previous
Lab
Work
DLF 0025-Z Z F 0025, zwf-DAS+4-bsdR Previous
Lab
Work
DLF 0025-U U DLF 0025, udhA-DAS+4-bsdR Previous
Lab
Work
DLF 0025-GU GU DLF 0025, gltA-DAS+4-
zeoR, udhA- Previous Lab
DAS+4-bsdR Work
DLF 0025-GZ GZ DLF 0025, gltA-DAS+4-zeoR, zwf- Previous
Lab
DAS+4-bsdR Work
DLF 0025-FG FG DLF 0025, fabI-DAS+4-gentR, gltA- Previous
Lab
DAS+4-zeoR Work
DLF 0025-FZ FZ DLF 0025, fabI-DAS+4-gentR, zwf- Previous
Lab
DAS+4-bsdR Work
DLF 0025-FU FU DLF 0025, fabI-DAS+4-gentR, udhA- Previous
Lab
DAS+4-bsdR Work
DLF 0025- FGU DLF 0025, fabI-DAS+4-gentR, gltA- Previous
Lab
FGU DAS+4-zeoR, udhA-DAS+4-bsdR Work
DLF 0025- FGZ DLF 0025, fabI-DAS+4-gentR, gltA- Previous
Lab
FGZ DAS+4-zeoR, zwf-DAS+4-bsdR Work
DLF 0025-FX FX DLF 0025, fabI-DAS+4-gentR, xy1A- This
Study
DAS+4-ampR
DLF 0025- FGX DLF 0025, fabI-DAS+4-gentR, gltA- This
Study
FGX DAS+4-zeoR, xy1A-DAS+4-ampR
DLF 0025- FZX DLF 0025, fabI-DAS+4-gentR, zwf- This
Study
FZX DAS+4-bsdR, xy1A-DAS+4-ampR
DLF 0025- FUX DLF 0025, fabI-DAS+4-gentR, udhA- This
Study
FUX DAS+4-bsdR, xy1A-DAS+4-ampR
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DLF 0025- FGUX DLF 0025, fabI-DAS+4-gentR, gltA- This Study
FGUX DAS+4-zeoR, udhA-DAS+4-bsdR,
xy1A-DAS+4-ampR
DLF 0025- FGZX DLF 0025, fabI-DAS+4-gentR, gltA- This Study
FGZX DAS+4-zeoR, zwf-DAS+4-bsdR,
xy1A-DAS+4-ampR
DLF 0025-UX UX DLF 0025, udhA-DAS+4-bsdR, xy1A- This Study
DAS+4-ampR
DLF 0025-ZX ZX DLF 0025, zwf-DAS+4-bsdR, xy1A- This Study
DAS+4-ampR
[00109] Table S2: Additional Strains used in the Examples:
Strain Genotype Source
DLF Z0025 F-, A(araD-araB)567, lacZ4787(del)(:=B-3) , rph-1, Previous Lab
Work
A(rhaD-rhaB)568, hsdR514, AackA-pta, ApoxB, ApflB,
AldhA, AadhE, Aic1R, AarcA, AsspB,
Acas3::tm-ugpb-sspB-pro-casA.
DLF Z0043 DLF Z0025, gltA-DAS+4-zeoR Previous Lab Work
DLF Z1002 DLF Z0025, zwf-DAS+4-bsdR Previous Lab Work
DLF Z0044 DLF Z0025, gltA-DAS+4-zeoR, zwf-DAS+4-bsdR Previous Lab Work
DLF Z0028 DLF Z0025, fabI-DAS+4-gentR This study
DLF Z0028 DLF Z0025, fabI-sfGFP-gentR This study
DLF Z0028 DLF Z0025, fabI-sfGFP-DAS+4-gentR This study
GD
DLF Z0763 DLF Z0025, udhA-DAS+4-bsdR This study
DLF Z0039 fDLF Z0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR This study
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DLF Z0040 DLF Z0025, fabI-DAS+4-gentR, zwf-DAS+4-bsdR This study
DLFZ 0045 DLF Z0025, fabI-DAS+4-gentR, udhA-DAS+4-bsdR This study
DLFZ 0046 DLF Z0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR, This study
zwf-DAS+4-bsdR
DLFZ 0047 DLF Z0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR, This study
udhA-DAS+4-bsdR
SL 0001 DLF Z0025, xylA-DAS+4-ampR This study
SL 0002 DLF Z0025, fabI-DAS+4-gentR, xylA -DAS+4-ampR This study
SL 0003 DLF Z0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR, This study
xylA -DAS+4-ampR
SL 0004 DLF Z0025, fabI-DAS+4-gentR, zwf-DAS+4-bsdR, This study
xylA -DAS+4-ampR
SL 0005 DLF Z0025, fabI-DAS+4-gentR, udhA-DAS+4-bsdR, This study
xylA -DAS+4-ampR
SL 0006 DLF Z0025, fabI-DAS+4-gentR, gltA-DAS+4- This study
zeoR,udhA-DAS+4-bsdR, xylA -DAS+4-ampR
SL 0007 DLF Z0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR, This study
zwf-DAS+4-bsdR, xylA -DAS+4-ampR
SL 0008 DLF Z0025, gltA-DAS+4-zeoR, udhA-DAS+4-bsdR This study
SL 0009 DLF Z0025, zwf-DAS+4,-bsdR xylA -DAS+4-ampR This study
SL 0010 DLF Z0025, fabI-DAS+4-gentR, zwf-DAS+4-bsdR, This study
Ay dbK
SL 0011 DLF Z0025, fabI-DAS+4-gentR, zwf-DAS+4-bsdR, This study
Afpr
On- origin of replication, Res - resistance marker, Sm - spectinomycin, Cm-
chloramphenicol,
Kan - kanamycin, Amp - ampicillin
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[00110] Chromosomal modifications were constructed using standard
recombineering
methodologies. A C-terminal DAS+4 tag on the xylA gene was added by direct
integration and
selected through integration of antibiotic resistance cassettes 3' of the
gene. All strains were
confirmed by PCR, agarose gel electrophoresis and confirmed by sequencing.
[00111] Table 3: Sequences of synthetic DNA:
xy1A-DAS4-ampR
GATACGATGGCACTGGC GCTGAAAATTGCAGC GC GCATGATTGAAGATGGC GAG
CTGGATAAACGCATC GC GCAGC GTTATTCC GGCTGGAATAGCGAATTGGGCCAG
CAAATCCTGAAAGGCCAAATGTCACTGGCAGATTTAGCCAAATATGCTCAGGAA
CATC ATTTGTC TC C GGTGCATC AGAGTGGTC GC CAGGAAC AAC TGGAAAATC TGG
TAAACCATTATCTGTTC GACAAAGCGGCCAACGATGAAAACTATTCTGAAAACTA
TGCGGATGCGTCTTAATGATAAGGACC GTGTTGACAATTAATCATC GGCATAGTA
TATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGAGTATTCAACA
TTTC C GTGTC GC C CTTATTC C CTTTTTTGC GGCATTTTGCCTTCCTGTTTTTGCTCA
CCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGT
GGGTTACATC GAACTGGATCTCAACAGC GGTAAGATC C TTGAGAGTTTAC GC C C C
GAAGAAC GTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGC GC GGTAT
TATC C C GTATTGAC GC C GGGCAAGAGCAAC TC GGTC GC C GCATACACTATTCTCA
GAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTCAC GGATGGCAT
GACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGC GGC
CAACTTACTTCTGGCAACGATCGGAGGACC GAAGGAGC TAAC C GCTTTTTTGC AC
AAC ATGGGGGATCATGTAACTC GC C TTGATC GTTGGGAAC C GGAGCTGAATGAA
GC C ATAC CAAAC GAC GAGC GTGACAC CAC GATGCCTGTAGCAATGGCAACAACG
TTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAA
TAGACTGGATGGAGGCGGATAAAGTTGCAGGATCACTTCTGC GCTCGGCCCTCCC
GGCTGGCTGGTTTATTGCTGATAAATCTGGAGCC GGTGAGC GTGGGTC TC GC GGT
ATCATTGCAGC AC TGGGGC CAGATGGTAAGC C CTC C C GCATC GTAGTTATCTAC A
CGACGGGGAGTCAGGCAACTATGGATGAAC GAAATAGACAGATC GC TGAGATAG
