KR20220164007A - Methods and compositions for producing xylitol from xylose using dynamic metabolic control - Google Patents

Abstract

The present invention relates to genetically engineered microbial strains and related biological processes for producing xylitol. More specifically, xylitol production is increased in a two-step process using dynamically regulated synthetic metabolic valves to lower the activity of certain enzymes.

Description

Methods and compositions for producing xylitol from xylose using dynamic metabolic control

Cross-reference to related applications

This application claims priority to U.S. Provisional Application No. 63/004,740, dated April 3, 2020, and U.S. Provisional Application No. 63/056,085, dated July 24, 2020, both of which are incorporated herein by reference in their entirety.

technical field of invention

The present invention relates to metabolically engineered microorganisms such as bacterial strains and bioprocesses using such strains. These strains provide dynamic regulation of the metabolic pathway that achieves the production of xylitol from xylose.

sequence listing

This application contains the electronically submitted sequence listing in ASCII format as 49196-46_ST25, 17051 bytes in size, created on March 29, 2021, which is incorporated herein by reference in its entirety.

Federally funded research or development

This invention is disclosed under Federal Grant No. EE0007563 awarded by the Department of Energy; Federal Contract No. HR0011-14-C-0075 awarded by the US Department of Defense; Federal Authorization No. ONR YIP 12043956 granted by the US Department of Defense; DARPA# HR0011-14-C-0075; ONR YIP #N00014-16-1-2558; DOE EERE approved #EE0007563; Made with government support under N00014-16-1-2558 awarded by NAVY/ONR and NIH Biotechnology Training Grant (T32GM008555). The government has certain rights in this invention.

Xylitol is an industrial sugar alcohol used primarily as a sweetener. It tastes similar to sucrose but has lower calories. The annual production of xylitol is ~125,000 tonnes and is produced through the reduction of xylose. Xylose is the second most abundant natural sugar (after glucose) and therefore an attractive feedstock. Many studies demonstrate the use of xylose as a feedstock for the biosynthesis of numerous products ranging from biofuels (ethanol) to chemicals including lactic acid, succinic acid, xylonates, 1,2,4-butanetriol and xylitol. It became.

The industrial production of xylitol is based 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. Attempts have been made to find more economical ways to produce xylitol from cheaper cellulosic sugar streams, including the development of biosynthetic processes. Biosynthetic production has the potential to reduce costs, utilize low-quality feedstocks, avoid the use of organic solvents, and eliminate the need for expensive reduction catalysts. However, most existing biosynthetic studies to produce xylitol from xylose rely on bioconversion that requires an additional sugar (usually glucose) as an electron donor. Oxidation of glucose (with gluconic acid as a by-product) produces NAD(P)H, which is utilized for xylose reduction. Although this process can provide xylitol in high titer and excellent yield if only xylose is considered, it is quite inefficient because it requires a molar level of glucose equal to that of xylose.

Perhaps the simplest conversion is to convert xylose to xylitol using just one enzyme, xylose reductase. Biosynthetic production of xylitol, compared to chemical conversion, avoids the use of organic solvents, does not require expensive reduction catalysts, and has the potential to improve product purity, while simultaneously reducing costs.

The present inventors have rationally designed genetically modified microbial strains to optimize the production of xylitol from xylose using two-step dynamic metabolic control. As illustrated in FIG. 1 , this design involves overexpression of xylose reductase and dynamic lowering of xylose isomerase ( xylA ) activity to reduce xylose metabolism competing with xylitol production. To this end, the present inventors have developed strains and plasmids that allow dynamic reduction of XylA activity and dynamic induction of xyrA upon phosphate depletion or upon occurrence of gene silencing, XylA proteolysis or other causative events through a combination of the two actions. built. The present invention provides a microbial strain for a scalable biofermentation process in which proliferation and product formation are separated using synthetic metabolic valves (SMVs). The mentioned strains, upon transformation, provide dynamic regulation of metabolic pathways, including pathways that negatively affect microbial growth under certain inducible conditions.

In addition, we fully describe an improved NADPH flux corresponding to xylitol biosynthesis in engineered E. coli . Xylitol is made from xylose by a NADPH-dependent reductase. We use two-step dynamic metabolic control to compare two ways to optimize xylitol biosynthesis: a stoichiometric way to reduce competing fluxes and a modulatory way to reduce levels of key regulatory metabolites. . The stoichiometric and controllable modes improve xylitol production 16-fold and 100-fold, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase develop new NADPH, leading to increased NADPH flux, despite activation of membrane-bound transhydrogenase and reduced NADPH pool. Activation of the generative pathway, i.e., the pyruvate ferredoxin oxidoreductase coupled with the NADPH-dependent ferredoxin reductase, results in an altered metabolite pool. This strain produces xylitol from xylose at a titer of 200 g/L, 86% of the theoretical yield in the bioreactor unit. Dynamic modulation of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase could achieve broadly NADPH dependent biotransformation improvements.

In addition, the present invention provides a multi-step xylitol production bioprocess using the mentioned genetically modified microorganisms having one or more synthetic metabolic valves, which provides dynamic flux control and achieves improved xylitol production. In certain embodiments, the carbon feedstock is xylose or may include a combination of xylose and glucose, arabinose, mannose, lactose, or alternatively carbon dioxide, carbon monoxide, methane, methanol, formaldehyde or oil. . Additional genetic modifications may be added to the microorganisms to provide additional conversion of xylitol into additional chemical or fuel products.

Other methods, features and/or advantages are or will become apparent upon review of the following drawings and detailed description. All of these additional methods, features and advantages are intended to be included in this description and protected by the appended claims.

