Patent Description:
This invention was made with government support under Federal Grant No. EE0007563 awarded by the Department of Energy; Federal Contract No. HR0011-<NUM>-C-<NUM> awarded by the United States Department of Defense; Federal Grant No. ONR YIP <NUM> awarded by the United States Department of Defense; DARPA# HR0011-<NUM>-C-<NUM>; ONR YIP #N00014-<NUM>-<NUM>-<NUM>; DOE EERE grant #EE0007563; N00014-<NUM>-<NUM>-<NUM> awarded by NAVY/ONR, and NIH Biotechnology Training Grant (T32GM008555). The government has certain rights in the invention.

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 ~<NUM>,<NUM> 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, <NUM>,<NUM>,<NUM>-butanetriol, and xylitol.

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 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.

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.

<NPL>) describes an analysis of NADPH supply during xylitol production by engineered E. <NPL>) describes efficient biosynthesis of xylitol from xylose by coexpression of xylose reductase and glucose dehydrogenase in E.

We rationally designed genetically modified microorganism strains to optimize xylitol production from xylose utilizing two stage dynamic metabolic control. As illustrated in <FIG>, 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 Xy1A 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.

We also fully describe improved NADPH flux coincident with xylitol biosynthesis in engineered E. 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 <NUM> fold and <NUM> fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-<NUM>-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and a new NADPH generation pathway, namely pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of <NUM>/L of xylitol from xylose at <NUM>% of theoretical yield in instrumented bioreactors. Dynamic control over enoyl-ACP reductase and glucose-<NUM>-phosphate dehydrogenase will broadly enable improved NADPH dependent bioconversions.

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.

Other methods, features and/or advantages is, or will become, apparent upon examination of the following Figures and detailed description.

The present invention is related to a genetically modified E. coli microorganism as defined in appended claim <NUM> and a multi-stage fermentation bioprocess as defined in appended claim <NUM>.

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.

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.

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.

As used herein, the term "gene disruption," or grammatical equivalents thereof (and 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.

Bio-production, Micro-fermentation (microfermentation) or Fermentation, as used herein, may be aerobic, microaerobic, or anaerobic.

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.

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.

Species and other phylogenic identifications are according to the classification known to a person skilled in the art of microbiology.

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. 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.

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.

The meaning of abbreviations is as follows: "C" means Celsius or degrees Celsius, as is 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), "µL'' 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, "µM" or "uM" means micromolar, "M" means molar, "mmol" means millimole(s), "µmol" or "uMol" means micromole(s)", "g" means gram(s), "µg'' or "ug" means microgram(s) and "ng" means nanogram(s), "PCR" means polymerase chain reaction, "OD" means optical density, "OD<NUM>" means the optical density measured at a photon wavelength of <NUM>, "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-µ-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.

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).

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.

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.

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, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces, and Pseudomonas. In some aspects the host microorganism is an E. coli microorganism.

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.

Also disclosed herein are media and culture conditions that comprise genetically modified microorganisms of the invention and optionally supplements.

Typically cells are grown at a temperature in the range of about <NUM>° C to about <NUM>° C in an appropriate medium, as well as up to <NUM>° 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 <NUM> to pH <NUM>, where pH <NUM> to pH <NUM> 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.

Fermentation systems utilizing methods and/or compositions are also disclosed herein. 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.

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).

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.

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.

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. coli, 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.

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 disclosed herein. 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.

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.

In various embodiments, to function more efficiently, a microorganism may comprise one or more gene deletions. For example, in E. coli, 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 iclR transcriptional regulator (iclR) 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.

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. coli may be designed as synthetic metabolic valves to alter metabolism and improve product formation. Representative genes in E. coli may include but are not limited to the following: fabI, zwf, gltA, ppc, udhA, lpd, sucD, aceA, pfkA, lon, 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.

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.

Accordingly, as described in various sections above, some compositions, methods and systems disclosed herein 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.

Aspects of the disclosure 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 specific productivity, volumetric productivity, titer and yield substantially over more basic combinations of genetic modifications.

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.

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: <NUM>) transcriptional gene silencing or repression technologies in combination with <NUM>) inducible and selective enzyme degradation and <NUM>) 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.

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.