GTGCCTCACTGATTAAGCATTGGTAGTAAGTAGGGATAACAGGGTAATCGGCTA
AC TGTGC AGTC C GTTGGC C C GGTTATCGGTAGC GATACCGGGCATTTTTTTAAGG
AAC GATC GATATGTATATCGGGATAGATCTTGGCACCTCGGGCGTAAAAGTTATT
TTGCTCAAC GAGCAGGGTGAGGTGGTTGCTGCGCAAAC GGAAAAGCTGACCGTT
TC GC GC C C GCATC C ACTCTGGTC GGAACAAGACCCGGAACAGTGGTGGCAGGCA
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ACTGATCGCGCAA (SEQ ID NO: 1)
fabI-DAS+4-gentR
CTATTGAAGATGTGGGTAACTCTGC GGCATTCCTGTGCTCC GATCTCTCTGC CGGT
ATCTCC GGTGAAGTGGTC CAC GTTGACGGC GGTTTCAGCATTGCTGCAATGAACG
AACTC GAACTGAAAGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATG
CGTCTTAATAGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCC GAATC CATG
TGGGAGTTTATTCTTGACACAGATATTTATGATATAATAACTGAGTAAGCTTAAC
ATAAGGAGGAAAAACATATGTTAC GCAGCAGCAACGATGTTAC GCAGCAGGGCA
GTC GC CCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAG
GCTC GGC C C TGAC CAAGTCAAATC C ATGC GGGC TGC TCTTGATC TTTTC GGTC GT
GAGTTC GGAGAC GTAGC C AC CTACTCC CAACATCAGCCGGACTC CGATTAC CTCG
GGAACTTGCTC C GTAGTAAGACATTCATC GC GC TTGCTGC CTTCGACCAAGAAGC
GGTTGTTGGC GC TC TC GC GGCTTAC GTTCTGC C CAAGTTTGAGCAGC C GC GTAGT
GAGATCTATATCTATGATCTC GCAGTCTCC GGCGAGCAC CGGAGGCAGGGCATTG
C CAC C GC GC TC ATC AATC TC CTCAAGCATGAGGCCAAC GC GC TTGGTGC TTATGT
GATCTACGTGCAAGCAGATTACGGTGACGATCC C GC AGTGGC TCTC TATAC AAAG
TTGGGCATAC GGGAAGAAGTGATGCACTTTGATATCGACC CAAGTAC C GC CAC CT
AAGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC CGTTCTGTTGGTAAAGATG
GGC GGC GTTC TGC C GC C C GTTATC TCTGTTATAC CTTTC TGATATTTGTTATC GC C
GATCCGTCTTTCTCCCCTTCCCGCCTTGCGTCAGG(SEQ ID NO: 2)
gltA-DAS+4-zeoR
GTATTC C GTCTTC C ATGTTCAC C GTC ATTTTC GCAATGGC AC GTAC CGTTGGCTGG
ATC GC C C AC TGGAGC GAAATGCACAGTGACGGTATGAAGATTGCCC GTC C GC GT
CAGCTGTATACAGGATATGAAAAAC GC GACTTTAAAAGC GATATC AAGC GTGC G
GC C AAC GATGAAAACTATTCTGAAAAC TATGC GGATGC GTCTTAATAGTTGACAA
TTAATCATC GGCATAGTATATCGGCATAGTATAATACGACTCACTATAGGAGGGC
CATCATGGC CAAGTTGAC CAGTGCCGTTC CGGTGCTCAC C GC GC GC GAC GTC GC C
GGAGC GGTCGAGTTCTGGACC GAC CGGCTC GGGTTCTCC CGGGACTTCGTGGAG
GAC GAC TTC GC CGGTGTGGTCCGGGAC GAC GTGACCCTGTTCATCAGC GC GGTC C
AGGACCAGGTGGTGCCGGACAACACC C TGGC C TGGGTGTGGGTGC GC GGC CTGG
AC GAGC TGTAC GC C GAGTGGTC GGAGGTC GTGTC C AC GAACTTC C GGGAC GC CT
CCGGGCC GGCCATGAC CGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTC GC CC
TGC GC GAC CC GGC CGGCAACTGCGTGCACTTTGTGGCAGAGGAGCAGGACTGAG
GATAAGTAATGGTTGATTGCTAAGTTGTAAATATTTTAACCCGCCGTTCATATGG
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CGGGTTGATTTTTATATGCCTAAACACAAAAAATTGTAAAAATAAAATCCATTAA
CAGACCTATATAGATATTTAAAAAGAATAGAACAGCTCAAATTATCAGCAACCC
AATACTTTCAATTAAAAACTTCATGGTAGTCGCATTTATAACCCTATGAAA(SEQ
ID NO: 3)
udhA-DAS+4-bsdR
TC TGGGTATTC AC TGCTTTGGC GAGC GC GCTGC C GAAATTATTCATATCGGTCAG
GCGATTATGGAACAGAAAGGTGGCGGCAACACTATTGAGTACTTCGTCAACACC
AC CTTTAAC TAC C C GAC GATGGCGGAAGCCTATCGGGTAGCTGC GTTAAACGGTT
TAAAC C GC CTGTTTGC GGC C AAC GATGAAAACTATTCTGAAAACTATGCGGATGC
GTCTTAATAGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACG
ACTCACTATAGGAGGGCCATCATGAAGACCTTCAACATCTCTCAGCAGGATCTGG
AGCTGGTGGAGGTCGCCACTGAGAAGATCACCATGCTCTATGAGGACAACAAGC
AC C ATGTC GGGGC GGCCATCAGGACCAAGACTGGGGAGATCATCTCTGCTGTCC
ACATTGAGGCCTACATTGGCAGGGTCACTGTCTGTGCTGAAGCCATTGCCATTGG
GTCTGCTGTGAGCAACGGGCAGAAGGACTTTGACACCATTGTGGCTGTCAGGCAC
CCCTACTCTGATGAGGTGGACAGATCCATCAGGGTGGTCAGCCCCTGTGGCATGT
GCAGAGAGCTCATCTCTGACTATGCTCCTGACTGCTTTGTGCTCATTGAGATGAA
TGGC AAGCTGGTC AAAAC CAC CATTGAGGAACTCATC C C C CTC AAGTAC AC CAG
GAACTAAAGTAAAACTTTATCGAAATGGCCATCCATTCTTGCGCGGATGGCCTCT
GC C AGCTGCTC ATAGC GGC TGC GCAGC GGTGAGC CAGGAC GATAAAC CAGGC CA
ATAGTGCGGCGTGGTTCCGGCTTAATGCACGG(SEQ ID NO: 4)
zwf-DAS+4-bsdR
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GAAGTGGAAGAAGCCTGGAAATGGGTAGACTCCATTACTGAGGCGTGGGCGATG
GACAATGATGCGCCGAAACCGTATCAGGCCGGAACCTGGGGACCCGTTGCCTCG
GTGGCGATGATTACCCGTGATGGTCGTTCCTGGAATGAGTTTGAGGCGGCCAACG
ATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATAGTTGACAATTAATCA
TCGGCATAGTATATCGGCATAGTATAATACGACTCACTATAGGAGGGCCATCATG
AAGACCTTCAACATCTCTCAGCAGGATCTGGAGCTGGTGGAGGTCGCCACTGAG
AAGATCACCATGCTCTATGAGGACAACAAGCACCATGTCGGGGCGGCCATCAGG
ACCAAGACTGGGGAGATCATCTCTGCTGTCCACATTGAGGCCTACATTGGCAGGG
TCACTGTCTGTGCTGAAGCCATTGCCATTGGGTCTGCTGTGAGCAACGGGCAGAA
GGACTTTGACACCATTGTGGCTGTCAGGCACCCCTACTCTGATGAGGTGGACAGA
TCCATCAGGGTGGTCAGCCCCTGTGGCATGTGCAGAGAGCTCATCTCTGACTATG
CTCCTGACTGCTTTGTGCTCATTGAGATGAATGGCAAGCTGGTCAAAACCACCAT
TGAGGAACTCATCCCCCTCAAGTACACCAGGAACTAAAGTAATATCTGCGCTTAT
CCTTTATGGTTATTTTACCGGTAACATGATCTTGCGCAGATTGTAGAACAATTTTT
ACACTTTCAGGCCTCGTGCGGATTCACCCACGAGGCTTTTTTTATTACACTGACTG
AAACGTTTTTGCCCTATGAGCTCCGGTTACAGGCGTTTCAGTCATAAATCCTCTGA
ATGAAACGCGTTGTGAATC(SEQ ID NO: 5)
[00112] Table S3: Additional synthetic DNA:
xy1A-DAS4-ampR
GATACGATGGCACTGGCGCTGAAAATTGCAGCGCGCATGATTGAAGATGGCGAGC
TGGATAAACGCATCGCGCAGCGTTATTCCGGCTGGAATAGCGAATTGGGCCAGCA
AATCCTGAAAGGCCAAATGTCACTGGCAGATTTAGCCAAATATGCTCAGGAACAT
CATTTGTCTCCGGTGCATCAGAGTGGTCGCCAGGAACAACTGGAAAATCTGGTAA
ACCATTATCTGTTCGACAAAGCGGCCAACGATGAAAACTATTCTGAAAACTATGC
GGATGCGTCTTAATGATAAGGACCGTGTTGACAATTAATCATCGGCATAGTATATC
GGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGAGTATTCAACATTTCC
GTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGA
AACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTAC
ATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTACGCCCCGAAGAAC
GTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGT
ATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT
TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTCACGGATGGCATGACAGTAAG
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AGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTC
TGGCAAC GATC GGAGGACCGAAGGAGCTAACC GC TTTTTTGC ACAAC ATGGGGGA
TCATGTAACTC GC C TTGATC GTTGGGAAC C GGAGC TGAATGAAGC CATAC C AAAC
GACGAGC GTGAC AC CAC GATGC CTGTAGCAATGGC AACAAC GTTGC GCAAAC TAT
TAACTGGCGAACTACTTACTCTAGCTTCCC GGCAACAATTAATAGACTGGATGGAG
GC GGATAAAGTTGCAGGATCACTTCTGCGCTCGGCCCTCCC GGCTGGCTGGTTTAT
TGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTG
GGGCCAGATGGTAAGCCCTCCCGCATCGTAGTTATCTACAC GACGGGGAGTCAGG
CAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAA
GCATTGGTAGTAAGTAGGGATAACAGGGTAATCGGCTAACTGTGCAGTCCGTTGG
CCCGGTTATC GGTAGC GATACCGGGCATTTTTTTAAGGAAC GATC GATATGTATAT
C GGGATAGATCTTGGCACCTCGGGCGTAAAAGTTATTTTGCTCAAC GAGCAGGGT
GAGGTGGTTGCTGC GCAAACGGAAAAGCTGACC GTTTC GC GC C C GC ATC CACTC T
GGTC GGAACAAGAC C C GGAAC AGTGGTGGCAGGCAAC TGATC GC GCAA (SEQ ID
NO: 1)
fabI-DAS+4-gentR
CTATTGAAGATGTGGGTAACTCTGC GGCATTCCTGTGCTCCGATCTCTCTGCCGGT
ATCTCC GGTGAAGTGGTC CAC GTTGACGGC GGTTTCAGCATTGCTGCAATGAAC GA
AC TC GAACTGAAAGCGGCCAAC GATGAAAACTATTCTGAAAACTATGCGGATGCG
TCTTAATAGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCC GAATCCATGTGG
GAGTTTATTCTTGACACAGATATTTATGATATAATAACTGAGTAAGCTTAACATAA
GGAGGAAAAACATATGTTAC GC AGCAGCAAC GATGTTAC GCAGCAGGGCAGTC GC
CCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCG
GC C CTGAC CAAGTC AAATC CATGC GGGC TGC TCTTGATCTTTTC GGTCGTGAGTTC
GGAGAC GTAGC C AC CTACTC C CAAC ATC AGC C GGAC TC C GATTAC CTC GGGAACT
TGCTCC GTAGTAAGACATTCATC GC GCTTGCTGC CTTC GACCAAGAAGCGGTTGTT
GGC GC TCTC GC GGCTTAC GTTCTGC C CAAGTTTGAGC AGC C GC GTAGTGAGATC TA
TATCTATGATCTC GC AGTC TC C GGC GAGCAC C GGAGGCAGGGC ATTGC CAC C GC
GC TC ATC AATC TC CTCAAGCATGAGGC C AAC GC GC TTGGTGCTTATGTGATCTAC G
TGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCAT
AC GGGAAGAAGTGATGCACTTTGATATC GAC C CAAGTAC C GC CAC CTAAGAAGTT
CCTATTCTCTAGAAAGTATAGGAACTTCC GTTCTGTTGGTAAAGATGGGC GGCGTT
C TGC C GC C C GTTATCTCTGTTATAC CTTTCTGATATTTGTTATC GC C GATC C GTCTTT
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CTCCCCTTCCCGCCTTGCGTCAGG(SEQ ID NO: 2)
fabI-sfGFP-gentR
AAAGACTTC C GCAAAATGCTGGCTCATTGC GAAGC C GTTAC C C C GATTC GC C GTAC
CGTTACTATTGAAGATGTGGGTAACTCTGCGGCATTCCTGTGCTCCGATCTCTCTG
C C GGTATC TC C GGTGAAGTGGTC C AC GTTGAC GGC GGTTTCAGC ATTGCTGCAATG
AACGAACTCGAACTGAAAGGGGGTTCAGGCGGGTCGGGTGGCgtgagcaagggcgaggagc
tgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgcgcggcgaggg
cgagggc
gatgccaccaacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtga
ccaccctga
cctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcgccacgacttcttcaagtccgccatgcccgaagg
ctacgtcca
ggagcgcaccatcagcncaaggacgacggcacctacaagacccgcgccgaggtgaagttcgagggcgacaccctggtga
accgc
atcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaacttcaacagccaca
acgtctata
tcaccgccgacaagcagaagaacggcatcaaggccaacttcaagatccgccacaacgtggaggacggcagcgtgcagct
cgccga
ccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgtg
ctgagcaa
agaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactcacggcatggacgag
ctgtacaa
gTAATGACGAATCCATGTGGGAGTTTATTCTTGACACAGATATTTATGATATAATA
ACTGAGTAAGCTTAACATAAGGAGGAAAAACATATGTTGCGTAGCTCTAACGATG
TGAC GCAAC AAGGTTC GC GTC CAAAGAC AAAATTGGGAGGC AGTAGC ATGGGGAT
C ATTC GCACTTGTC GC C TGGGGC CAGAC CAGGTGAAGTC AATGC GTGC GGC TC TG
GACTTATTC GGGC GC GAATTTGGAGATGTAGC CAC TTACTCACAGCAC CAAC C GG
ACAGTGATTACTTGGGGAATTTACTTCGCAGTAAAACTTTTATCGCTTTGGCCGCT
TTC GAC C AGGAGGCTGTAGTAGGTGC GTTGGCAGC CTATGTTCTTC CTAAATTC GA
GC AAC C GC GTAGC GAAATTTACATC TATGATCTTGCAGTCTC C GGC GAACATC GC C
GTCAGGGGATC GC CACAGCTTTAATC AAC CTTTTGAAGCATGAGGCTAATGC ACTT
GGAGCGTACGTGATTTATGTGCAGGCTGACTACGGTGATGATCCTGCAGTCGCTCT
GTACAC CAAACTGGGTATC C GC GAGGAGGTCATGCACTTTGATATTGAC CC GTC G
AC GGC TAC CTAAGTTC TGTTGGTAAAGATGGGC GGC GTTCTGC C GC C C GTTATCTC
TGTTATACCTTTCTGATATTTGTTATCGCCGATCCGTCTTTCTCCCCTTCCCGCCTTG
CGTCAGG(SEQ ID NO: 21)
fabI-sfGFP-DAS+4- gentR
AAAGACTTC C GCAAAATGCTGGCTCATTGC GAAGC C GTTAC C C C GATTC GC C GTAC
CGTTACTATTGAAGATGTGGGTAACTCTGCGGCATTCCTGTGCTCCGATCTCTCTG
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CCGGTATCTCCGGTGAAGTGGTCCACGTTGACGGCGGTTTCAGCATTGCTGCAATG
AACGAACTCGAACTGAAAGGGGGTTCAGGCGGGTCGGGTGGCgtgagcaagggcgaggagc
tgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgcgcggcgaggg
cgagggc
gatgccaccaacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtga
ccaccctga
cctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcgccacgacttcttcaagtccgccatgcccgaagg
ctacgtcca
ggagcgcaccatcagcttcaaggacgacggcacctacaagacccgcgccgaggtgaagttcgagggcgacaccctggtg
aaccgc
atcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaacttcaacagccaca
acgtctata
tcaccgccgacaagcagaagaacggcatcaaggccaacttcaagatccgccacaacgtggaggacggcagcgtgcagct
cgccga
ccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgtg
ctgagcaa
agaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactcacggcatggacgag
ctgtacaa
gGGTGGGGGTGGGAGC GGC GGC GGTGGC TC C GC GGC CAAC GATGAAAAC TATTC T
GAAAACTATGCGGATGCGTCTTAATGACGAATCCATGTGGGAGTTTATTCTTGACA
CAGATATTTATGATATAATAACTGAGTAAGCTTAACATAAGGAGGAAAAACATAT
GTTGCGTAGCTCTAACGATGTGACGCAACAAGGTTCGCGTCCAAAGACAAAATTG
GGAGGCAGTAGCATGGGGATCATTCGCACTTGTCGCCTGGGGCCAGACCAGGTGA
AGTCAATGCGTGCGGCTCTGGACTTATTCGGGCGCGAATTTGGAGATGTAGCCACT
TACTCACAGCACCAACCGGACAGTGATTACTTGGGGAATTTACTTCGCAGTAAAA
CTTTTATCGCTTTGGCCGCTTTCGACCAGGAGGCTGTAGTAGGTGCGTTGGCAGCC