The novel features of the invention are specifically pointed out 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 and accompanying drawings, which set forth exemplary embodiments in which the principles of the present invention are applied:
1 shows the design of a metabolic valve for bioproducing xylitol. Biosynthetic process of xylitol in E. coli by xylose reductase (XyrA) and NADPH as cofactor (solid arrow). A major competitive pathway for consuming xylose is xylose consumption by xylose isomerase (XylA, valve).
2A-C show xylose reductase expression and enzyme kinetics. Figure 2A, XyrA expression in BL21 using media combinations of SM10++ (for propagation) and SM10-No phos (for expression). After expression, postproduction cells are subjected to cell lysis through a freeze-thaw cycle. Then, a His-tag on XyrA was designed into the plasmid sequence to extract the xyrA protein with NN resin. 2B, xyrA activity using NADPH as a cofactor. Graph the reaction rate as a function of xylose concentration. In these assays, NADPH is maintained at a constant initial level of 50 μM. Figure 2C, Kinetic parameters for XyrA obtained from this experiment and other studies as a comparison.
3 shows xylitol titer/OD (g/L-OD) measured using different combinations of xylA silencing and xylA proteolysis. The specific productivity of the different strains shows significant differences from the control strain DLF-0025-EV. Statistically significantly higher levels were achieved when all three valves were combined compared to the DLF25-EV control, but xylA silencing or proteolysis alone was superior to this combination.
4 depicts xylitol production in E. coli using two-step dynamic regulation. Strain metabolic network design. Major metabolic pathways include fatty acid biosynthesis, citric acid cycle (TCA), NADPH supply, pentose phosphate pathway transhydrogenase and glycolysis. Valves that can be 'switched off' in the metabolic system are xylose isomerase (xylA-X), soluble transhydrogenase (udhA-U) enoyl-ACP reductase (fabI-F), citrate synthase ( gltA-G) and glucose-6-phosphate dehydrogenase (zwf-Z). These valves are all highlighted with red valves. Xylose reductase (xyrA) can be dynamically 'switched on' to produce xylitol with NADPH as a cofactor.
Figures 5A-B show (5A) Rank order plots for the average xylitol titer and standard deviation of all valve strains investigated in two-stage micro-fermentation. Xylitol production in the control strain is shown in red. A post hoc Dunnett's test confirms that these combinations are significantly different from the DLF025-empty vector control at p < 0.05, which is indicated by shading (instead of gray bars indicating non-significance) in titers sorted per unit OD plot . (5B) Heatmap showing xylitol titers, from 0 g/L (white) to 12 g/L (shaded) in two-step production following different combinations of proteolysis and silencing. The x-axis shows different proteolytic valves, and the y-axis shows different pCASCADE silencing. A DLF_25 empty valve adjustment is indicated by a red circle. Gray dots indicate combinations that did not analyze or did not adequately propagate cells in the entire replicate. According to the heatmap results, for combinations with titer/OD >3, replication is performed 6 times to avoid false positive results.
FIG. 6 shows the micro-expression results of FIG. 5 as a p-value map.
Figures 7A-B show (7A) exemplary production strain Z-FZ (zwf("Z") silenced, fabI and proteolysis of zwf ("FZ")) and (7B) control strain (DLF-0025-EV). A graph of device fermentation in a 1 L bioreactor is shown. Blue lines represent OD600 values and orange lines represent xylitol titers at various time points. The Z-FZ combination achieves a titer of 104+/- 11.31 g/L after 160 hours of production, whereas the control strain (DLF_0025-EV) produces only -3 g/L at the same production time point. The present inventors reproduced the Z-FZ tank fermentation using the same spawn and fermentation conditions, and the result is the average of two replicate tanks, and the standard deviation is indicated at the sample point of the graph.
8 depicts a conceptual model of two-step NADPH production in the operating system of the present invention. Glucose-6-phosphate dehydrogenase (encoded by the zwf gene) is usually responsible for the biosynthesis of most of NADPH. This irreversible response imposes a NADPH setpoint that stops the SoxRS oxidative stress response (gray area). A dynamic decrease in Zwf levels activates the SoxRS response to reduce the NADPH pool, followed by expression of pyruvate ferredoxin oxidoreductase (Pfo, encoded by the ydbK gene) and NADPH-dependent ferredoxin reductase (Fpr). Activated. Pfo and Fpr (working in reverse) together constitute a new pathway to produce NADPH, but also to allow continued pyruvate oxidation and production of acetyl-CoA to enter the tricarboxylic acid cycle (TCA cycle) . NADPH flux is further enhanced by lowering fatty acid biosynthesis, the product of which inhibits membrane-bound transhydrogenase (encoded by the pntAB gene). Activated PntAB uses proton drive to convert NADH from the TCA cycle into NADPH. NADPH can be used for bioconversion, such as in xylitol production.
Figures 9A-B show enzyme levels of 9A) XylA and 9B) UdhA in response to inducible proteolysis and/or gene silencing during phosphate depleted plateau phases. ev - empty vector, x- xylA promoter, u-udhA promoter.
Figure 10 AC : Engineered to dynamically control the levels of 10A) xylose isomerase (XylA), 10B) soluble transhydrogenase (UdhA), and 10C) a combination of xylose isomerase and soluble transhydrogenase. Specific xylitol production in strains. ev - empty vector, x-xylA promoter, u-udhA promoter. All results were obtained from micro ferments.
Figure 11: XyrA expression and purification in BL21 (DE3). Left: expression over time after phosphate depletion, whole cell lysates show XyrA expression. Densitometry confirms the expression level to be -20%. Middle: XyrA purification (containing an N-terminal 6X histidine tag) by IMAC. Right: Kinetic analysis of purified XyrA. Initial velocity (μM/s) is plotted as a function of substrate (xylose) concentration.
12 AD : 12A) Overview of xylitol production and metabolic valve location in central metabolism. Xylitol is produced from xylose by xylose reductase (xyrA). The valve contains inducible proteolysis and/or silencing of five enzymes: citrate synthase (gltA), xylose isomerase (xylA), glucose-6-phosphate dehydrogenase (zwf), eno yl-ACP reductase (fabI) and soluble transhydrogenase (udhA). Membrane-bound transhydrogenase (pntAB) is also indicated. 12B) Specific xylitol production (g/L-OD600 nm) in micro-fermentation following silencing and/or proteolysis. 12C) p-value for data in 12B comparing each strain to non-valve control using Welch's t-test. 12D) Rank-order plots of panel data. Bars indicate 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: phosphoenolpyruvate, OAA: oxaloacetic acid, X5P: xylul Ros-5-phosphate, Fd: ferredoxin. Silence: ev: empty vector, g2: gltAp2 promoter, z: zwf promoter, x: xylA promoter, u: udhA promoter. Protein degradation: F: fabI-DAS+4, G: gltA-DAS+4, Z: zwf-DAS+4, U: udha_DAS+4, X: xylA-DAS+4. All results were obtained from micro-fermentation.
13 AD : Agarose gel electrophoretic analysis of gRNA array stability. After transformation into host strains engineered for dynamic metabolic regulation, gRNA arrays from 8 clones were amplified using colony PCR. "Guide" indicates that PCR products were taken from sequence-verified gRNA arrays containing 0, 1 or 2 gRNAs. 13A) strain DLF_Z0025, white marking: pCASCADE-ev, yellow marking: pCASCADE-g2, 13B) strain DLF_Z0025, white marking: pCASCADE-z, yellow marking: pCASCADE-fg2, 13C) white marking: strain DLF_Z0044, pCASCADE-fg2, Yellow mark: DLF_Z0025, pCASCADE-fg2, 13D) White mark: strain DLF_Z0046 , pCASCADE-g2, gray mark: DLF_Z1002 pCASCADE-fg2.
Figure 14: Dynamic regulation of FabI (enoyl-ACP reductase) levels due to inducible proteolysis with the DAS+4 degron tag. A C-terminal sfGFP tag was attached to the chromosomal fabI gene. Protein levels were measured by ELISA 24 hours after induction of phosphate depletion in micro-fermentation.
Figure 15 AD: Identification of pathways responsible for NADPH and xylitol production in the "FZ" valve strain. 15A) Effect of deletion of ydbK and fpr on specific xylitol production, 15B) Effect of pntAB overexpression on xylitol production. (15C-D) "FZ" valve strain further modified for dynamic regulation of 15C) GltA levels and 15D) UdhA levels. ev-empty vector, z-zwf promoter, g2-gltAp2 promoter, u-udhA promoter. All results were obtained from micro-fermentation.
Figure 16 AB : Stoichiometric flux model for 16A) cell proliferation and 16B) stationary phase xylitol production in the "FZ" valve strain. Path flux is relative to xylose uptake rate. During proliferation, most of the flux is through the pentose phosphate pathway (PPP), pyruvate dehydrogenase polyenzyme complex (PDH), and minimal flux is through the pentose membrane-bound transhydrogenase. Upon winter regulation, a 4-fold increase in membrane-bound transhydrogenase flux is accompanied by an increase in 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.
Figure 17 AB NADPH production reaction modeling and xylitol production pathway in various production strains. 17A) Specific reaction fluxes during xylitol production. 17B) % pathway flux in xylitol production.
Figure 18 AC: 18A) Control strain expressing xylose reductase (DLF_Z0025, pCASCADE-ev, pHCKan-xyrA), 18B) "FZ" valve strain (DLF_Z0025-fabI-DAS+4-zwf-DAS+4, pCASCADE -z, pHCKan-xyrA), 18C) also overexpressing membrane-bound transhydrogenase pntAB (DLF_Z0025-fabI-DAS+4-zwf-DAS+4, pCASCADE-z, pHCKan-xyrA, pCDF-pntAB) Xylitol production via minimal medium feed batch fermentation in a bioreactor apparatus by the "FZ" valve strain. Biomass (black) and xylitol (blue) are shown as a function of time. In Figures 18B and 18C, x and triangles represent the measured values of experiments performed in duplicate with two plates.
19 depicts plateau phase NADPH pools in engineered strains. Pools were measured 24 hours after phosphate depletion.

The present invention relates to a variety of genetically modified microorganisms having utility in producing xylitol or related chemical products, and to methods for preparing the above chemical products using such microorganisms.

Justice

As used in this specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. That is, for example, reference to “an expression vector” encompasses not only one expression vector but also a plurality of expression vectors, either the same (eg, the same operon) or different; Reference to “a microorganism” encompasses a plurality of microorganisms as well as a single microorganism; Others are like this.

As used herein, the terms "heterologous DNA", "heterologous nucleic acid sequence" and the like refer to a nucleic acid sequence that is one or more of the following: (a) the sequence of the nucleic acid is foreign to a given host microorganism, or (ie, not found naturally in a given host microorganism); (b) the sequence may be found naturally in a given host microorganism, but may be present in non-natural (eg, higher than expected) amounts; or (c) the nucleic acid sequence comprises two or more subsequences that are not found in nature in the same interrelationship. For example, with respect to case (c), the heterologous nucleic acid sequence produced by recombination is derived from non-related genes aligned to create a new functional nucleic acid, such as non-natural promoter driven gene expression. will have more than one sequence. The term "heterologous" is a term commonly used in the art and is intended to encompass "exogenous". An enzyme-encoding nucleic acid sequence is heterologous (whether or not the heterologous nucleic acid sequence has been introduced into the genome) relative to the host microorganism's genome prior to introduction of the heterologous nucleic acid sequence. As used herein, chromosomes and natural and endogenous refer to the genetic material of a host microorganism.

As used herein, the term "synthetic metabolic valve" and the like means altering metabolic flux through the regulation of proteolysis, gene silencing or a combination of proteolysis and gene silencing.

As used herein, the term "gene disruption" or its grammatical equivalents ("to disrupt enzymatic function", "disruption of enzymatic function", etc.) refers to a microbial cell in which the encoded gene product is not modified, or It is intended to mean the genetic modification of a microorganism such that the polypeptide activity is reduced compared to the polypeptide activity derived from that microorganism. Genetic alterations include, for example, deletion of an entire gene, deletion or other alteration of a regulatory sequence necessary for transcription or translation, deletion of a portion of a gene that results in a truncated gene product (eg, enzyme), or a gene that is encoded It can be by any of a variety of mutagenesis strategies that lower the activity of the product (including to undetectable levels of activity). Disruption broadly includes deletion of all or part of the nucleic acid sequence encoding this enzyme, and may also include other types of genetic alterations, such as introduction of stop codons, frameshift mutations, introduction or removal of portions of genes. , and the introduction of degradation signals, but are not limited to, such genetic modifications affect mRNA transcription levels and/or stability, and cause changes in promoters or repressors upstream of genes encoding enzymes.

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

Where genetic modification of a gene product, ie enzyme, is recited herein, such as in a claim, genetic modification is understood to be a genetic modification to a nucleic acid sequence, such as or including a gene that normally encodes the referenced gene product, ie enzyme.

As used herein, the term "metabolic flux" and the like refers to a change in metabolism that results in a change in product and/or by-product production, including production rate, production titer, and production yield from a given substance.

Species and other phylogenetic identifications conform to classifications known to those skilled in the art of microbiology.

Enzymes are described herein by UniProt identification numbers well known to those skilled in the art. The UniProt database can be accessed at http://www.UniProt.org/. Where genetic modification of a gene product, ie enzyme, is recited herein, including in the claims, genetic modification is understood to be a genetic modification to a nucleic acid sequence, such as or including a gene that normally encodes the referenced gene product, ie enzyme.

Where methods and steps recited herein describe specific instances in which a particular order is performed, it will be appreciated by those skilled in the art that the order of the specific steps may be changed and such modifications are in accordance with variations of the present invention. In addition, certain steps may be performed sequentially as well as concurrently in a parallel process where possible.

The meanings of the abbreviations are as follows: "C" means degrees Celsius or degrees Celsius, DCW means dry cell weight, "s" means second(s), and "min" means minute(s), “h”, “hr” or “hrs” means hour(s), “psi” means pounds per square inch, “nm” means nanometers, “d” means one(s), “μL” or “μL” or “ul” means microliter(s), “mL” means milliliter(s), and “L” means liter(s) means (s), “mm” means millimeter(s), “nm” means nanometer, “mM” means millimolar concentration, “uM” or “μM” means micromolar "M" means molar concentration, "mmol" means millimole(s), "μmol" or "μMol" means micromole(s), and "g" means gram(s) , "μg" or "ug" means microgram(s), "ng" means nanogram(s), "PCR" means polymerase chain reaction, and "OD" means means optical density, “OD ₆₀₀ ” means optical density measured at a light wavelength of 600 nm, “kDa” means kilodalton, “g” means gravitational constant, and “bp” means base pair means (s), "kbp" means kilo base pair(s), "% w/v" means weight/volume %, "% v/v" means volume/volume % , "IPTG" means isopropyl-μ-D-thiogalactopyranoside, "aTc" means anhydrous tetracycline, "RBS" means ribosome binding site, and "rpm" means revolutions per minute , "HPLC" means high performance liquid chromatography, and "GC" means gas chromatography.