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 <NPL>. Methodologies and mechanisms for CRISRPR interference are taught by <NPL>. In addition, methodologies, and mechanisms for CRISRPR interference using the native E. coli CASCADE system are taught by <NPL>. In additional numerous transcriptional repressor systems are well known in the art and can be used to turn off gene expression.

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. coli clpXP protease for controlled protein degradation are taught by <NPL>. This methodology relies upon adding a specific C-terminal peptide tag such as a DAS4 (or DAS+<NUM>) 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 <NPL>), 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.

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. 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 <NPL>, teaches the expression and use of the E. coli clpXP protease in the yeast Saccharomyces cerevisiae. Such approaches can be used to transfer the methodology for synthetic metabolic valves to any genetically tractable host.

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-<NUM>-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.

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.

Within the scope of the invention are genetically modified microorganisms as defined in the appended claims, wherein the microorganism is capable of producing xylitol at a specific rate selected from the rates of greater than <NUM>/gDCW-hr, <NUM>/gDCW-hr, greater than <NUM>/gDCW-hr, greater than <NUM>/gDCW-hr, greater than <NUM>/gDCW-hr, greater than <NUM>/gDCW-hr, greater than <NUM>/gDCW-hr, greater than <NUM>/gDCW-hr, greater than <NUM>/gDCW-hr, greater than <NUM>/gDCW-hr, greater than <NUM>/gDCW-hr, greater than <NUM>/gDCW-hr, or greater than <NUM>/gDCW-hr.

Within the scope of the invention are genetically modified microorganisms as defined in the appended claims, wherein the microorganism is capable of producing xylitol from xylose or another sugar source at a yield greater than <NUM> product /g xylose, greater than <NUM> product /g xylose, greater than <NUM> product /g xylose, greater than <NUM> product /g xylose, greater than <NUM> product /g xylose, greater than <NUM> product /g xylose, or greater than <NUM> product /g xylose.

Also disclosed herein is 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 <NUM> gDCW/L, <NUM> gDCW/L, greater than <NUM> gDCW/L, greater than <NUM> gDCW/L, greater than <NUM> gDCW/L, greater than <NUM> gDCW/L or greater than <NUM> gDCW/L, such as when the volume of the aqueous medium is selected from greater than <NUM>, greater than <NUM>, greater than <NUM>, greater than <NUM>, greater than <NUM>, greater than <NUM>, greater than <NUM>, greater than <NUM>, greater than <NUM>,<NUM>, greater than <NUM>,<NUM>, greater than <NUM>,<NUM> or greater than <NUM>,<NUM>, and such as when the volume of the aqueous medium is greater than <NUM> and contained within a steel vessel.

In one aspect, a genetically modified microorganism for producing xylitol comprising is provided, wherein the genetically modified microorganism is as defined in appended claim <NUM>.

In one aspect, the xylose reductase of the genetically modified microorganism is an NADPH dependent xylose reductase or the xylose reductase may be the xyrA gene of A.

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.

In one aspect, the gene-silencing synthetic metabolic valve or the enzyme degradation synthetic metabolic valve of the genetically modified microorganism may be directed to control of the gene encoding glucose-<NUM>-phosphate dehydrogenase (zwf) or the glucose-<NUM>-phosphate dehydrogenase (zwf) enzyme.

In one aspect, the gene-silencing synthetic metabolic valve or the enzyme degradation synthetic metabolic valve of the genetically modified microorganism may be directed to control more than one gene, for example a gene encoding glucose-<NUM>-phosphate dehydrogenase (zwf) or the glucose-<NUM>-phosphate dehydrogenase (zwf) enzyme; and a gene encoding enoyl-ACP reductase (fabI) or the enoyl-ACP reductase (fabI) enzyme.

In yet another aspect, the gene-silencing synthetic metabolic valve or the enzyme degradation synthetic metabolic valve of the genetically modified microorganism may be directed to control silencing of a gene encoding glucose-<NUM>-phosphate dehydrogenase (zwf) and enzyme degradation of glucose-<NUM>-phosphate dehydrogenase (zwf) enzyme; and enoyl-ACP reductase (fabI) enzyme.

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.

In one aspect, the genetically modified microorganism may further comprise a chromosomal deletion.

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.

According to the present invention, the genetically modified microorganism produces a xylitol product titer of greater than <NUM>/L at twenty four in a biofermentation process.