TATGTTCTTCCTAAATTCGAGCAACCGCGTAGCGAAATTTACATCTATGATCTTGC
AGTCTC C GGC GAACATC GC C GTCAGGGGATC GC CACAGC TTTAATCAAC CTTTTGA
AGCATGAGGCTAATGCACTTGGAGCGTACGTGATTTATGTGCAGGCTGACTACGG
TGATGATCCTGCAGTCGCTCTGTACACCAAACTGGGTATCCGCGAGGAGGTCATGC
AC TTTGATATTGAC CC GTC GAC GGCTAC CTAAGTTC TGTTGGTAAAGATGGGC GGC
GTTCTGCCGCCCGTTATCTCTGTTATACCTTTCTGATATTTGTTATCGCCGATCCGT
CTTTCTCCCCTTCCCGCCTTGCGTCAGG(SEQ ID NO: 22)
udhA-DAS+4-bsdR
TCTGGGTATTCACTGCTTTGGCGAGCGCGCTGCCGAAATTATTCATATCGGTCAGG
CGATTATGGAACAGAAAGGTGGCGGCAACACTATTGAGTACTTCGTCAACACCAC
CTTTAACTACCCGACGATGGCGGAAGCCTATCGGGTAGCTGCGTTAAACGGTTTAA
AC C GC C TGTTTGC GGC CAAC GATGAAAACTATTC TGAAAACTATGC GGATGC GTC T
TAATAGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACTCA
CTATAGGAGGGCCATCATGAAGACCTTCAACATCTCTCAGCAGGATCTGGAGCTG
GTGGAGGTCGCCACTGAGAAGATCACCATGCTCTATGAGGACAACAAGCACCATG
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TCGGGGCGGCCATCAGGACCAAGACTGGGGAGATCATCTCTGCTGTCCACATTGA
GGCCTACATTGGCAGGGTCACTGTCTGTGCTGAAGCCATTGCCATTGGGTCTGCTG
TGAGCAACGGGCAGAAGGACTTTGACACCATTGTGGCTGTCAGGCACCCCTACTC
TGATGAGGTGGACAGATCCATCAGGGTGGTCAGCCCCTGTGGCATGTGCAGAGAG
CTCATCTCTGACTATGCTCCTGACTGCTTTGTGCTCATTGAGATGAATGGCAAGCT
GGTCAAAACCACCATTGAGGAACTCATCCCCCTCAAGTACACCAGGAACTAAAGT
AAAACTTTATCGAAATGGCCATCCATTCTTGCGCGGATGGCCTCTGCCAGCTGCTC
ATAGCGGCTGCGCAGCGGTGAGCCAGGACGATAAACCAGGCCAATAGTGCGGCG
TGGTTCCGGCTTAATGCACGG(SEQ ID NO: 4)
[00113] The xyrA gene from Aspergillus niger was codon optimized for E.
coil and the
plasmid, pHCKan-INS: yibDp-6xhis-xyrA, enabling the low phosphate induction of
xylose
reductase, was constructed by TWIST Biosciences. pCASCADE guide RNA array
plasmids
were prepared by the combination of PCR and Gibson assembly as previously
described.
[00114] Primers used for assembly are given in Table 4:
Plasmids/p sequences
rimers
name
gltAl
TCGAGTTCCCCGCGCCAGCGGGGATAAACCGAAAAGCATATAATG
CGTAAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGATAAA
CCG (SEQ ID NO: 6)
gltAl -FOR CCGGATGAGCATTCATCAGGCGGGCAAG (SEQ ID NO: 7)
gltAl -REV C GGTTTATC CCC GCTGGC GC GGGGAACTC GAACTTCATAACTTTTA
C (SEQ ID NO: 8)
gltA2
TATTGACCAATTCATTCGGGACAGTTATTAGTTCGAGTTCCCCGCG
CCAGCGGGGATAAACCG (SEQ ID NO: 9)
gltA2-FOR CCGGATGAGCATTCATCAGGCGGGCAAG (SEQ ID NO: 10)
gltA2-REV CGGTTTATCCCCGCTGGCGCGGGGAACTCGAACTAATAACTGTC
(SEQ ID NO: 11)
udhA
TTACCATTCTGTTGCTTTTATGTATAAGAATCGAGTTCCCCGCGCCA
GCGGGGATAAACCG (SEQ ID NO: 12)
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udhA-FOR CCGGATGAGCATTCATCAGGCGGGCAAG (SEQ ID NO: 13)
udhA-REV CGGTTTATCCCCGCTGGCGCGGGGAACTCGATTCTTATACATAAAA
GC (SEQ ID NO: 14)
zwf CTCGTAAAAGCAGTACAGTGCACCGTAAGATCGAGTTCCCCGCGC
CAGCGGGGATAAACCG (SEQ ID NO: 15)
zwf-FOR CCGGATGAGCATTCATCAGGCGGGCAAG (SEQ ID NO: 16)
zwf-REV CGGTTTATCCCCGCTGGCGCGGGGAACTCGATCTTACGGTGCACTG
TAC (SEQ ID NO: 17)
xylA GGAGTGCCCAATATTACGACATCATCCATCTCGAGTTCCCCGCGCC
AGCGGGGATAAACCG (SEQ ID NO: 18)
xy1A-FOR CCAGCGGGGATAAACCGGGAGTGCCCAATATTAC (SEQ ID NO: 19)
xy1A-REV CTTGCCCGCCTGATGAATGCTCATCCGG (SEQ ID NO: 20)
[00115] Table S4: Additional oligonucleotides:
Plasmids/pr Sequences
imers name
fabI int F GCAAAATGCTGGCTCATTG(SEQ ID NO: 23)
gentR intR GCGATGAATGTCTTACTACGGA(SEQ ID NO: 24)
gltA int F TATCATCCTGAAAGCGATGG(SEQ ID NO: 25)
zeo intR ACTGAAGCCCAGACGATC(SEQ ID NO: 26)
zwf intF CTGCTGGAAACCATGCG(SEQ ID NO: 27)
udhA intF CAAAAGAGATTCTGGGTATTCACT(SEQ ID NO: 28)
bsdR intR GAGCATGGTGATCTTCTCAGT(SEQ ID NO: 29)
xylA intF AGATGGCGAGCTGGATA(SEQ ID NO: 30)
ampR intR AGTACTCAACCAAGTCATTCTG(SEQ ID NO: 31)
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Plasmids/pr Sequences
imers name
g1tA2 TATTGACCAATTCATTCGGGACAGTTATTAGTTCGAGTTCCCCGCGC
CAGCGGGGATAAACCG(SEQ ID NO: 6)
g1tA2-FOR CCGGATGAGCATTCATCAGGCGGGCAAG(SEQ ID NO: 7)
gltA2-REV CGGTTTATCCCCGCTGGCGCGGGGAACTCGAACTAATAACTGTC(SE
Q ID NO: 8)
udhA TTACCATTCTGTTGCTTTTATGTATAAGAATCGAGTTCCCCGCGCCA
GCGGGGATAAACCG(SEQ ID NO: 12)
udhA-FOR CCGGATGAGCATTCATCAGGCGGGCAAG(SEQ ID NO: 13)
udhA-REV CGGTTTATCCCCGCTGGCGCGGGGAACTCGATTCTTATACATAAAA
GC(SEQ ID NO: 14)
xylA GGAGTGCCCAATATTACGACATCATCCATCTCGAGTTCCCCGCGCCA
GCGGGGATAAACCG(SEQ ID NO: 18)
xy1A-FOR CCAGCGGGGATAAACCGGGAGTGCCCAATATTAC(SEQ ID NO: 19)
xy1A-REV CTTGCCCGCCTGATGAATGCTCATCCGG(SEQ ID NO: 20)
[00116] Micro and 1L Fermentations
[00117] Micro-fermentations and 1L fermentations in instrumented
bioreactors were
performed as previously reported, except that xylose was substituted for
glucose (1gram xylose
for lgram glucose) in all media formulations. Guide array stability was
confirmed after
transformation of pCASCADE vector by PCR prior to evaluation in 96 well plate
micro-
fermentations.
[00118] Xylose and Xylitol Quantification
In micro-fermentations, xylose and xylitol were quantified by commercial
bioassays from
Megazyme (Wicklow, Ireland, Catalog #K-XYLOSE and K-SORB), according to the
manufacturer's instructions. All the results were tested by measuring the
absorbance at 492nm. For
the quantification of tank fermentations, an HPLC method coupled with a
refractive index detector
was used to measure both xylose as well as xylitol. At 55 C, a Rezex ROA-
Organic Acid H+
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(8%) Analysis HPLC Column (CAT#: #00H-0138-KO, Phenomenex, Inc., Torrance, CA,
300 x
7.8 mme;) was employed for the compound's separation. According to reference,
we chose sulfuric
acid as the isocratic eluent solvents, and the flow rate was set at 0.5mL/min.