I. Carbon Source

A bio-production medium used in the present invention using a recombinant microorganism must contain a carbon source or carbon material suitable for both the growth and production stages. Suitable substances may include, but are not limited to, xylose or a combination of xylose and glucose, sucrose, xylose, mannose, arabinose, oil, carbon dioxide, carbon monoxide, methane, methanol, formaldehyde, or glycerol. it is not going to be All of the aforementioned carbon materials and mixtures thereof are considered suitable in the present invention as carbon source(s).

II. microbe

The features described and claimed herein may be provided to a microorganism selected from those listed herein, or other suitable microorganisms, which also include one or more natural, introduced or enhanced product bio-production pathways. Thus, in some embodiments, such microorganism(s) comprise an endogenous product production pathway (which, in some such embodiments, may be enriched), and in other embodiments do not comprise an endogenous product production pathway.

More specifically, based on the various criteria mentioned herein, microbial hosts suitable for bio-producing chemical products include, but are not limited to, the organisms mentioned in the methods section.

The host microorganism or source microorganism for any gene or protein mentioned herein may be selected from the following list of microorganisms: Citrobacter , Enterobacter , Clostridium , Klebsiella ( Klebsiella ), Aerobacter ( Aerobacter ), Lactobacillus ( Lactobacillus ), Aspergillus ( Aspergillus ), Saccharomyces ( Saccharomyces ), Schizosaccharomyces ( Schizosaccharomyces ), Zygosaccharomyces ( Zygosaccharomyces ), blood Kia ( Pichia ), Kluyveromyces ( Kluyveromyces ), Candida ( Candida ), Hansenula ( Hansenula ), Devariomyces ( Debaryomyces ), Mucor ( Mucor ), Torulopsis ( Torulopsis ), Methylobacter ( Methylobacter ), Escherichia , Salmonella , Bacillus , Streptomyces and Pseudomonas . In some aspects, the host microorganism is an E. coli microorganism.

III. Media and culture conditions

In addition to a suitable carbon source, such as one selected from one of the types described herein, a bio-production medium suitable for facilitating growth of cultures and bio-production of chemical products in the present invention is known to those skilled in the art. It should contain known and suitable minerals, salts, cofactors, buffers and other ingredients.

Another aspect of the present invention relates to a medium and culture conditions comprising the genetically modified microorganism of the present invention, and optionally a supplement.

Typically, the cells grow in a suitable medium at temperatures ranging from about 25° C. to about 40° C., as well as up to 70° C. for thermophilic microorganisms. Suitable culture media are well characterized and known in the art. A suitable pH range for bio-production is pH 2.0 to pH 10.0, and pH 6.0 to pH 8.0 in initial conditions is a typical pH range. However, actual culture conditions in certain embodiments are not intended to be limited to these pH ranges. Bio-production can be carried out with or without agitation under aerobic, microaerobic or anaerobic conditions.

IV. Bio-production reactors and production

Fermentation systems using methods and/or compositions according to the present invention are also within the scope of the present invention. Any of the recombinant microorganisms described and/or referenced herein can be introduced into an industrial bio-production system where the microorganism converts a carbon source into a product in a commercially feasible operation. The bio-production system may place such recombinant microorganisms in a bioreactor vessel at an appropriate temperature range for a time suitable to grow the recombinant microorganisms and achieve the desired conversion of the bio-production system to convert some of the substrate molecules into selected chemical products (and in the range of dissolved oxygen if the reaction is aerobic or microaerobic) with a carbon source substrate and a bio-production medium suitable for maintenance. Bio-production can be carried out 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 art of chemical engineering and bioprocess engineering.

The amount of product produced in the bio-production medium is generally measured using a number of methods known in the art, for example high performance liquid chromatography (HPLC), gas chromatography (GC) or GC/mass spectrometry (MS). ) can be used to determine

V. Genetic Modifications, Nucleotide Sequences and Amino Acid Sequences

Embodiments of the invention may result from introducing into the host microorganism an expression vector containing a nucleic acid sequence encoding an enzyme that is or is not normally found in the host microorganism.

The ability of a host cell to genetically modify is essential to producing any genetically modified (recombinant) microorganism. The mode of gene transfer technique can be by electroporation, conjugation, transduction or natural transformation. A wide range of host conjugation plasmids and drug resistance markers are available. Cloning vectors are tailored to the host organism based on the properties of antibiotic resistance markers capable of functioning in the host cell. Also, as described herein, genetically modified (recombinant) microorganisms may include other modifications, such as modifications to the microbial genomic DNA, other than through introduction of a plasmid.

More generally, a nucleic acid construct is a polypeptide having an enzymatic activity operably linked with one or more (several) regulatory sequences that directs expression of a coding sequence in a microorganism, such as E. coli , under conditions compatible with the regulatory sequences. It can be prepared by including an isolated polynucleotide encoding a. An isolated polynucleotide can be engineered to provide expression of the polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide sequence prior to insertion into the vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well established in the art.

The regulatory sequence may be a suitable promoter sequence, ie, a nucleic acid sequence recognized by a host cell for expressing a polynucleotide encoding a polypeptide of the present invention. Promoter sequences may include transcriptional regulatory sequences that mediate expression of the polypeptide. A promoter sequence can be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, including mutant, truncated and hybrid promoters, which can be derived from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. can be obtained. Techniques for modifying and using recombinant DNA promoter sequences are well established in the art.

In various embodiments of the present invention, genetic manipulations may include directed manipulations to alter the control, i.e., the ultimate activity, of an enzyme identified in any given pathway or the enzymatic activity of an enzyme. can Such genetic modifications may be for transcriptional modifications, translational modifications and post-translational modifications, resulting in changes in enzyme activity and/or selectivity under selected culture conditions. Genetic manipulation of nucleic acid sequences may be copy number increase, and/or may involve the use of mutations of enzymes involved in product production. Specific methodologies and approaches for achieving such genetic modifications are well known to those skilled in the art.

In various embodiments, a microorganism may contain one or more genetic deletions in order to function more efficiently. For example, in E. coli , lactate dehydrogenase (ldhA), phosphate acetyltransferase (pta), pyruvate oxidase (poxB), pyruvate-formate lyase (pflB), methylglyoxal synthase (mgsA), acetate kinase (ackA), alcohol dehydrogenase (adhE), clpXP protease specificity enhancer (sspB), ATP-dependent Lon protease (lon), outer membrane protease (ompT), arcA transcriptional dual regulator ( arcA) and the genes encoding the iclR transcriptional regulator (iclR) can be disrupted, including deletion. Genetic disruptions such as deletions are not meant to be limited, and may be implemented in various combinations in various embodiments. Gene deletion can be achieved by a number of strategies well known in the art, such as methods for integrating foreign DNA into the host chromosome.

In various embodiments, a microorganism may contain one or more synthetic metabolic valves composed of enzymes that target regulated proteolysis, expression silencing or a combination of regulated proteolysis and expression silencing in order to operate more efficiently. have. For example, in E. coli one enzyme encoded by one gene or a combination of several enzymes encoded by several genes can be designed as a synthetic metabolic valve to induce a change in metabolism to improve product formation. . Representative genes in E. coli include fabI, zwf, gltA, ppc, udhA, lpd, sucD, aceA, pfkA, lon, rpoS, pykA, pykF, tktA or tktB , but are not limited thereto. It is understood that methods for identifying homologues of these genes and/or other genes in additional microbial species are well known to those skilled in the art.

For all nucleic acid and amino acid sequences provided herein, it is understood that conservatively modified variants of these sequences are included and, in their various embodiments, are within the scope of the present invention. Conservatively modified variants as well as functionally equivalent nucleic acid and amino acid sequences (functional variants) that may contain extensively altered sequences are well within the skill of the person skilled in the art, and microorganisms comprising such sequences and/or or methods and systems involving microorganisms are as such included within the scope of various embodiments of the present invention.

Thus, as described in various sections above, some compositions, methods and systems of the present invention include both production pathways for producing desired products from central intermediates in combination with synthetic metabolic valves for redistributing flux. It includes providing a genetically modified microorganism that does.

Aspects of the present invention also relate to providing a plurality of genetic modifications to improve overall microbial efficiency in converting a select carbon source to a select product. Certain combinations, as in the examples, are found to substantially increase specific productivity, large-scale productivity, titer and yield compared to more basic genetically modified combinations.

In addition to the genetic modification described above, in various embodiments, genetic modification, including synthetic metabolic valves, is also provided to increase the pool and availability of the cofactors NADPH and/or NADH that can be consumed to make a product. .

VI. synthetic metabolic valve

The use of synthetic metabolic valves allows for simpler models of metabolic fluxes and physiological requirements during the production phase that converts proliferating cells to stationary phase biocatalysts. These synthetic metabolic valves can be used to shut down essential genes in a multi-step fermentation process and direct carbon, electron and energy fluxes towards product formation. One or more of the following synthetic valves are provided: 1) 2) inducible and selective enzymatic degradation in combination with transcriptional gene silencing or suppression techniques and 3) nutrient restriction to induce a stationary or non-proliferative state of the cell. . SMV is generalizable in any pathway and microbial hosts. These synthetic metabolic valves enable novel and rapid metabolic engineering strategies useful for producing renewable chemicals and fuels and any product producible by whole-cell catalysis.

In particular, the present invention describes the construction of a synthetic metabolic valve comprising one or more of, or a combination of, regulated gene silencing and regulated proteolysis. It is understood that one skilled in the art will be aware of several methods for gene silencing and controlled protein degradation.

VI.A gene silencing

Specifically, the present invention describes the use of controlled gene silencing to modulate metabolic flux through a controlled multi-stage fermentation process. Several methods are known in the art for modulatory gene silencing, such as mRNA silencing or RNA interference, transcriptional repressor silencing and CRISPR interference, but are not limited thereto. Methods and mechanisms of RNA interference are described in Agrawal et al. "RNA Interference: Biology, Mechanism, and Applications" Microbiology and Molecular Biology Reviews, December 2003; 67(4) p657-685. DOI: 10.1128/MMBR.67.657-685.2003. CRISRPR interference methods and mechanisms are described in Qi et al. "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression" Cell February 2013; 152(5) p1173-1183. DOI: 10.1016/j.cell.2013.02.022. In addition, the CRISRPR interference method and mechanism using the natural E. coli cascade system were described by Luo et al. "Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression" NAR. October 2014; DOI: 10.1093. In addition, a number of transcriptional repressor systems are well known in the art and can be used to shut down gene expression.