In one aspect, the invention provides for a multi-stage fermentation bioprocess for producing xylitol from a genetically modified microorganism, wherein the multi-stage fermentation bioprocess is as defined in appended claim <NUM>.

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-<NUM>-phosphate dehydrogenase (zwf) or the glucose-<NUM>-phosphate dehydrogenase (zwf) enzyme; and a gene encoding enoyl-ACP reductase (fabI) or the enoyl-ACP reductase (fabI) enzyme.

In some aspects, the multi-stage fermentation bioprocess will produce a xylitol product titer of greater than <NUM>/L at twenty four in a biofermentation process.

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.

In one aspect, the genetically modified microorganism for producing xylitol, the microorganism comprises: inducible reduction of xylose isomerase; inducible reduction of glucose-<NUM>-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.

Also disclosed herein is a multi-stage fermentation bioprocess for producing xylitol from a genetically modified microorganism including inducible reduction of xylose isomerase and inducible reduction of glucose-<NUM>-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.

In one aspect, the genetically modified microorganism for producing xylitol, the microorganism comprises: inducible reduction of xylose reductase; inducible reduction of glucose-<NUM>-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.

Also disclosed herein is a multi-stage fermentation bioprocess for producing xylitol from a genetically modified microorganism including inducible reduction of xylose reductase; inducible reduction of glucose-<NUM>-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.

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 ferredoxin reductase is increased; and wherein the microorganism produces at least one chemical product whose biosynthesis requires NADPH.

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. 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 <FIG> and <FIG>), 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.

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., <NPL>.

The following published resources are 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 (<NPL> for biological reactor design; <NPL>, e.g., for process and separation technologies analyses;<NPL>, e.g., for separation technologies teachings).

Publications, patents, and patent applications mentioned in this specification also include <CIT>, and <CIT>, and <CIT> and <CIT>.

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.

All reagents and chemicals were obtained from Sigma Aldrich (St. Louis, MO) unless otherwise noted. MOPS (<NUM>-(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 (<NPL>)) except that xylose was substituted for glucose (<NUM> gram xylose for <NUM> gram glucose) in all media formulations. LB, Lennox formulation, was used for routine strain propagation. Working antibiotic concentrations were as follows: kanamycin: <NUM>µg/mL, chloramphenicol: <NUM>µg/mL, gentamicin:<NUM>µg/mL, <NUM> zeocin: <NUM>µg/mL, blasticidin: <NUM>µg/mL, spectinomycin: <NUM>µg/mL, tetracycline: <NUM>µg/mL.

Refer to Supplemental Table S1 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 (<NPL>)). The recombineering plasmid pSIM5 was a kind gift from Donald Court (NCI, https://redrecombineering. gov/court-lab. <NUM>,<NUM> C-terminal DAS+<NUM> tags were added by direct integration and selected through integration of antibiotic resistance cassettes <NUM>' of the gene as previously described. <NUM> 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.

The xyrA gene from Aspergillus niger was codon optimized for expression in E. coli and the plasmid, pHCKan-xyrA (Addgene #<NUM>), enabling the low phosphate induction of xylose reductase, was constructed by TWIST Biosciences (San Francisco, CA). pCDF-pntAB (Addgene # <NUM>) was constructed using PCR and Gibson Assembly from pCDF-ev <NUM> to drive expression of the pntAB operon from the low phosphate inducible ugpBp promoter (<NPL>). 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.

Chromosomal modifications were constructed using standard recombineering methodologies. A C-terminal DAS+<NUM> tag on the xylA gene was added by direct integration and selected through integration of antibiotic resistance cassettes <NUM>' of the gene. All strains were confirmed by PCR, agarose gel electrophoresis and confirmed by sequencing.

The xyrA gene from Aspergillus niger was codon optimized for E. coli 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.

Primers used for assembly are given in Table <NUM>:.

Micro-fermentations and <NUM> fermentations in instrumented bioreactors were performed as previously reported, except that xylose was substituted for glucose (1gram xylose for 1gram glucose) in all media formulations. Guide array stability was confirmed after transformation of pCASCADE vector by PCR prior to evaluation in <NUM> well plate micro-fermentations.