Waters Acquity H-
Class UPLC integrated with a Waters 2414 Refractive Index (RI) detector
(Waters Corp., Milford,
MA. USA) was used for the chromatographic detection. Injection volume of
sample and standard
was set as 10 uL. Samples were diluted in 20 times using filtered ultrapure
water to make all the
sample points appear within the standards linear range. The standard variation
range was between
0.01 to 20 g/L. MassLynx v4.1 software was used for all the peak integration
and concentration
analysis.
[00119] Fermentations.
[00120] Minimal media microfermentations were performed as previously
reported
(Moreb, E. A. et al. Media Robustness and scalability of phosphate regulated
promoters useful
for two-stage autoinduction in E. coli. ACS Synthetic Biology (2020)
doi:10.1021/acssynbio.0c00182 and Menacho-Melgar, R. etal. Scalable, two-
stage,
autoinduction of recombinant protein expression in E. coli utilizing phosphate
depletion.
Biotechnol. Bioeng. 26, 44 (2020)) except that xylose was substituted for
glucose (1 gram xylose
for 1 gram glucose) in all media formulations. Guide array stability was
confirmed after
transformation of pCASCADE plasmids by PCR prior to evaluation according to Li
et al.' Fed
batch fermentations were performed as previously reported, again with xylose
instead of glucose
(Menacho-Melgar, R. et al. Scalable, two-stage, autoinduction of recombinant
protein expression
in E. coli utilizing phosphate depletion. Biotechnol. Bioeng. 26, 44 (2020).
Xylose feeding was
as modified as follows. The starting batch glucose concentration was 25 g/L.
Concentrated sterile
filtered xylose feed (500 g/L) was added to the tanks at an initial rate of 10
g/h when cells
entered mid-exponential growth. This rate was then increased exponentially,
doubling every
1.083 hours (65 min) until 40 g total glucose had been added, after which the
feed was
maintained at 1.75g/hr. The feed was reduced to 0.875 g/hr due to xylose
accumulation at 85 hrs
post inoculation, and stopped at 120hrs post inoculation.
[00121] (XyrA) Xylose reductase Purification and Activity assays
[00122] E. coli BL21(DE3) (New England Biolabs, Ipswich, MA) with plasmid
pHCKan-
xyrA (bearing a 6x his tag) was cultured overnight in Luria Broth (Lenox
formulation). The
overnight culture was used to inoculate SM10++ media (with xylose as a carbon
source instead
of glucose) with appropriate antibiotics. Cells were cultured at 37 C for 16
hours, then cells were
centrifuged, and the pellet was washed with SM10 No phosphate media. Next, the
washed pellet
was resuspended and cultured in SM10 No Phosphate media again with the
appropriate
antibiotics. After the expression, the postproduction cells were lysed by a
freeze-thaw cycle.
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XyrA protein was purified using Ni-NTA Resin (G-Biosciences, Cat # 786-939)
according to
manufacturer's instructions. Kinetics assays for XyrA were performed in a
reaction buffer
composed of 50 mM sodium phosphate (pH 7.6, 5mM MgCl2) with NADPH as cofactor
(Suzuki, T. et al. Expression of xyrA gene encoding for D-Xylose reductase of
Candida
tropicalis and production of xylitol in Escherichia coli. I Biosci. Bioeng.
87, 280-284 (1999)).
In these assays, NADPH was held at a constant initial level of 50 M. Results
of the assay were
measured through monitoring the absorbance of NADPH at 340nm for 1.5 hours
(15s per read)
using a SpectraMax Plus 384 microplate reader (Molecular Devices). Reaction
velocity is plotted
as a function of xylose concentration. Using the Eadie-hofstee equation, we
got the parameters:
Vmax=22.6 1.01 U, kcat=13.56 3.05 s-1 and Km: 35.12 3.05 mM.
[00123] (XylA) Xylose isomerase quantification
[00124] Xylose isomerase activities from cell extracts were quantified with
a D-xylose
reductase coupled enzyme assay, similar to methods previously described, and
following a
decrease in absorbance of NADPH at 340nm (Guaman, L. P. etal. xylA and xylB
overexpression as a successful strategy for improving xylose utilization and
poly-3-
hydroxybutyrate production in Burkholderia sacchari. I Ind. Microbiol.
Biotechnol. 45, 165-173
(2018) and Lee, S.-M., Jellison, T. & Alper, H. S. Directed evolution of
xylose isomerase for
improved xylose catabolism and fermentation in the yeast Saccharomyces
cerevisiae. App!.
Environ. Microbiol. 78, 5708-5716 (2012)). Cultures were grown in shake flasks
in SM10++
media and harvested in mid exponential phase, washed and resuspended in SM 10
No phosphate
media. After 16 hours of phosphate depletion, cells were pelleted by 10
minutes of
centrifugation (4122 RCF, 4 degrees C) and lysed with BugBuster protein
extraction reagent
(Millipore Sigma, Catalog #70584) according to the manufacturer's protocol.
Cell debris was
removed by two rounds of centrifugation, 20 minutes (4122 RCF, 4 degrees C)
followed by a 20
minute hard spin (14000 RCF, 4 degrees C). The lysate was filtered with Amicon
30MWCO
filters (Millipore Sigma, Catalog #UFC8030) according to the manufacturer's
protocol and
washed three times to exchange the buffer with the reaction buffer (45mM
sodium phosphate,
10mM MgCl2, pH 7.6) and remove metabolites. Samples were assayed in triplicate
in a 96 well
plate with 100uL of the filtered cell extract per well containing 31.25mM
xyulose, 0.5mM
NADPH, and lug/mL of purified D-xylose reductase (see above). The absorbance
at 340nm was
measured every 15seconds for 1.5hours and the slope of the linear region was
used to quantify
XylA activity. Total protein concentration of each sample was determined with
a standard
Bradford assay. Kinetic parameters were as follows: kcat: 13.56 3.05 s-1,
Km: 35.12 3.05
mM.
[00125] (UdhA) Soluble transhydrogenase quantification
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[00126] The activity of the soluble transhydrogenase was quantified by
method previously
reported (Chou, H.-H., Marx, C. J. & Sauer, U. Transhydrogenase promotes the
robustness and
evolvability of E. coli deficient in NADPH production. PLoS Genet. 11,
e1005007 (2015) and
Sauer, U., Canonaco, F., Hen, S., Perrenoud, A. & Fischer, E. The soluble and
membrane-bound
transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism
of
Escherichia coli. I Biol. Chem. 279, 6613-6619 (2004)). The process of UdhA
expression and
cell lysis was carried out using the same method as the XyrA expression
mentioned above. The
lysates were centrifuged for 15 minutes (4200 RPM, 4 C) to remove large
debris. A second hard
spin was performed for 30 minutes (14000 RPM, 4 C) to remove remaining debris
and further
separate the membrane fraction from the soluble transhydrogenase. Lysates were
diluted 1:5 with
the assay reaction buffer (50mM Tris-HC1, 2mM MgCl, pH 7.6) and transferred to
an Amicon
Ultra centrifugal filter (10kDa MWCO). The samples were centrifuged for 30
minutes (4200
RPM, 4 C) and this step was repeated 3 times to remove metabolites and
exchange the lysis
buffer for the assay buffer. After filtration the protein concentrations of
the samples were
quantified with a standard Bradford assay.
[00127] Then soluble transhydrogenase activity was assayed at room
temperature. Assays
were performed in black 96 well plates by mixing equal volumes of lysate and
reaction buffer for
a final volume of 100uL per well and a final concentration of 0.5mM NADPH and
1mM 3-
acetylpyridine adenine dinucleotide (APAD+). Changes in absorbance at 400nm
and 310nm due
to the reduction of APAD+ and the oxidation of NADPH, respectively, were
monitored
simultaneously by Spectramax Plus 384 microplate reader at 30 second intervals
for 30 minutes.
A standard curve was used to calculate the molar absorptivity of NADPH
(3.04*103 A4-1 cm-1).
The molar absorptivity was used to convert the measured slope of the linear
region to the change
in concentration per minute. The specific activity (Units per mg of total
protein) was determined
by dividing the change in concentration per minute by the protein
concentration.
[00128] FabI Quantification
[00129] Quantification of FabI via a C-terminal GFP tags was performed
using a GFP
quantification kit from AbCam (Cambridge, UK, Cat # ab171581) according to
manufacturer's
instructions.
[00130] Xylose and Xylitol Quantification
[00131] In micro-fermentations, xylose and xylitol were quantified by
commercial
bioassays from Megazyme (Wicklow, Ireland, Cat # K-XYLOSE and K-SORB),
according to
the manufacturer's instructions. An HPLC method coupled with a refractive
index detector was
used to quantify both xylose as well as xylitol from instrumented
fermentations. Briefly, a Rezex
ROA-Organic Acid H+ (8%) Analysis HPLC Column (Cat #: #00H-0138-KO,
Phenomenex, Inc.,
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Torrance, CA, 300 x 7.8 mme;) was employed for the separation of xylose and
xylitol. 5 mM
Sulfuric acid as the isocratic mobile phase at a flow rate of 0.5mL/min, at 55
C,. A Waters
Acquity H-Class UPLC integrated with a Waters 2414 Refractive Index (RI)
detector (Waters
Corp., Milford, MA. USA) was used for detection. The injection volume of
samples and
standards was 10 pL. Samples were diluted 20 fold in water in order to be in
the linear range
(0.01 to 20 g/L). MassLynx v4.1 software was used for all the peak integration
and analyses.