VI.B Regulated protein degradation

Specifically, the present invention describes the use of controlled protein degradation or proteolysis to modulate metabolic flux in a controlled multi-stage fermentation process. In the art, several methods are known for regulatory protein denaturation, such as cleavage of a protein target by a specific protease and targeted denaturation of a protein by a specific peptide tag, but are not limited thereto. A system using the E. coli clpXP protease to control protein denaturation is described in McGinness et al, "Engineering controllable protein degradation", Mol Cell. June 2006; 22(5) p701-707. This method is based on adding a specific C-terminal peptide tag, such as a DAS4 (or DAS+4) tag. This tagged protein is not degraded by the clpXP protease until the specificity enhancing chaperone sspB is expressed. sspB induces degradation of DAS4-bound proteins by the clpXP protease. In addition, several site-specific protease systems are well known in the art. A protein can be engineered to contain the specific target site of a given protease and then cleaved after regulated expression of the protease. In some embodiments, cleavage can be expected to result in inactivation or denaturation of the protein. For example, in 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), clpS-dependent It teaches that an N-terminal sequence can be added to a protein of interest to provide clpAP denaturation. In addition, this sequence can also be additionally masked by an additional N-terminal sequence that can be controllably cleaved, such as by ULP hydrolase. This allows controlled N-rule degradation dependent on hydrolase expression. Thus, it is possible to tag proteins at the N-terminus or C-terminus to control protein degradation. The preference for using a C-terminal tag versus an N-terminal tag will largely depend on which tag affects the function of the protein prior to the onset of controlled denaturation.

The present invention describes the use of modulatory protein denaturation or proteolysis to control metabolic flux in a controlled multi-stage fermentation process in E. coli . Several methods are known in the art for controlling protein denaturation in different microbial hosts, including a wide variety of gram-negative and gram-positive bacteria, enzymes and even archaea. Specifically, systems for controlling protein degradation can be delivered from natural microbial hosts and can be used in non-natural hosts. 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 describes the expression of the E. coli clpXP protease and the yeast Saccharomyces cerevisiae. It teaches use in Saccharomyces cerevisiae . Using this approach, methods of synthetic metabolic valves can be delivered to any tractable host.

Regulation of VI.C synthetic metabolic valves

Specifically, the present invention describes the use of synthetic metabolic valves to regulate metabolic flux in multi-stage fermentation processes. A number of methods are known in the art for inducing expression that can be used to transition between stages in a multistage fermentation. These include man-made chemicals such as tetracycline, anhydrous tetracycline, lactose, IPTG (isopropyl-β-D-1-thiogalactopyranoside), arabinose, rapitose, tryptophan, and many others. Inducing substances include, but are not limited to. Systems that link the use of these well-known inducers with modulated gene expression silencing and/or regulated proteolysis can be integrated into genetically modified microbial systems, enabling a multi-stage fermentation process to establish a transition between growth and production phases. transition can be controlled.

In addition, it may be desirable to control the transition between the growth phase and the production phase in a multistage fermentation by depleting one or more limiting nutrients consumed during growth. Limiting nutrients may include, but are not limited to, phosphate, nitrogen, sulfur and magnesium. Using these natural gene expression systems in response to nutrient limitation, regulation of gene expression silencing and/or regulated proteolysis can be operatively linked to the transition between the proliferative and productive phases in a multi-step process.

Within the scope of the present invention, genetically modified microorganisms may contain xylitol greater than 0.05 g/g DCW-hr, greater than 0.08 g/g DCW-hr, greater than 0.1 g/g DCW-hr, greater than 0.13 g/g DCW-hr. greater than hr, greater than 0.15 g/g DCW-hr, greater than 0.175 g/g DCW-hr, greater than 0.2 g/g DCW-hr, greater than 0.25 g/g DCW-hr, 0.3 g/g DCW-hr higher than hr, higher than 0.35 g/g DCW-hr, higher than 0.4 g/g DCW-hr, higher than 0.45 g/g DCW-hr, or higher than 0.5 g/g DCW-hr. A microorganism that can produce at a specific rate.

Within the scope of the present invention, genetically modified microorganisms can convert xylitol from xylose or other sugar sources to greater than 0.5 g product/g xylose, greater than 0.6 g product/g xylose, greater than 0.7 g product/g xylose. , is a microorganism capable of producing yields greater than 0.8 g product/g xylose, greater than 0.9 g product/g xylose, greater than 0.95 g product/g xylose, and greater than 0.98 g product/g xylose.

In various embodiments, the present invention provides a genetically modified microorganism according to any one of the claims herein and a carbon source in an aqueous medium, wherein the genetically modified organism has a volume of aqueous medium greater than 5 mL, for example. , greater than 100 mL, greater than 0.5 L, greater than 1 L, greater than 2 L, greater than 10 L, greater than 250 L, greater than 1000 L, greater than 10,000 L, greater than 50,000 L, greater than 100,000 L ; present in an amount greater than g DCW/L, greater than 5 g DCW/L, greater than 10 g DCW/L, greater than 15 gDCW/L, or greater than 20 gDCW/L.

Overview of Aspects of the Invention

In one aspect, a genetically modified microorganism for producing xylitol is provided. A genetically modified microorganism characterized by an inducible modification of the expression of xylose reductase (xyrA) and an inducible synthetic metabolic valve is provided. Synthetic metabolic valves include gene expression-silencing synthetic metabolic valves characterized by silencing of gene expression of one or more genes encoding one or more enzymes; or an enzymatically modified synthetic metabolic valve characterized by induction of enzymatic modification of one or more enzymes, or a combination thereof.

In one aspect, the xylose reductase of the genetically modified microorganism is a NADPH dependent xylose reductase or potentially the xyrA gene of Aspergillus niger ( A. niger ) xylose reductase.

In one aspect, the genetically modified microorganism produces xylitol from a xylose feedstock. Of course, the genetically modified microorganism may use a feedstock in which xylose and a second sugar are mixed in any ratio.

In one aspect, the gene-silent synthetic metabolic valve or enzyme-modified synthetic metabolic valve of a genetically modified microorganism comprises xylose isomerase or a gene encoding a xylose isomerase enzyme; or the regulation of a gene encoding a glucose-6-phosphate dehydrogenase ( zwf ) or glucose-6-phosphate dehydrogenase ( zwf ) enzyme.

In one aspect, the gene-silent synthetic metabolic valve or enzyme-modified synthetic metabolic valve of the genetically modified microorganism is two or more genes, such as glucose-6-phosphate dehydrogenase ( zwf ) or glucose-6-phosphate dehydrogenase the gene encoding the genase ( zwf ) enzyme; and regulation of xylose isomerase or a gene encoding a xylose isomerase enzyme.

In another aspect, in one aspect, the gene-silent synthetic metabolic valve or enzyme-modified synthetic metabolic valve of the genetically modified microorganism is two or more genes, for example, glucose-6-phosphate dehydrogenase ( zwf ) or glucose -6-phosphate dehydrogenase ( zwf ) gene encoding enzyme; and regulation of genes encoding enoyl-ACP reductase ( fabI ) or enoyl-ACP reductase ( fabI ) enzymes.

In another aspect, in one aspect, the gene-silenced synthetic metabolic valve or enzyme-modified synthetic metabolic valve of the genetically modified microorganism is silencing the gene encoding glucose-6-phosphate dehydrogenase ( zwf ), and glucose-6 -phosphate dehydrogenase ( zwf ) enzyme; and regulation of enzymatic denaturation of the enoyl-ACP reductase ( fabI ) enzyme.

In another aspect, expression of xylose reductase, gene expression-silenced synthetic metabolic valve, and enzymatically modified synthetic metabolic valve are induced under conditions of a transition phrase of a multi-step biofermentation process. Induction can occur by nutrient depletion or phosphate depletion.

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

In one aspect, the silencing of gene expression comprises CRISPR interference, and the genetically modified microorganism also expresses a cascade guide array, wherein the array has each small guide specific for targeting different genes for simultaneous silencing of multiple genes. It contains two or more genes encoding RNA.

In one aspect, the genetically modified microorganism produces xylitol at a titer greater than 0.08 g/L in 24 hours in a biofermentation process.

In one aspect, the present invention provides a multi-step fermentation bioprocess for producing xylitol from a genetically modified microorganism comprising the step of (a) providing a genetically modified microorganism. Genetically modified microorganisms are characterized by altered expression of xylose reductase and synthetic metabolic valves, which include gene expression-silenced synthetic metabolic valves characterized by silencing of expression of one or more genes encoding one or more enzymes; or an enzymatically modified synthetic metabolic valve characterized by induction of enzymatic modification of one or more enzymes, or a combination thereof. One or more enzymes of each synthetic metabolic valve may be the same or different. The method further comprises growing the genetically modified microorganism in a medium containing the xylose feedstock, and transitioning from the growing phase to the xylitol producing phase. The transition phase induces the synthetic metabolic valve(s) to slow or stop the growth of the microbe; and inducing expression of xylose reductase to produce xylitol.

In some aspects, the multi-step fermentation bioprocess comprises a gene-silent synthetic metabolic valve or an enzyme-modified synthetic metabolic valve encoding a glucose-6-phosphate dehydrogenase ( zwf ) or glucose-6-phosphate dehydrogenase ( zwf ) enzyme. a gene that does; and enoyl-ACP reductase ( fabI ) or a gene encoding an enoyl-ACP reductase ( fabI ) enzyme, characterized by directing the regulation of two or more genes, genetically modified microorganisms can be used.

In some aspects, a multi-stage fermentation bioprocess will produce titers greater than 0.08 g/L of xylitol at 24 hours in the biofermentation process.

In some aspects, the transition phase of a multi-stage fermentation bioprocess is caused by phosphate depletion of the culture medium. In some aspects, the genetically modified microorganisms of the multi-step fermentation bioprocess are further characterized by chromosomal deletions.

In one aspect, a microorganism genetically modified to produce xylitol is such that, when the microorganism is induced, xylitol is produced from feedstock xylose by reducing the inducibility of xylose isomerase; inducible reduction of glucose-6-phosphate dehydrogenase activity. In another aspect, the microorganism is an E. coli microorganism. In one aspect, induction of microorganisms occurs by nutrient depletion. In one aspect, induction of microorganisms occurs by phosphate depletion.