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 <NUM>. 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 <NUM>, a Rezex ROA-Organic Acid H+ (<NUM>%) Analysis HPLC Column (CAT#: #<NUM>-<NUM>-K0, Phenomenex, Inc. , Torrance, CA, <NUM> x <NUM> 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 <NUM>/min. Waters Acquity H-Class UPLC integrated with a Waters <NUM> Refractive Index (RI) detector (Waters Corp. , Milford, MA. USA) was used for the chromatographic detection. Injection volume of sample and standard was set as <NUM>µL. Samples were diluted in <NUM> times using filtered ultrapure water to make all the sample points appear within the standards linear range. The standard variation range was between <NUM> to <NUM>/L. MassLynx v4. <NUM> software was used for all the peak integration and concentration analysis.

Minimal media microfermentations were performed as previously reported (<NPL> and <NPL>)) except that xylose was substituted for glucose (<NUM> gram xylose for <NUM> 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. <NUM> Fed batch fermentations were performed as previously reported, again with xylose instead of glucose (<NPL>). Xylose feeding was as modified as follows. The starting batch glucose concentration was <NUM>/L. Concentrated sterile filtered xylose feed (<NUM>/L) was added to the tanks at an initial rate of <NUM>/h when cells entered mid-exponential growth. This rate was then increased exponentially, doubling every <NUM> hours (<NUM>) until <NUM> total glucose had been added, after which the feed was maintained at <NUM>/hr. The feed was reduced to <NUM>/hr due to xylose accumulation at <NUM> hrs post inoculation, and stopped at 120hrs post inoculation.

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 <NUM> for <NUM> 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. XyrA protein was purified using Ni-NTA Resin (G-Biosciences, Cat # <NUM>-<NUM>) according to manufacturer's instructions. Kinetics assays for XyrA were performed in a reaction buffer composed of <NUM> sodium phosphate (pH <NUM>, <NUM> MgCl2) with NADPH as cofactor (<NPL>)). In these assays, NADPH was held at a constant initial level of <NUM>. Results of the assay were measured through monitoring the absorbance of NADPH at <NUM> for <NUM> hours (<NUM> per read) using a SpectraMax Plus <NUM> microplate reader (Molecular Devices). Reaction velocity is plotted as a function of xylose concentration. Using the Eadie-hofstee equation, we got the parameters: Vmax=<NUM> ± <NUM> U, kcat=<NUM> ± <NUM>-<NUM> and Km: <NUM> ± <NUM>.

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 <NUM> (<NPL>) and <NPL>)). Cultures were grown in shake flasks in SM10++ media and harvested in mid exponential phase, washed and resuspended in SM <NUM> No phosphate media. After <NUM> hours of phosphate depletion, cells were pelleted by <NUM> minutes of centrifugation (<NUM> RCF, <NUM> degrees C) and lysed with BugBuster protein extraction reagent (Millipore Sigma, Catalog #<NUM>) according to the manufacturer's protocol. Cell debris was removed by two rounds of centrifugation, <NUM> minutes (<NUM> RCF, <NUM> degrees C) followed by a <NUM> minute hard spin (<NUM> RCF, <NUM> 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 (<NUM> sodium phosphate, <NUM> MgCl<NUM>, pH <NUM>) and remove metabolites. Samples were assayed in triplicate in a <NUM> well plate with 100uL of the filtered cell extract per well containing <NUM> xyulose, <NUM> NADPH, and 1ug/mL of purified D-xylose reductase (see above). The absorbance at <NUM> was measured every 15seconds for <NUM>. 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: <NUM> ± <NUM>-<NUM>, Km: <NUM> ± <NUM>.

The activity of the soluble transhydrogenase was quantified by method previously reported (<NPL>) and <NPL>)). 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 <NUM> minutes (<NUM> RPM, <NUM>) to remove large debris. A second hard spin was performed for <NUM> minutes (<NUM> RPM, <NUM>) to remove remaining debris and further separate the membrane fraction from the soluble transhydrogenase. Lysates were diluted <NUM>:<NUM> with the assay reaction buffer (<NUM> Tris-HCl, <NUM> MgCl, pH <NUM>) and transferred to an Amicon Ultra centrifugal filter (10kDa MWCO). The samples were centrifuged for <NUM> minutes (<NUM> RPM, <NUM>) and this step was repeated <NUM> 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.