[00132] NADPH Pool Quantification
[00133] NADPH pools were measured t using an NADPH Assay Kit (AbCam,
Cambridge, UK, Cat # ab186031) according to manufacturer's instructions.
Cultures and
phosphate depletion were performed as described above for XyrA expression
(except there was
no xyrA plasmid in the cell). Cells were lysed using the lysis buffer in the
assay kit.
[00134] Metabolic Modeling
[00135] In silica analyses were performed implementing Constraint-based
(COBRA)
models for E. coli, developed employing the COBRApy Python package with a
previously
reported reconstruction as a starting point. This curated E. coli K-12 MG1655
reconstruction
includes 2,719 metabolic reactions and 1,192 unique metabolites. This model
was adapted as
follows. First, missing reactions and metabolites for xylitol production and
export were added as
shown in Table S5:
[00136] Table S5. Xylitol Specific reactions added to the metabolic model
Name Reaction Identifier
Xylose reductase h c + nadph c + xyl D c # nadp c + xylt c XYLR
Xylitol exchange xylt e # EX xylt e
Xylitol transport xylt e # xylt c XYLTt
via passive
diffusion
Flavodoxin 2.0 flxso c + nadph c # 2.0 flxr c + h c + nadp c FLDR2
reductase
(NADPH)
[00137] All reactions, metabolites stoichiometry and identificators were
extracted from
the BiGG Models database. The resulting model was validated for mass balances
and metabolite
compartment formulas with COBRApy validation methods. Once properly balanced,
a growth
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model was created and analyzed. Specific evaluated conditions and biomass
fluxes are shown in
Table S6.
[00138] Table S6. Metabolic model validation with different carbon sources
and oxygen
levels. (All flux values are expressed in nmol/gDW*hr).
Only C.S Flux Oxygen Flux Biomass Flux Previously reported
Carbon obtained from Biomass Flux*
Source model
Glucose -8.8 -30 (Aerobic) 0.771914 0.70 0.01
Glucose -8.8 0 (Anaerobic) 0.253285 0.33 0.02
Xylose -9.5 -30 (Aerobic) 0.645282 0.50 0.02
Xylose -9.5 0 (Anaerobic) 0.115445 0.13 0.02
[00139] *Numbers from (Prasad, S., Singh, A. & Joshi, H. C. Ethanol as an
alternative
fuel from agricultural, industrial and urban residues. Resour. Conserv.
Recycl. 50, 1-39 (2007)).
[00140] Next, experimental data obtained from the xylitol micro-
fermentations was used
to constrain the model. Specific constraints included: i) setting the ratio
for pyruvate
consumption through Pyruvate Dehydrogenase (PDH) and Pyruvate-flavodoxin
Oxidoreductase
(ybdk), with 10% and 90% of total flux respectively and ii) setting
Ferredoxin/flavodoxin
reductase to a reversible reaction and iii) using xylose as a sole carbon
source with an input flux
of 10 mmol/gCDW*hr under minimal media conditions. Finally a set of specific
xylitol
production strains were constructed and evaluated in silico using Flux Balance
Analysis (FBA)
to obtain xylitol yields, analyze cofactor and/or metabolites of interest as
well as production and
consumption fluxes. Specific cases that were analyzed included reduction or
increased activity
of: Zwf, FabI, GltA, XylA, PntAB and UdhA as shown in Table S7. For each
case/condition the
following data was obtained: Xylitol yield, NADPH producing and consuming g
reaction fluxes
and escher maps of central metabolism for flux distribution visualization.
Finally, major changes
in fluxes between the most relevant strains were analyzed.
[00141] Table S7. Strains modeled
Individual Valves Combinations of Valves
Strain Valves Strain Valves Silencing
Silencing
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WT Z-F zwf & fabI
zwf Z-G zwf & gltA
fabI Z-X zwf & xylA
gltA Z-pAB zwf & pntAB
X xylA Z-F-G zwf, fabI & gltA
pAB pntAB Z-G-U zwf, gltA & udhA
udhA Z-F-pAB zwf, fabI & pntAB
[00142] Example 1: Characterization of XyrA Xylose reductase
[00143] Referring now to FIG 2(A), Expression of XyrA in BL21 using media
combination
of SM10++(for growth) and SM10-No phos(for expression). After the expression,
the
postproduction cells were lysed by freeze-thawing cycle. Next, the xyrA
protein was extracted by
N-N Resin because of the His-tag on XyrA which was design into plasmid
sequence. In FIG2(B),
Activity of xyrA with NADPH as co-factor. Reaction velocity is plotted as
function of xylose
concentration. In these assays, NADPH was held at a constant initial level of
50 uM. FIG 2(C)
Kinetic Parameters for XyrA from this project and from other research sources
as comparison.
Kinetics for XyrA were measured using 50 mM sodium phosphate, pH 7.6
(containing 5mM
MgC12).26 50 uM NADPH. Results of the assay were measured through monitoring
the absorbance
of NADPH at 340nm. Using Eadie-Hofstee equation, the parameters Vmax=22.6U,
kcat=13.5s-1
and km=35.12mM were established thus confirming protein enzyme activity that
could be used in
the tank fermentation process. Knowing the Vmax, the minimal expression level
needed to hit a
desired specific production rate can then be established.
Example 2: Design of metabolic valves for bioproduction of xylitol
[00144] Rationally designed strains to optimize xylitol production from
xylose utilizing
two stage dynamic metabolic control, in a phosphate depleted stationary phase
were developed.
As illustrated in FIG 1, this design included overexpression of xylose
reductase and the dynamic
reduction in xylose isomerase (xylA) activity to reduce xylose metabolism
which competes with
xylitol production. Toward this goal we constructed strains and plasmids to
enable the dynamic
induction of xyrA, and dynamic reduction in XylA activity upon phosphate
depletion, either
through gene silencing, proteolysis of XylA or the combination. Refer to
Tables 1 and 2 for
plasmids and strains used. These strains were evaluated in 96 well plate micro-
fermentations as
reported by Moreb et al and results are given in FIG 3.
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Example 3: Xylitol production utilizing 2-stage dynamic control
[00145] Since dynamic control over XylA ("X") activity only led to modest
improvements
in xylitol production, FIG 3, we evaluated the potential impact of a larger
set of valves on xylitol
production. We constructed a set of strains with valves in key metabolic
pathways, FIG 4. These
valves included: citrate synthase (G1tA-"G"), glucose-6-phosphate
dehydrogenase (Zwf-"Z"),
enoyl-ACP reductase (FabI-"F") and soluble transhydrogenase (udhA-"U") which
control flux
through the tricarboxylic acid cycle, pentose phosphate pathway, fatty acid
biosynthesis and
NADPH supply respectively. Strains were constructed with combinations of X, U,
G, Z and F
valves and evaluated for xylitol production. As described above, dynamic
metabolic control was
accomplished by adding C-terminal DAS+4 degron tags to the xylA, udhA, zwf,
gltA and fabI
genes as well as the overexpression of guide RNAs enabling silencing of their
transcription.
Refer to the Methods section for detailed chromosomal modification and plasmid
construction.
[00146] The panel consisted of ¨370 valve combinations of X, U, G, Z and F
that were
evaluated for xylitol production in two stage 96 well plate micro-
fermentations in at least
triplicate. Results of these experiments are given in FIG 5Aand 5B. Xylitol
titers ranged from ¨
Og/L-OD(600nm) to ¨9.35g/L-OD(600nm). Approximately ¨80% of the silencing and
proteolysis combinations performed better than the control strain, which only
produced 0.106
g/L-OD. Significant differences in specific xylitol production (xylitol (g/L)
per unit OD600nm)
between valve strains and the control strain were determined by one-way ANOVA
(F(414,851)=7.598, p<0.0001).
[00147] P-values were used to generate a p-value heatmap (FIG 6), where
only
combinations with a p value less than 0.05 are highlighted .Combinations not
assayed or with
less than 2 successful replicates (lack of success is due to lack of cell
growth) are indicated by a
gray dot since they are not qualified for statistical analysis. While the
incorporation of X valves
generally led to increase xylitol production, to surprisingly the two highest
xylitol producers had
neither X or U valves (which should increase NADPH levels) but rather
combinations of F, G
and Z valves. The highest producer had a combination of F and Z valves, which
the xylitol
specific productivity could reach 9.35g/L-0D600nm. The performance of this
genetic
combination was also synergistic above either F or Z valves alone. This was
surprising since
these two enzymes have no direct or predictable impact of xylitol biosynthesis
as can be seen in
FIG 4.