In one aspect, the present invention provides a multi-step fermentation biological process for producing xylitol from a genetically modified microorganism comprising inducible reduction of xylose isomerase and inducible reduction of glucose-6-phosphate dehydrogenase activity. do. The bioprocess includes (a) providing a genetically modified microorganism, (b) growing the genetically modified microorganism in a medium containing a xylose feedstock; (c) transitioning from the proliferative phase to the xylitol producing phase by inducing the synthetic metabolic valve(s) to slow or stop the growth of the microorganism; and inducing expression of xylose isomerase to (d) produce xylitol.

In one aspect, a microorganism genetically modified for producing xylitol has reduced inducibility of xylose reductase; an inducible decrease in glucose-6-phosphate dehydrogenase activity; including inducible reduction of enoyl-ACP reductase; Here, the strain produces xylitol from feedstock xylose upon induction. In one aspect, the microorganism is an E. coli microorganism. In some aspects, induction of microorganisms occurs by nutrient depletion or phosphate depletion.

In one aspect, the present invention reduces the inducibility of xylose reductase; an inducible decrease in glucose-6-phosphate dehydrogenase activity; A multi-step fermentation bioprocess for producing xylitol from genetically modified microorganisms comprising inducible reduction of enoyl-ACP reductase is provided. The bioprocess includes (a) providing a genetically modified microorganism; (b) growing the genetically modified microorganism in a medium containing a xylose feedstock; (c) transitioning from the proliferative phase to the xylitol producing phase by inducing the synthetic metabolic valve(s) to slow or stop the growth of the microorganism; and (d) producing xylitol by inducing expression of xylose reductase.

In one aspect, a microorganism genetically modified to produce xylitol includes increased activity of membrane-bound transhydrogenase; Increased activity of pyruvate ferredoxin oxidoreductase; NADPH-dependent increase in the activity of ferredoxin reductase, wherein the microorganism produces one or more chemical products that require NADPH for biosynthesis.

The disclosed embodiments are non-limiting.

Although various embodiments of the present invention have been shown and mentioned herein, it is emphasized that these embodiments are provided as simplistic examples. Numerous modifications, changes and substitutions may be made in various embodiments without departing from the invention. Specifically, for any reason, a particular protein, including a compound, nucleic acid sequence, functional enzyme, metabolic pathway enzyme or intermediate, mentioned or otherwise presented herein in a list, table or other grouping (eg, metabolic pathway enzymes shown in FIGS . 1 and 4 ). In any group for a polypeptide, factor or other composition, or concentration, unless expressly stated otherwise, each such group is defined by excluding one or more members (or subsets) of each recited group. It is intended to serve to identify and provide a basis for various subset embodiments in the broadest sense, including all subsets of the group. In addition, where any range is set forth herein, the range encompasses all numbers and all subranges subsumed therein, unless expressly stated otherwise.

Also, more generally, conventional molecular biology, cell biology, microbiology and recombinant DNA techniques within the skill of the art may be employed in the description, discussion, examples and embodiments herein. These techniques are fully described in the literature. See, eg, Sambrook and Russell, "Molecular Cloning: A Laboratory Manual", Third Edition 2001 (volumes 1 - 3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; See Animal Cell Culture, R. I. Freshney, ed., 1986. These published materials are incorporated herein by reference.

The material published below is based on the content available in connection with the invention described herein, e.g., industrial bio-production methods of chemical product(s) from sugar sources and industrial systems available to achieve this conversion. (Biochemical Engineering Fundamentals, 2 ^nd Ed. JE Bailey and DF Ollis, McGraw Hill, New York, 1986, e.g. on biological reactor design, Chapter 9, pages 533-657; Unit Operations of Chemical Engineering, 5 ^th Ed., WL McCabe et al., McGraw Hill, New York 1993, eg for analysis of process and separation techniques; Equilibrium Staged Separations, PC Wankat, Prentice Hall, Englewood Cliffs, NJ USA, 1988, eg, for teachings on separation techniques).

All publications, patents and patent applications mentioned herein are U.S. Provisional Application No. 62/010,574, filed June 11, 2014, U.S. Provisional Application No. 62/461,436, filed February 21, 2017, and PCT/US2015/035306, filed June 11, 2015. and PCT/US2018/019040, dated Feb. 21, 2018, the entirety of which is incorporated herein by reference.

Example

This embodiment provides some examples, but is not meant to be limiting. All reagents are purchased commercially unless otherwise noted. Species and other phylogenetic identifications follow classifications known to those skilled in the microbiology, molecular biology, and biochemistry arts.

common method

reagents and media

All reagents and chemicals were purchased from Sigma Aldrich (St. Louis, Mo.) unless otherwise noted. MOPS (3-(N-morpholino)propanesulfonic acid) is manufactured by 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 published (Menacho -Melgar, R. et al. Scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion. Biotechnol. Bioeng. 26 , 44 (2020)). LB, Lennox formulation was used for routine strain growth. Working antibiotic concentrations were as follows: kanamycin: 35 μg/mL, chloramphenicol: 35 μg/mL, gentamicin: 10 μg/mL, 10 zeoxin: 100 μg/mL, blasticidin: 100 μg/mL, specifications Actinomycin: 25 μg/mL, Tetracycline: 5 μg/mL.

Strains & Plasmid Construction

See Appendix Table S1 for a list of strains and plasmids used in this experiment. The synthetic DNA sequences used in this experiment are presented in Appendix Table S2. Chromosomal alterations were constructed using standard recombination engineering methods (Liochev, SI et al Proc. Natl. Acad. Sci. USA 91 , 1328-1331 (1994)). Plasmid ^pSIM5 for recombination engineering was kindly gifted from Donald Court (NCI, https://redrecombineering.ncifcrf.gov/court-lab.html) 53,54. As previously described, the C-terminal DAS+4 tag was added by direct integration and selected through integration of genes into the antibiotic resistance cassette 3'. All strains were verified by PCR, agarose gel electrophoresis, and verified by sequencing. Oligos used for strain validation and sequencing see Table S3.

The xyrA gene of Aspergillus niger was codon-optimized to be expressed in E. coli , and a plasmid capable of inducing xylose reductase with a low concentration of phosphate, pHCKan-xyrA (Addgene #58613), was developed by TWIST Biosciences (San Francisco, CA). ) was built from. pCDF-pntAB (Addgene # 158609) was constructed from pCDF-ev 30 using PCR and Gibson assembly to drive expression of the pntAB operon from the ugpBp promoter inducible by low concentrations of phosphate (Moreb, EA 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). The pCASCADE guide RNA array plasmid was prepared as previously described by a combination of PCR and Gibson assembly. See Table S4 for the oligos used to construct the pCASCADE plasmid.

Table 1: Plasmids used in the examples:

plasmid promoter Ori Res Addgene
number source pSMART-HC-Kan None colE1 Kan NA Lucigen pHC-Kan-yibDp-xyrA yibDp colE1 Kan TBD Existing experimental studies pCASCADE-EV ugpBp p15A cm TBD Existing experimental studies pCASCADE-gltA1 ugpBp p15A cm TBD Existing experimental studies pCASCADE-gltA2 ugpBp p15A cm 65817 Existing experimental studies pCASCADE-zwf ugpBp p15A cm 65825 Existing experimental studies pCAcade-udhA ugpBp p15A cm 65818 Existing experimental studies pCASCADE-xylA ugpBp p15A cm TBD Existing experimental studies pCASCADE-gltA1-zwf ugpBp p15A cm TBD Existing experimental studies pCASCADE-gltA2-zwf ugpBp p15A cm TBD Existing experimental studies pCASCADE-gltA1-udhA ugpBp p15A cm TBD Existing experimental studies pCAcade-gltA2-udhA ugpBp p15A cm 65819 Existing experimental studies pCASCADE-gltA1-gltA2 ugpBp p15A cm TBD Existing experimental studies pCASCADE-gltA1-gltA2-zwf ugpBp p15A cm TBD Existing experimental studies pCASCADE-gltA1-gltA2-udhA ugpBp p15A cm TBD Existing experimental studies pCASCADE-zwf-xylA ugpBp p15A cm TBD this experiment pCASCADE-udhA-xylA ugpBp p15A cm TBD this experiment pCASCADE-gltA1-xylA ugpBp p15A cm TBD this experiment pCASCADE-gltA2-xylA ugpBp p15A cm TBD this experiment pCASCADE-gltA1-zwf-xylA ugpBp p15A cm TBD this experiment pCASCADE-gltA2-zwf-xylA ugpBp p15A cm TBD this experiment pCASCADE-gltA1-udhA-xylA ugpBp p15A cm TBD this experiment pCAcade-gltA2-udhA-xylA ugpBp p15A cm TBD this experiment pCASCADE-gltA1-gltA2-xylA ugpBp p15A cm TBD this experiment pCASCADE-gltA1-gltA2-zwf-xylA ugpBp p15A cm TBD this experiment pCASCADE-gltA1-gltA2-udhA-xylA ugpBp p15A cm TBD this experiment

Table S1: Additional plasmids used in the examples :

plasmid insertion promoter Ori Res Addgene source pSMART-HC-Kan doesn't exist doesn't exist colE1 Kan NA Lucigen pCDF-ev doesn't exist doesn't exist cloDF13 SM 89596 Existing experimental studies pHCKan-xyrA xyrA yibDp ² colE1 Kan 158610 this experiment pCDF-pntAB pntAB ugpBp ² cloDF13 SM 158609 this experiment pCASCADE-ev doesn't exist ugpBp ² p15 cm 65821 Existing experimental studies pCASCADE-g2 gltAp2 gRNAs ugpBp ² p15 cm 65817 Existing experimental studies pCASCADE-f fabIp gRNA ugpBp ² p15 cm 66635 this experiment pCASCADE-z zwfp gRNAs ugpBp ² p15 cm 65825 Existing experimental studies pCASCADE-u udhAp gRNAs ugpBp ² p15 cm 65818 this experiment pCASCADE-x xylAp gRNAs ugpBp ² p15 cm 158611 this experiment pCASCADE-g2z gltAp2, zwfp gRNA array ugpBp ² p15 cm 71338 Existing experimental studies pCASCADE-g2u gltAp2 udhAp gRNA array ugpBp ² p15 cm 65819 this experiment pCASCADE-g2x gltAp2, xylAp gRNA array ugpBp ² p15 cm 158613 this experiment pCASCADE-zx zwfp xylAp gRNA array ugpBp ² p15 cm 158614 this experiment pCASCADE-ux udhAp, xylAp gRNA array ugpBp ² p15 cm 158612 this experiment pCASCADE-fg2 fabIp, gltAp2 gRNA array ugpBp ² p15 cm 71341 this experiment pCASCADE-fz fabIp, zwf gRNA array ugpBp ² p15 cm 71335 this experiment