Then soluble transhydrogenase activity was assayed at room temperature. Assays were performed in black <NUM> well plates by mixing equal volumes of lysate and reaction buffer for a final volume of 100uL per well and a final concentration of <NUM> NADPH and <NUM> <NUM>-acetylpyridine adenine dinucleotide (APAD+). Changes in absorbance at <NUM> and <NUM> due to the reduction of APAD+ and the oxidation of NADPH, respectively, were monitored simultaneously by Spectramax Plus <NUM> microplate reader at <NUM> second intervals for <NUM> minutes. A standard curve was used to calculate the molar absorptivity of NADPH (<NUM>*<NUM><NUM> M-<NUM> cm-<NUM>). 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.

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.

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+ (<NUM>%) Analysis HPLC Column (Cat #: #<NUM>-<NUM>-K0, Phenomenex, Inc. , Torrance, CA, <NUM> x <NUM> mme;) was employed for the separation of xylose and xylitol. <NUM> Sulfuric acid as the isocratic mobile phase at a flow rate of <NUM>/min, at <NUM>,. A Waters Acquity H-Class UPLC integrated with a Waters <NUM> Refractive Index (RI) detector (Waters Corp. , Milford, MA. USA) was used for detection. The injection volume of samples and standards was <NUM>µL. Samples were diluted <NUM> fold in water in order to be in the linear range (<NUM> to <NUM>/L). MassLynx v4. <NUM> software was used for all the peak integration and analyses.

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.

In silico 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-<NUM> MG1655 reconstruction includes <NUM>,<NUM> metabolic reactions and <NUM>,<NUM> 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:.

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 model was created and analyzed. Specific evaluated conditions and biomass fluxes are shown in Table S6.

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 <NUM>% and <NUM>% 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 <NUM> 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.

Referring now to <FIG>, 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 <FIG>, 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 <NUM>. <FIG> Kinetic Parameters for XyrA from this project and from other research sources as comparison. Kinetics for XyrA were measured using <NUM> sodium phosphate, pH <NUM> (containing <NUM> MgCl<NUM>). <NUM> <NUM> NADPH. Results of the assay were measured through monitoring the absorbance of NADPH at <NUM>. Using Eadie-Hofstee equation, the parameters Vmax=<NUM>. 6U, kcat=<NUM>-<NUM> and km=<NUM>. <NUM> 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.

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>, 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 <NUM> and <NUM> for plasmids and strains used. These strains were evaluated in <NUM> well plate micro-fermentations as reported by Moreb et al and results are given in <FIG>.

Since dynamic control over XylA ("X") activity only led to modest improvements in xylitol production, <FIG>, 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>. These valves included: citrate synthase (GltA-"G"), glucose-<NUM>-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+<NUM> 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.

The panel consisted of ~<NUM> valve combinations of X, U, G, Z and F that were evaluated for xylitol production in two stage <NUM> well plate micro-fermentations in at least triplicate. Results of these experiments are given in <FIG>. Xylitol titers ranged from ~ <NUM>/L-OD(<NUM>) to ~<NUM>/L-OD(<NUM>). Approximately ~<NUM>% of the silencing and proteolysis combinations performed better than the control strain, which only produced <NUM>/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(<NUM>,<NUM>)=<NUM>, p<<NUM>).

P-values were used to generate a p-value heatmap (<FIG>), where only combinations with a p value less than <NUM> are highlighted. Combinations not assayed or with less than <NUM> 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 <NUM>/L-OD600nm. 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>.

Based on the results from the micro-fermentations (<FIG> and <FIG>), we chose the "Z-FZ" valve strain (silencing of zwf "Z" and proteolysis of fabI and zwf "FZ") which has titer of <NUM>/L-OD600 as well as the control for evaluation in instrumented bioreactors. Fermentations 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>. Results of these fermentations are given in <FIG> below. The Z-FZ strain (FIG 6A) enabled xylitol production up to <NUM>+/-<NUM>/L with <NUM> hours, while xyrA expression in our control strain DLF_0025-EV (FIG 6B) led to only ~<NUM>/L of xylitol within the same time.

Most previous studies producing xylitol from xylose rely on a bioconversion requiring an additional sugar (usually glucose) as an electron donor (<NPL>). ; <NPL>); and <NPL>)). Oxidation of glucose (producing the byproduct gluconic acid) generates NADPH which is then used for xylose reduction (<NPL>). 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.