[00148] Example 4: Xylitol Production in Instrumented Bioreactors
[00149] Based on the results from the micro-fermentations (FIGs 5 and 6),
we chose the "Z-
FZ" valve strain (silencing of zwf "Z" and proteolysis of fabI and zwf "FZ")
which has titer of
9.35 g/L-0D600 as well as the control for evaluation in instrumented
bioreactors. Fermentations
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were performed according to Menacho-Melgar et al, where phosphate is limiting
in the media
leading to phosphate depletion and xylitol production in stationary phase as
illustrated above in
FIG 5. Results of these fermentations are given in FIG 6 below. The Z-FZ
strain (FIG 6A) enabled
xylitol production up to 104+/-11.31 g/L with 160 hours, while xyrA expression
in our control
strain DLF 0025-EV (FIG 6B) led to only ¨3g/L of xylitol within the same time.
[00150] Example 5: Improvement of NADPH flux and xylitol biosynthesis
[00151] Most previous studies producing xylitol from xylose rely on a
bioconversion
requiring an additional sugar (usually glucose) as an electron donor
(Albuquerque, T. L. de, da
Silva, I. J., de Macedo, G. R. & Rocha, M. V. P. Biotechnological production
of xylitol from
lignocellulosic wastes: A review. Process Biochem. 49, 1779-1789 (2014).;
Cirino, P. C., Chin, J.
W. & Ingram, L. 0. Engineering Escherichia coli for xylitol production from
glucose-xylose
mixtures. Biotechnol. Bioeng. 95, 1167-1176 (2006); and Su, B., Wu, M., Zhang,
Z., Lin, J. &
Yang, L. Efficient production of xylitol from hemicellulosic hydrolysate using
engineered
Escherichia coli. Metab. Eng. 31, 112-122 (2015)). Oxidation of glucose
(producing the
byproduct gluconic acid) generates NADPH which is then used for xylose
reduction (Jin, L.-Q.,
Xu, W., Yang, B., Liu, Z.-Q. & Zheng, Y.-G. Efficient Biosynthesis of Xylitol
from Xylose by
Coexpression of Xylose Reductase and Glucose Dehydrogenase in Escherichia
coli. App!.
Biochem. Biotechnol. 187, 1143-1157 (2019). While these processes offer high
xylitol titers and
a good yield when considering xylose, the requirement for glucose at equimolar
levels to xylose
is a significant inefficiency. More broadly, improving NADPH availability or
flux, useful in the
synthesis of numerous metabolites as well as cell based bioconversions, has
been a long standing
challenge in metabolic engineering.
[00152] We applied two-stage dynamic metabolic control (DMC) to improve
NADPH
flux and xylitol production using xylose as a sole feedstock (Burg, J. M.,
Cooper, C. B., Ye, Z.,
Reed, B. R. & Moreb, E. A. Large-scale bioprocess competitiveness: the
potential of dynamic
metabolic control in two-stage fermentations. Current opinion in (2016)).
Dynamic control over
metabolism has become a popular approach in metabolic engineering, and has
been used for the
production of various products from 3-hydroxypropionic acid to myo-inositol
and many others.
We have recently reported an extension of dynamic metabolic control to two-
stage bioprocesses,
where products are made in a metabolically productive phosphate depleted
stationary phase. The
implementation of this approach relies on combined use of controlled
proteolysis and gene
silencing, using degron tags and CRISPR interference respectively.
Importantly, in these initial
studies we demonstrated that improved metabolic fluxes resulting from dynamic
metabolic
control, can be a consequence of reducing levels of central metabolites which
are feedback
regulators of other key metabolic pathways. Specifically, we have recently
shown that
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decreasing glucose-6-phosphate dehydrogenase levels activates the SoxRS
regulon increasing
expression and activity of pyruvate ferredoxin/flavodoxin oxidoreductase
(Pfo). Pfo leads to
improved acetyl-CoA production in stationary phase. (Refer to FIG 11(A)). In
this work we
report the evaluation of combinations of synthetic metabolic valves on xylitol
production from
xylose. Firstly, increased Pfo activity not only leads to improved acetyl-CoA
flux but also
NADPH production. NADPH is produced from reduced flavodoxin/ferredoxin via the
action of
NADPH dependent flavodoxin/ferredoxin reductase (Fpr). We also identify a key
regulatory
mechanism controlling NADPH fluxes, namely the inhibition of the membrane
bound
transhydrogenase (PntAB) by fatty acid metabolites. By dynamically disrupting
fatty acid
biosynthesis, we alleviate inhibition of PntAB. This mechanism is synergistic
with activating Pfo
and greatly increases NADPH flux and xylitol production. We compare this
"regulatory"
approach with a more intuitive stoichiometric strategy where the levels of key
enzymes
competing with xylitol production are dynamically reduced. Importantly,
improved NADPH
fluxes are, in part, a consequence of reduced NADPH pools. Reduced NADPH pools
drive
changes in expression and activity that result in increased NADPH fluxes,
presumably a
regulatory mechanism which has evolved to restore set point NADPH levels.
These results are a
reminder that pools and flux are not equivalent and not necessarily
correlated.
[00153] Stoichiometric Strategy
[00154] We initially rationally designed strains to optimize xylitol
production from xylose
utilizing two stage dynamic metabolic control, reliant on decreasing levels of
key competitive
pathways. As illustrated in FIG 1, this design included dynamic reduction in
xylose isomerase
(xylA) and soluble transhydrogenase (udhA) activities. These modifications
were designed to
reduce xylose metabolism which competes with xylitol production and increases
NADPH
supply. NADPH can be consumed by the soluble transhydrogenase. Toward this
goal we
constructed strains and plasmids to enable the dynamic reduction in XylA and
UdhA levels upon
phosphate depletion. Refer to Supplemental Table 51 for strains and plasmids
used in this study.
As described above and previously reported dynamic reduction in activity was
accomplished by
adding C-terminal DAS+4 degron tags to the chromosomal xylA and udhA genes as
well as the
overexpression of guide RNAs aimed at silencing their expression. The impact
of these
modifications on enzyme levels in two stage cultures is given in FIG 9A-B. In
the case of XylA,
proteolysis led to ¨ 60% reduction in activity. To our surprise the silencing
gRNA actually led to
an increased XylA activity level. The mechanism behind this is currently
unknown and requires
further study. The combination of silencing and proteolysis resulted in no
further reduction in
activity when compared to proteolysis alone. In the case of UdhA, proteolysis
resulted in a ¨30%
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reduction in activity, whereas silencing had no detectable impact on activity
levels with or
without proteolysis.
[00155] The combination of proteolysis and silencing for XylA or "X valves"
and
proteolysis alone in the case of UdhA, a "U Valve", are evaluated for xylitol
production.
Specifically strains were engineered with these metabolic valves as well as
for overexpression of
a xylose reductase (xyrA from A. niger) and evaluated in two-stage minimal
media
microfermentations as reported by Moreb et al. Results are given in FIG 10A-C.
Additionally, a
confirmatory analysis of XyrA kinetics was performed, and results are given in
Supplemental
FIG 11. The combination of modifications resulted in a 16 fold increase in
xylitol production
compared to the control.
[00156] Regulatory Strategy
[00157] To investigate the impact of a regulatory strategy, we next sought
to evaluate the
potential impact of a larger set of valves on xylitol production as
illustrated in FIG 12A. We
constructed a set of strains with valves in citrate synthase (G1tA), glucose-6-
phosphate
dehydrogenase (Zwf) and enoyl-ACP reductase (FabI) which control flux through
the
tricarboxylic acid cycle, pentose phosphate pathway and fatty acid
biosynthesis, respectively.
We have previously reported the construction of metabolic valves in GltA ("G
Valves"), and
Zwf ("Z Valves") which comprised either proteolytic degradation (DAS+4 tags),
gene silencing
(either the zwf promoter or gltAp2 promoter) or both. In the case of FabI, we
constructed new
strains and plasmids to evaluate two-stage dynamic control on FabI levels.
Toward this goal, as
similarly reported by Li et al., we appended a superfolder GFP to the C-
terminus of the fabI
allele to enable quantification of protein levels by an ELISA. Unfortunately
and unexpectedly,
when plasmids silencing fabI expression were evaluated, guide RNA protospacer
loss was
observed (FIG 13A-D) and as a result we could not reliably obtain results
where fabI is silenced.