Table 2: Host strains used in the examples:

strain Abbreviation for proteolysis genotype source DLF_0025 control/none F-, λ-, Δ(araD-araB)567, lacZ4787(del)(::rrnB-3), rph-1, Δ(rhaD-rhaB)568, hsdR514, ΔackA-pta, ΔpoxB, ΔpflB, ΔldhA, ΔadhE, ΔsspB, ΔiclR, ΔarcA, Δcas3::tm-ugpb-sspB-pro [casA*] Existing experimental studies DLF_0025-X X DLF_0025, xylA-DAS+4-ampR this experiment DLF_0025-F F DLF_0025, fabI-DAS+4-gentR Existing experimental studies DLF_0025-G G DLF_0025, gltA-DAS+4-zeoR Existing experimental studies DLF_0025-Z Z F_0025, zwf-DAS+4-bsdR Existing experimental studies DLF_0025-U U DLF_0025, udhA-DAS+4-bsdR Existing experimental studies DLF_0025-GU GU DLF_0025, gltA-DAS+4-zeoR, udhA-DAS+4-bsdR Existing experimental studies DLF_0025-GZ GZ DLF_0025, gltA-DAS+4-zeoR, zwf-DAS+4-bsdR Existing experimental studies DLF_0025-FG FG DLF_0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR Existing experimental studies DLF_0025-FZ FZ DLF_0025, fabI-DAS+4-gentR, zwf-DAS+4-bsdR Existing experimental studies DLF_0025-FU FU DLF_0025, fabI-DAS+4-gentR, udhA-DAS+4-bsdR Existing experimental studies DLF_0025-FGU FGU DLF_0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR, udhA-DAS+4-bsdR Existing experimental studies DLF_0025-FGZ FGZ DLF_0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR, zwf-DAS+4-bsdR Existing experimental studies DLF_0025-FX FX DLF_0025, fabI-DAS+4-gentR, xylA-DAS+4-ampR this experiment DLF_0025-FGX FGX DLF_0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR, xylA-DAS+4-ampR this experiment DLF_0025-FZX FZX DLF_0025, fabI-DAS+4-gentR, zwf-DAS+4-bsdR, xylA-DAS+4-ampR this experiment DLF_0025-FUX FUX DLF_0025, fabI-DAS+4-gentR, udhA-DAS+4-bsdR, xylA-DAS+4-ampR this experiment DLF_0025-FGUX FGUX DLF_0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR, udhA-DAS+4-bsdR, xylA-DAS+4-ampR this experiment DLF_0025-FGZX FGZX DLF_0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR, zwf-DAS+4-bsdR, xylA-DAS+4-ampR this experiment DLF_0025-UX UX DLF_0025, udhA-DAS+4-bsdR, xylA-DAS+4-ampR this experiment DLF_0025-ZX ZX DLF_0025, zwf-DAS+4-bsdR, xylA-DAS+4-ampR this experiment

Table S2: Additional Strains Used in Examples:

strain genotype source DLF_Z0025 F-, λ-, Δ(araD-araB)567, lacZ4787(del)(::rrnB-3), rph-1, Δ(rhaD-rhaB)568, hsdR514, ΔackA-pta, ΔpoxB, ΔpflB, ΔldhA, ΔadhE, ΔiclR, ΔarcA, ΔsspB,
Δcas3::tm-ugpb-sspB-pro-casA. Existing experimental studies DLF_Z0043 DLF_Z0025, gltA-DAS+4-zeoR Existing experimental studies DLF_Z1002 DLF_Z0025, zwf-DAS+4-bsdR Existing experimental studies DLF_Z0044 DLF_Z0025, gltA-DAS+4-zeoR, zwf-DAS+4-bsdR Existing experimental studies DLF_Z0028 DLF_Z0025, fabI-DAS+4-gentR this experiment DLF_Z0028G DLF_Z0025, fabI-sfGFP-gentR this experiment DLF_Z0028GD DLF_Z0025, fabI-sfGFP-DAS+4-gentR this experiment DLF_Z0763 DLF_Z0025, udhA-DAS+4-bsdR this experiment DLF_Z0039 fDLF_Z0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR this experiment DLF_Z0040 DLF_Z0025, fabI-DAS+4-gentR, zwf-DAS+4-bsdR this experiment DLFZ_0045 DLF_Z0025, fabI-DAS+4-gentR, udhA-DAS+4-bsdR this experiment DLFZ_0046 DLF_Z0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR, zwf-DAS+4-bsdR this experiment DLFZ_0047 DLF_Z0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR, udhA-DAS+4-bsdR this experiment SL_0001 DLF_Z0025, xylA-DAS+4-ampR this experiment SL_0002 DLF_Z0025, fabI-DAS+4-gentR, xylA-DAS+4-ampR this experiment SL_0003 DLF_Z0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR, xylA-DAS+4-ampR this experiment SL_0004 DLF_Z0025, fabI-DAS+4-gentR, zwf-DAS+4-bsdR, xylA-DAS+4-ampR this experiment SL_0005 DLF_Z0025, fabI-DAS+4-gentR, udhA-DAS+4-bsdR, xylA-DAS+4-ampR this experiment SL_0006 DLF_Z0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR,udhA-DAS+4-bsdR, xylA-DAS+4-ampR this experiment SL_0007 DLF_Z0025, fabI-DAS+4-gentR, gltA-DAS+4-zeoR, zwf-DAS+4-bsdR, xylA-DAS+4-ampR this experiment SL_0008 DLF_Z0025, gltA-DAS+4-zeoR, udhA-DAS+4-bsdR this experiment SL_0009 DLF_Z0025, zwf-DAS+4,-bsdR xylA -DAS+4-ampR this experiment SL_0010 DLF_Z0025, fabI-DAS+4-gentR, zwf-DAS+4-bsdR, ΔydbK this experiment SL_0011 DLF_Z0025, fabI-DAS+4-gentR, zwf-DAS+4-bsdR, Δfpr this experiment Ori - origin of replication, Res - resistance marker, Sm - spectinomycin, Cm - chloramphenicol, Kan - kanamycin, Amp - ampicillin

Chromosomal alterations were constructed using standard recombination engineering methods. A C-terminal DAS+4 tag on the xylA gene was added by direct integration and selection was made by integrating an antibiotic resistance cassette 3' of the gene. All strains were verified by PCR, agarose gel electrophoresis and sequencing.

Table 3: Synthetic DNA Sequences:

xylA-DAS4-ampR GATACGATGGCACTGGCGCTGAAAATTGCAGCGCGCATGATTGAAGATGGCGAGCTGGATAAACGCATCGCGCAGCGTTATTCCGGCTGGAATAGCGAATTGGGCCAGCAAATCCTGAAAGGCCAAATGTCACTGGCAGATTTAGCCAAATATGCTCAGGAACATCATTTGTCTCCGGTGCATCAGAGTGGTCGCCAGGAACAACTGGAAAATCTGGTAAACCATTATCTGTTCGACAAAGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATGATAAGGACCGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTACGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTCACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGGCAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCG GATAAAGTTGCAGGATCACTTCTGCGCTCGGCCCTCCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGCATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAGTAAGTAGGGATAACAGGGTAATCGGCTAACTGTGCAGTCCGTTGGCCCGGTTATCGGTAGCGATACCGGGCATTTTTTTAAGGAACGATCGATATGTATATCGGGATAGATCTTGGCACCTCGGGCGTAAAAGTTATTTTGCTCAACGAGCAGGGTGAGGTGGTTGCTGCGCAAACGGAAAAGCTGACCGTTTCGCGCCCGCATCCACTCTGGTCGGAACAAGACCCGGAACAGTGGTGGCAGGCAACTGATCGCGCAA (서열번호 1) fabI-DAS+4-gentR CTATTGAAGATGTGGGTAACTCTGCGGCATTCCTGTGCTCCGATCTCTCTGCCGGTATCTCCGGTGAAGTGGTCCACGTTGACGGCGGTTTCAGCATTGCTGCAATGAACGAACTCGAACTGAAAGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATAGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCCGAATCCATGTGGGAGTTTATTCTTGACACAGATATTTATGATATAATAACTGAGTAAGCTTAACATAAGGAGGAAAAACATATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAAGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAAGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCCGTTCTGTTGGTAAAGATGGGCGGCGTTCTGCCGCCCGTTATCTCTGTTATACCTTTCTGATATTTGTTATCGCCGATCCGTCTTTCTCCCCTTCCCGCCTTGCGTCAGG(서열번호 2) gltA-DAS+4-zeoR GTATTCCGTCTTCCATGTTCACCGTCATTTTCGCAATGGCACGTACCGTTGGCTGGATCGCCCACTGGAGCGAAATGCACAGTGACGGTATGAAGATTGCCCGTCCGCGTCAGCTGTATACAGGATATGAAAAACGCGACTTTAAAAGCGATATCAAGCGTGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATAGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACTCACTATAGGAGGGCCATCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTTGTGGCAGAGGAGCAGGACTGAGGATAAGTAATGGTTGATTGCTAAGTTGTAAATATTTTAACCCGCCGTTCATATGGCGGGTTGATTTTTATATGCCTAAACACAAAAAATTGTAAAAATAAAATCCATTAACAGACCTATATAGATATTTAAAAAGAATAGAACAGCTCAAATTATCAGCAACCCAATACTTTCAATTAAAAACTTCATGGTAGTCGCATTTATAACCCTATGAAA(서열번호 3) udhA-DAS+4-bsdR TCTGGGTATTCACTGCTTTGGCGAGCGCGCTGCCGAAATTATTCATATCGGTCAGGCGATTATGGAACAGAAAGGTGGCGGCAACACTATTGAGTACTTCGTCAACACCACCTTTAACTACCCGACGATGGCGGAAGCCTATCGGGTAGCTGCGTTAAACGGTTTAAACCGCCTGTTTGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATAGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACTCACTATAGGAGGGCCATCATGAAGACCTTCAACATCTCTCAGCAGGATCTGGAGCTGGTGGAGGTCGCCACTGAGAAGATCACCATGCTCTATGAGGACAACAAGCACCATGTCGGGGCGGCCATCAGGACCAAGACTGGGGAGATCATCTCTGCTGTCCACATTGAGGCCTACATTGGCAGGGTCACTGTCTGTGCTGAAGCCATTGCCATTGGGTCTGCTGTGAGCAACGGGCAGAAGGACTTTGACACCATTGTGGCTGTCAGGCACCCCTACTCTGATGAGGTGGACAGATCCATCAGGGTGGTCAGCCCCTGTGGCATGTGCAGAGAGCTCATCTCTGACTATGCTCCTGACTGCTTTGTGCTCATTGAGATGAATGGCAAGCTGGTCAAAACCACCATTGAGGAACTCATCCCCCTCAAGTACACCAGGAACTAAAGTAAAACTTTATCGAAATGGCCATCCATTCTTGCGCGGATGGCCTCTGCCAGCTGCTCATAGCGGCTGCGCAGCGGTGAGCCAGGACGATAAACCAGGCCAATAGTGCGGCGTGGTTCCGGCTTAATGCACGG(서열번호 4) zwf-DAS+4-bsdR GAAGTGGAAGAAGCCTGGAAATGGGTAGACTCCATTACTGAGGCGTGGGCGATGGACAATGATGCGCCGAAACCGTATCAGGCCGGAACCTGGGGACCCGTTGCCTCGGTGGCGATGATTACCCGTGATGGTCGTTCCTGGAATGAGTTTGAGGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATAGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACTCACTATAGGAGGGCCATCATGAAGACCTTCAACATCTCTCAGCAGGATCTGGAGCTGGTGGAGGTCGCCACTGAGAAGATCACCATGCTCTATGAGGACAACAAGCACCATGTCGGGGCGGCCATCAGGACCAAGACTGGGGAGATCATCTCTGCTGTCCACATTGAGGCCTACATTGGCAGGGTCACTGTCTGTGCTGAAGCCATTGCCATTGGGTCTGCTGTGAGCAACGGGCAGAAGGACTTTGACACCATTGTGGCTGTCAGGCACCCCTACTCTGATGAGGTGGACAGATCCATCAGGGTGGTCAGCCCCTGTGGCATGTGCAGAGAGCTCATCTCTGACTATGCTCCTGACTGCTTTGTGCTCATTGAGATGAATGGCAAGCTGGTCAAAACCACCATTGAGGAACTCATCCCCCTCAAGTACACCAGGAACTAAAGTAATATCTGCGCTTATCCTTTATGGTTATTTTACCGGTAACATGATCTTGCGCAGATTGTAGAACAATTTTTACACTTTCAGGCCTCGTGCGGATTCACCCACGAGGCTTTTTTTATTACACTGACTGAAACGTTTTTGCCCTATGAGCTCCGGTTACAGGCGTTTCAGTCATAAATCCTCTGAATGAAACGCGTTGTGAATC(서열번호 5)

Table S3: Additional synthetic DNA:

xylA-DAS4-ampR GATACGATGGCACTGGCGCTGAAAATTGCAGCGCGCATGATTGAAGATGGCGAGCTGGATAAACGCATCGCGCAGCGTTATTCCGGCTGGAATAGCGAATTGGGCCAGCAAATCCTGAAAGGCCAAATGTCACTGGCAGATTTAGCCAAATATGCTCAGGAACATCATTTGTCTCCGGTGCATCAGAGTGGTCGCCAGGAACAACTGGAAAATCTGGTAAACCATTATCTGTTCGACAAAGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATGATAAGGACCGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTACGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTCACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGGCAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCG GATAAAGTTGCAGGATCACTTCTGCGCTCGGCCCTCCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGCATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAGTAAGTAGGGATAACAGGGTAATCGGCTAACTGTGCAGTCCGTTGGCCCGGTTATCGGTAGCGATACCGGGCATTTTTTTAAGGAACGATCGATATGTATATCGGGATAGATCTTGGCACCTCGGGCGTAAAAGTTATTTTGCTCAACGAGCAGGGTGAGGTGGTTGCTGCGCAAACGGAAAAGCTGACCGTTTCGCGCCCGCATCCACTCTGGTCGGAACAAGACCCGGAACAGTGGTGGCAGGCAACTGATCGCGCAA (서열번호 1) fabI-DAS+4-gentR
GCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAAGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCCGTTCTGTTGGTAAAGATGGGCGGCGTTCTGCCGCCCGTTATCTCTGTTATACCTTTCTGATATTTGTTATCGCCGATCCGTCTTTCTCCCCTTCCCGCCTTGCGTCAGG(서열번호 2) fabI-sfGFP-gentR AAAGACTTCCGCAAAATGCTGGCTCATTGCGAAGCCGTTACCCCGATTCGCCGTACCGTTACTATTGAAGATGTGGGTAACTCTGCGGCATTCCTGTGCTCCGATCTCTCTGCCGGTATCTCCGGTGAAGTGGTCCACGTTGACGGCGGTTTCAGCATTGCTGCAATGAACGAACTCGAACTGAAAGGGGGTTCAGGCGGGTCGGGTGGCgtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgcgcggcgagggcgagggcgatgccaccaacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcgccacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcagcttcaaggacgacggcacctacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaacttcaacagccacaacgtctatatcaccgccgacaagcagaagaacggcatcaaggccaacttcaagatccgccacaacgtggaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgtgctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactcacggcatggacgagctgtacaagTAATGACGAATCCATGTGGGAGTTTATTCTTGACACAGATATTTATGATATAATAACTGAGTAAGCTTAACATAAG GAGGAAAAACATATGTTGCGTAGCTCTAACGATGTGACGCAACAAGGTTCGCGTCCAAAGACAAAATTGGGAGGCAGTAGCATGGGGATCATTCGCACTTGTCGCCTGGGGCCAGACCAGGTGAAGTCAATGCGTGCGGCTCTGGACTTATTCGGGCGCGAATTTGGAGATGTAGCCACTTACTCACAGCACCAACCGGACAGTGATTACTTGGGGAATTTACTTCGCAGTAAAACTTTTATCGCTTTGGCCGCTTTCGACCAGGAGGCTGTAGTAGGTGCGTTGGCAGCCTATGTTCTTCCTAAATTCGAGCAACCGCGTAGCGAAATTTACATCTATGATCTTGCAGTCTCCGGCGAACATCGCCGTCAGGGGATCGCCACAGCTTTAATCAACCTTTTGAAGCATGAGGCTAATGCACTTGGAGCGTACGTGATTTATGTGCAGGCTGACTACGGTGATGATCCTGCAGTCGCTCTGTACACCAAACTGGGTATCCGCGAGGAGGTCATGCACTTTGATATTGACCCGTCGACGGCTACCTAAGTTCTGTTGGTAAAGATGGGCGGCGTTCTGCCGCCCGTTATCTCTGTTATACCTTTCTGATATTTGTTATCGCCGATCCGTCTTTCTCCCCTTCCCGCCTTGCGTCAGG(서열번호 21) fabI-sfGFP-DAS+4-gentR AAAGACTTCCGCAAAATGCTGGCTCATTGCGAAGCCGTTACCCCGATTCGCCGTACCGTTACTATTGAAGATGTGGGTAACTCTGCGGCATTCCTGTGCTCCGATCTCTCTGCCGGTATCTCCGGTGAAGTGGTCCACGTTGACGGCGGTTTCAGCATTGCTGCAATGAACGAACTCGAACTGAAAGGGGGTTCAGGCGGGTCGGGTGGCgtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgcgcggcgagggcgagggcgatgccaccaacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcgccacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcagcttcaaggacgacggcacctacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaacttcaacagccacaacgtctatatcaccgccgacaagcagaagaacggcatcaaggccaacttcaagatccgccacaacgtggaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgtgctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactcacggcatggacgagctgtacaagGGTGGGGGTGGGAGCGGCGGCGGTGGCTCCGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTT AATGACGAATCCATGTGGGAGTTTATTCTTGACACAGATATTTATGATATAATAACTGAGTAAGCTTAACATAAGGAGGAAAAACATATGTTGCGTAGCTCTAACGATGTGACGCAACAAGGTTCGCGTCCAAAGACAAAATTGGGAGGCAGTAGCATGGGGATCATTCGCACTTGTCGCCTGGGGCCAGACCAGGTGAAGTCAATGCGTGCGGCTCTGGACTTATTCGGGCGCGAATTTGGAGATGTAGCCACTTACTCACAGCACCAACCGGACAGTGATTACTTGGGGAATTTACTTCGCAGTAAAACTTTTATCGCTTTGGCCGCTTTCGACCAGGAGGCTGTAGTAGGTGCGTTGGCAGCCTATGTTCTTCCTAAATTCGAGCAACCGCGTAGCGAAATTTACATCTATGATCTTGCAGTCTCCGGCGAACATCGCCGTCAGGGGATCGCCACAGCTTTAATCAACCTTTTGAAGCATGAGGCTAATGCACTTGGAGCGTACGTGATTTATGTGCAGGCTGACTACGGTGATGATCCTGCAGTCGCTCTGTACACCAAACTGGGTATCCGCGAGGAGGTCATGCACTTTGATATTGACCCGTCGACGGCTACCTAAGTTCTGTTGGTAAAGATGGGCGGCGTTCTGCCGCCCGTTATCTCTGTTATACCTTTCTGATATTTGTTATCGCCGATCCGTCTTTCTCCCCTTCCCGCCTTGCGTCAGG(서열번호 22) udhA-DAS+4-bsdR TCTGGGTATTCACTGCTTTGGCGAGCGCGCTGCCGAAATTATTCATATCGGTCAGGCGATTATGGAACAGAAAGGTGGCGGCAACACTATTGAGTACTTCGTCAACACCACCTTTAACTACCCGACGATGGCGGAAGCCTATCGGGTAGCTGCGTTAAACGGTTTAAACCGCCTGTTTGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATAGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACTCACTATAGGAGGGCCATCATGAAGACCTTCAACATCTCTCAGCAGGATCTGGAGCTGGTGGAGGTCGCCACTGAGAAGATCACCATGCTCTATGAGGACAACAAGCACCATGTCGGGGCGGCCATCAGGACCAAGACTGGGGAGATCATCTCTGCTGTCCACATTGAGGCCTACATTGGCAGGGTCACTGTCTGTGCTGAAGCCATTGCCATTGGGTCTGCTGTGAGCAACGGGCAGAAGGACTTTGACACCATTGTGGCTGTCAGGCACCCCTACTCTGATGAGGTGGACAGATCCATCAGGGTGGTCAGCCCCTGTGGCATGTGCAGAGAGCTCATCTCTGACTATGCTCCTGACTGCTTTGTGCTCATTGAGATGAATGGCAAGCTGGTCAAAACCACCATTGAGGAACTCATCCCCCTCAAGTACACCAGGAACTAAAGTAAAACTTTATCGAAATGGCCATCCATTCTTGCGCGGATGGCCTCTGCCAGCTGCTCATAGCGGCTGCGCAGCGGTGAGCCAGGACGATAAACCAGGCCAATAGTGCGGCGTGGTTCCGGCTTAATGCACGG(서열번호 4)