We applied two-stage dynamic metabolic control (DMC) to improve NADPH flux and xylitol production using xylose as a sole feedstock (<NPL>)). Dynamic control over metabolism has become a popular approach in metabolic engineering, and has been used for the production of various products from <NUM>-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 decreasing glucose-<NUM>-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 <NUM>(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.

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>, 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 S1 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+<NUM> 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>. In the case of XylA, proteolysis led to ~ <NUM>% 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 ~<NUM>% reduction in activity, whereas silencing had no detectable impact on activity levels with or without proteolysis.

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>. Additionally, a confirmatory analysis of XyrA kinetics was performed, and results are given in Supplemental <FIG>. The combination of modifications resulted in a <NUM> fold increase in xylitol production compared to the control.

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>. We constructed a set of strains with valves in citrate synthase (GltA), glucose-<NUM>-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+<NUM> 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>) and as a result we could not reliably obtain results where fabI is silenced. FabI proteolysis led to a ~<NUM> % reduction in FabI levels (<FIG>), 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>. 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>). This was surprising in that these two enzymes have no direct or predictable impact of xylitol biosynthesis as can be seen in <FIG>. 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). Using deletions in ydbK and fpr, as shown in <FIG>, 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>). Palmitoyl-CoA, specifically, has a reported Ki of <NUM>-<NUM> µM. 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.

We next evaluated several additional modifications on top of the "FZ" valves, with a potential to impact xylitol production. 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>). 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>). This suggests that citrate synthase (GltA) activity, and flux through the TCA cycle, is required for optimal NADPH flux.

Using results from these experiments, we were able to estimate boundary conditions for several intracellular fluxes. For example from <FIG>, we can estimate that flux through the Pfo/Fpr pathway accounts for at most ~ <NUM>% of the NADPH/xylitol production. As a result we are able to build stoichiometric metabolic models, as illustrated in <FIG>, comparing an optimal growth phase and xylitol production phase. Importantly, this model confirms that indeed TCA flux is critical for xylitol production <FIG>) and that a <NUM>-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 ~<NUM>/g of xylose, in line with yields measured in fed batch fermentations as discussed below.

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>. While xyrA expression in our control strain (DLF_Z0025) led to only a few grams per liter of xylitol (<FIG>), the incorporation of "FZ" valves led to titers over <NUM>/L in <NUM> hours of production (<FIG>). The additional overexpression of pntAB (<FIG>) resulted in maximal titers over <NUM>/L (<NUM>-<NUM>/L) in <NUM> hrs. In these duplicate fermentations the average overall xylitol yield was <NUM> +/- <NUM>/g xylose, and the average production yield (in stationary phase) was <NUM> +/- <NUM>/g xylose.

Lastly, we measured the levels of NADPH in a set of our engineered strains. Results are given in <FIG>. 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-<NUM>-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 of pntAB increased xylitol production rates and fluxes it did not improve NADPH pools in the "FZ" background.

The use of <NUM>-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 <NUM> 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 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.

In order to explain the lack of correlation between NADPH pools and our results, we developed a conceptual model as illustrated in <FIG>. 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>) 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>) levels leads to increased NADPH pools (<FIG>) 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.

Claim 1:
A genetically modified E. coli microorganism for producing xylitol from xylose comprising:
inducible overexpression of xylose reductase and inducible synthetic metabolic valves comprising:
an enzymatic degradation synthetic metabolic valve characterized by inducing enzymatic degradation of the xylose isomerase enzyme; and
a gene expression-silencing synthetic metabolic valve characterized by silencing gene expression or an enzymatic degradation synthetic metabolic valve characterized by inducing enzymatic degradation of at least one gene or enzyme selected from the group consisting of:
a gene encoding glucose-<NUM>-phosphate dehydrogenase (zwf) or the glucose-<NUM>-phosphate dehydrogenase (zwf) enzyme; and
a gene encoding enoyl-ACP reductase (fabI) or the enoyl-ACP reductase (fabI) enzyme;
wherein the genetically modified E. coli microorganism produces xylitol at a product titer of greater than <NUM>/L/<NUM> hours in a biofermentation process.