FabI proteolysis led to a ¨75 % reduction in FabI levels (FIG 14), and as a
result proteolytic
degradation alone will be referred to as an "F Valve". Strains were
constructed with
combinations of "X", "U", "G", "Z" and "F" valves and evaluated for xylitol
production, again
in minimal media microfermentations. Results are given in FIG 12A-D. To our
surprise the
highest xylitol producer had neither "X" or "U" valves but rather a
combination of "F" and "Z"
valves. Xylitol production in the "FZ" valve strain was synergistic above
either "F" or "Z"
valves alone (FIG 12B). This was surprising in that these two enzymes have no
direct or
predictable impact of xylitol biosynthesis as can be seen in FIG 12A. We have
recently reported
that the "Z" valve results in increased acetyl-CoA fluxes by leading to the
activation of Pfo
(encoded by the ydbK gene). With increased flux through Pfo we hypothesized
that NADPH
could be generated from reduced ferredoxin/flavodoxin through ferredoxin
reductase (Fpr).
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Using deletions in ydbK and fpr , as shown in FIG 15, we confirmed that this
pathway, and
specifically Fpr is indeed in part responsible for the elevated xylitol
production and NADPH flux
observed in our "FZ" valve strain. This is, to our knowledge, the first
confirmation that Fpr is
reversible in vivo. This reverse flux through Fpr may be dependent on low
NADPH pools (as
discussed below). The synergistic impact of the "F" valves was somewhat
unanticipated.
However, elevated NADPH fluxes due to dynamic control over FabI (enoyl-ACP
reductase) can
be attributed to reduced levels of fatty acid metabolites, specifically acyl-
CoAs (and potentially
their precursors acyl-ACPs). Fatty acyl-CoAs are competitive inhibitors of the
membrane bound
transhydrogenase encoded by the pntAB genes (FIG 12A). Palmitoyl-CoA,
specifically, has a
reported Ki of 1-5 M. Control over FabI levels and/or activity has been
previously shown to
reduce acyl-ACP pools and as a result alleviate feedback inhibition of acetyl-
CoA carboxylase
and malonyl-CoA synthesis. To our knowledge this is the first study
demonstrating the
importance of these metabolites in controlling NADPH fluxes. While previous
reports
demonstrate the inhibition of PntAB by acyl-CoAs (which in minimal media are
derived from
fatty acid biosynthesis there remains a possibility that acyl-ACPs also act as
inhibitors, although
future work is needed to confirm this hypothesis.
[00158] We next evaluated several additional modifications on top of the
"FZ" valves,
with a potential to impact xylitol production. (FIG 15B-D). Specifically we
evaluated the
addition of "G" and "U" valves as well as overexpression of pntAB. Plasmid
based
overexpression of the pntAB genes (using a low phosphate inducible promoter
led to a significant
improvement in xylitol production (FIG 15B). In contrast, the addition of
either the "G" or "U"
valve to the "FZ" combination did not increase xylitol synthesis but rather
led to a significant
decrease in xylitol production (FIG 15C-D). This suggests that citrate
synthase (G1tA) activity,
and flux through the TCA cycle, is required for optimal NADPH flux.
[00159] Using results from these experiments, we were able to estimate
boundary
conditions for several intracellular fluxes. For example from FIG 15B, we can
estimate that flux
through the Pfo/Fpr pathway accounts for at most ¨ 55% of the NADPH/xylitol
production. As a
result we are able to build stoichiometric metabolic models, as illustrated in
FIG 16A-B,
comparing an optimal growth phase and xylitol production phase. Importantly,
this model
confirms that indeed TCA flux is critical for xylitol production FIG 17A-B)
and that a 4-fold
increase in PntAB activity, in addition to flux through the Pfo/Fpr pathways
is needed to explain
increases in NADPH flux and xylitol production. The model predicts an overall
maximal xylitol
yield in this metabolic state of ¨0.864g/g of xylose, in line with yields
measured in fed batch
fermentations as discussed below.
[00160] Production in Instrumented Bioreactors
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[00161] Next, we compared xylitol production in instrumented bioreactors
using the "FZ"
valve strain with and without pntAB overexpression with a control strain.
Minimal media fed
batch fermentations were performed as described by Menacho-Melgar et al.,
wherein the media
has enough batch phosphate to support target biomass levels ( 25gCDW/L) prior
to phosphate
depletion and induction of xylitol biosynthesis in stationary phase. Results
of these studies are
given in FIG 18. While xyrA expression in our control strain (DLF Z0025) led
to only a few
grams per liter of xylitol (FIG 18A), the incorporation of "FZ" valves led to
titers over 100g/L in
160 hours of production (FIG 18B). The additional overexpression ofpntAB (FIG
18C) resulted
in maximal titers over 200g/L (185-204g/L) in 170 hrs. In these duplicate
fermentations the
average overall xylitol yield was 0.873 +/- 0.026 g/g xylose, and the average
production yield (in
stationary phase) was 0.935 +/- 0.011 g/g xylose.
[00162] Improved NADPH Flux is not Correlated with NADPH pools.
[00163] Lastly, we measured the levels of NADPH in a set of our engineered
strains.
Results are given in FIG 19A. Interestingly, there was no correlation between
specific xylitol
production and NADPH pools. In this case, the three strains having the highest
NADPH pools
were the control strain and the strains with dynamic control over enoyl-ACP
levels ("F" valve)
or soluble transhydrogenase ("U" valve) levels. The addition of the "Z" valve
(reduced levels of
glucose-6-phosphate dehydrogenase) led to a decrease in NADPH pools but an
increase in
NADPH flux. Deletions of either ydbK and or, fpr , also led to decreases in
NADPH levels, and
while overexpression ofpntAB increased xylitol production rates and fluxes it
did not improve
NADPH pools in the "FZ" background.
[00164] The use of 2-stage dynamic control generated an usual metabolic
state leading to
enhanced NADPH fluxes and xylitol production. To our knowledge this is the
highest titer and
yield of xylitol produced to date in engineered E. coli, particularly with
xylose as a sole carbon
source. Additionally, the productive stationary phase generated with these
modifications can be
extended to at least 170 hours. While the focus of this work has been on
xylitol production, the
identification of "F" and "Z" valves impacting NADPH flux has applicability to
other NADPH
dependent processes including more complicated pathways, and may represent a
facile method
for routine NADPH dependent bioconversions. The impact of FabI activity and
fatty acid
metabolite pools, on transhydrogenase activity, is consistent with previous
biochemical studies,
and has likely evolved to balance NADPH supply with fatty acid synthesis
demand.
Unfortunately, this feedback regulatory mechanism has been lost in the past
several decades of
metabolic engineering studies in E. coli, yet represents a powerful approach
to improving
NADPH fluxes. The unpredictable combination of "F" and "Z" valves is at odds
with standard
thinking regarding NADPH flux, where Zwf is often considered one of the
primary sources of
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NADPH in the cell and reducing Zwf activity would not be high on a list of
changes to make in
order to increase NADPH supply.
[00165] In order to explain the lack of correlation between NADPH pools and
our results,
we developed a conceptual model as illustrated in FIG 1. The "Z" valve leads
to a decrease in
NADPH pools which activate the SoxRS regulon, which is sensitive to oxidant
and NADPH
levels. SoxRS activation leads to increased expression of Pfo, which is
required to maintain a
high rate of pyruvate oxidation, generating NADPH via Fpr. Uniquely, this
study identifies a
previously unreported pathway for NADPH production utilizing Pfo and Fpr and
supports that
Fpr catalyzes a reversible reaction in vivo. Pfo expression is required, not
only for pyruvate
oxidation and sugar consumption but also NADH generation via the TCA cycle.
Increased TCA
flux produces excess NADH which is needed as a substrate for PntAB for maximal
NADPH
flux. Disruption of the TCA cycle ("G" Valve, FIG 15B) eliminates NADH
production and
acetyl-CoA consumption, greatly reducing NADPH flux. Increased NADPH levels
due to the
"F" valve make sense in light of the results discussed and are attributable to
increased activity of
the membrane bound transhydrogenase, PntAB. Reduced soluble transhydrogenase
(UdhA, FIG
15D) levels leads to increased NADPH pools (FIG 19) which presumably reduce
SoxRS
activation and Pfo expression. Simply put, the metabolic network responds to
decreased NADPH
and acyl-CoA pools by increasing sugar consumption and NADPH flux to
compensate. If "set"
point NADPH pools are regained or if continued sugar catabolism stops,
continued NADPH flux
is halted.
[00166] Lastly, the metabolic state leading to enhanced NADPH flux and
xylitol
production would be hard to identify and/or engineer in a growth coupled
process as it relies on
the manipulation of feedback inhibition due to central metabolites. These
central metabolic
regulatory circuits have evolved to balance fluxes to both optimize growth and
enable adaptive
responses to environmental and physiological perturbations. Dynamic metabolic
control, and in
particular two-stage dynamic metabolic control, is uniquely suited to
manipulate central
metabolite levels without impacting cell growth or survival. This approach can
lead to the
discovery as well as the manipulation of central regulatory mechanisms, which
in turn have a
high potential to enhance metabolic fluxes and drive future metabolic
engineering strategies.