The xyrA gene of Aspergillus niger was codon-optimized for E. coli , and a plasmid capable of inducing xylose reductase with a low concentration of phosphate, pHCKan-INS: yibDp-6xhis-xyrA, was constructed at TWIST Biosciences. The pCASCADE guide RNA array plasmid was prepared as previously described by a combination of PCR and Gibson assembly.

Primers used for assembly are shown in Table 4.

Table 4

Plasmid/primer name order gltA1 (SEQ ID NO: 6) gltA1-FOR CCGGATGAGCATTCATCAGGCGGGCAAG (SEQ ID NO: 7) gltA1-REV CGGTTTATCCCCGCTGGCGCGGGGAACTCGAACTTCATAACTTTTAC (SEQ ID NO: 8) gltA2 TATTGACCAATTCATTCGGGACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACCG (SEQ ID NO: 9) gltA2-FOR CCGGATGAGCATTCATCAGGCGGGCAAG (SEQ ID NO: 10) gltA2-REV CGGTTTATCCCCGCTGGCGCGGGGAACTCGAACTAATAACTGTC (SEQ ID NO: 11) udhA TTACCATTCTGTTGCTTTTATGTATAAGAATCGAGTTCCCCGCGCCAGCGGGGATAAACCG (SEQ ID NO: 12) udhA-FOR CCGGATGAGCATTCATCAGGCGGGCAAG (SEQ ID NO: 13) udhA-REV CGGTTTATCCCCGCTGGCGCGGGGAACTCGATTCTTATACATAAAAGC (SEQ ID NO: 14) zwf CTCGTAAAAGCAGTACAGTGCACCGTAAGATCGAGTTCCCCGCGCCAGCGGGGATAAACCG (SEQ ID NO: 15) zwf-FOR CCGGATGAGCATTCATCAGGCGGGCAAG (SEQ ID NO: 16) zwf-REV CGGTTTATCCCCGCTGGCGCGGGGAACTCGATCTTACGGTGCACTGTAC (SEQ ID NO: 17) xylA GGAGTGCCCAATATTACGACATCATCCATCTCGAGTTCCCCGCGCCAGCGGGGATAAACCG (SEQ ID NO: 18) xylA-FOR CCAGCGGGGATAAACCGGGAGTGCCCAATATTAC (SEQ ID NO: 19) xylA-REV CTTGCCCGCCTGATGAATGCTCATCCGG (SEQ ID NO: 20)

Table S4: Additional oligonucleotides:

Plasmid/primer name order 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) gltA2 TATTGACCAATTCATTCGGGACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACCG (SEQ ID NO: 6) gltA2-FOR CCGGATGAGCATTCATCAGGCGGGCAAG (SEQ ID NO: 7) gltA2-REV CGGTTTATCCCCGCTGGCGCGGGGAACTCGAACTAATAACTGTC (SEQ ID NO: 8) udhA TTACCATTCTGTTGCTTTTATGTATAAGAATCGAGTTCCCCGCGCCAGCGGGGATAAACCG (SEQ ID NO: 12) udhA-FOR CCGGATGAGCATTCATCAGGCGGGCAAG (SEQ ID NO: 13) udhA-REV CGGTTTATCCCCGCTGGCGCGGGGAACTCGATTCTTATACATAAAAGC (SEQ ID NO: 14) xylA GGAGTGCCCAATATTACGACATCATCCATCTCGAGTTCCCCGCGCCAGCGGGGATAAACCG (SEQ ID NO: 18) xylA-FOR CCAGCGGGGATAAACCGGGAGTGCCCAATATTAC (SEQ ID NO: 19) xylA-REV CTTGCCCGCCTGATGAATGCTCATCCGG (SEQ ID NO: 20)

Micro fermentation and 1L fermentation

Micro-fermentation and 1L fermentation in a bioreactor apparatus were performed as previously published, except that glucose was replaced with xylose in all media formulations (glucose 1 g -> xylose 1 g). After transforming the pCASCADE vector, guide array stability was verified by PCR and then evaluated by 96-well plate micro-fermentation.

Xylose and xylitol quantification

In the micro-fermentation, xylose and xylitol were quantified using a commercially available bioassay from Megazyme (Wicklow, Ireland, Catalog #K-XYLOSE and K-SORB) according to the manufacturer's instructions. All results were checked by measuring the absorbance at 492 nm. To quantify the tank fermentation, both xylose and xylitol were measured using the HPLC method coupled with a refractive index detector. A Rezex ROA-Organic Acid H+ (8%) Analytical HPLC column (CAT#: #00H-0138-K0, Phenomenex, Inc., Torrance, CA, 300 x 7.8 mme;) at 55°C was used for compound separation. According to the reference literature, sulfuric acid was chosen as the isocratic eluent solvent, and the flow rate was set at 0.5 mL/min. A Waters Acquity H-Class UPLC with integrated Waters 2414 refractive index (RI) detector (Waters Corp., Milford, MA. USA) was used for chromatographic detection. The injection volume of samples and standards was set at 10 μl. Samples were diluted 20-fold with filtered ultrapure water, and all sample points were set to fall within the standard linear range. Standard deviations ranged from 0.01 to 20 g/L. MassLynx v4.1 software was used for all peak integration and concentration analyses.

fermentation

Minimal medium micro-fermentation was performed as previously published (Moreb, EA 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. et al. Scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion. Biotechnol. Bioeng. 26 , 44 (2020)), but xylose in all medium formulations Replaced glucose with (glucose 1 g -> xylose 1 g). After transforming the pCASCADE plasmid and verifying guide array stability by PCR, Li et al. ²⁴ was evaluated. Feed batch fermentation was performed as previously published, again using 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 feed was modified as follows: Starting batch glucose concentration was 25 g/L When cells entered mid-log phase, a concentrated sterile filtered xylose feed (500 g/L) was added to the tank at an initial rate of 10 g/h, which was increased exponentially by a factor of 2 every 1.083 hours (65 minutes) until 40 g total glucose had been added, after which The feed was maintained at 1.75 g/hr, the feed was reduced to 0.875 g/hr due to xylose accumulation at 85 hours after inoculation and stopped at 120 hours after inoculation.

(XyrA) xylose reductase purification and activity assay

E. coli BL21(DE3) (New England Biolabs, Ipswich, Mass.) with the plasmid pHCKan-xyrA (containing a 6x his tag) was cultured overnight in Luria broth (Lenox formulation). Overnight cultures were inoculated into SM10++ medium (xylose instead of glucose as carbon source) supplemented with appropriate antibiotics. Cells were incubated for 16 hours at 37° C., then cells were centrifuged and the pellet was rinsed with SM10 No Phosphate Medium. Then, the rinsed pellet was resuspended in SM10 No Phosphate medium supplemented with appropriate antibiotics and cultured. After expression, post-production cells were lysed by freeze-thaw cycles. XyrA protein was purified using Ni-NTA resin (G-Biosciences, Cat # 786-939) according to the manufacturer's instructions. Kinetic analysis for XyrA was performed in reaction buffer consisting of 50 mM sodium phosphate (pH 7.6, 5 mM MgCl2) with NADPH as a cofactor (Suzuki, T. et al. Expression of xyrA gene encoding for D-Xylose reductase of Candida tropicalis and production of xylitol in Escherichia coli.J. Biosci.Bioeng.87 , 280-284 (1999)). In these assays, NADPH was fixed at a constant initial level of 50 μM. Assay results were measured by monitoring NADPH absorbance at 340 nm for 1.5 hours (15 seconds/reading) using a spectraMax Plus 384 microplate reader (Molecular Devices). The reaction rate according to the xylose concentration was plotted. By the Edie-Hofsty equation, the parameters: Vmax=22.6±1.01 U, kcat=13.56±3.05 s ⁻¹ and Km: 35.12±3.05 mM were obtained.

(XylA) xylose isomerase quantification

Xylose isomerase activity in cell extracts was quantified by the D-xylose reductase coupled enzyme assay as previously described, and the decrease in absorbance of NADPH at 340 nm was followed (Guamαn, LP et al. xylA and xylB overexpression as a successful strategy for improving xylose utilization and poly-3-hydroxybutyrate production in Burkholderia saccari.J . Ind. Microbiol.Biotechnol.45 , 165-173 (2018) and Lee, S.-M., Jellison, T & Alper, HS Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae. Appl. Environ. Microbiol. 78 , 5708-5716 (2012)). Cultures were grown in shake flasks in SM10++ medium, harvested in mid-logarithmic phase, rinsed and resuspended in SM 10 No phosphate medium. After 16 hours of phosphate depletion, cells were pelleted by centrifugation (4122 RCF, 4°C) for 10 minutes, and cells were lysed using BugBuster protein extraction reagent (Millipore Sigma, Catalog #70584) according to the manufacturer's protocol. Cell debris was removed by centrifugation (4122 RCF, 4°C) for 20 minutes twice followed by hard spin (14000 RCF, 4°C) for 20 minutes. The cell lysate was filtered through an Amicon 30MWCO filter (Millipore Sigma, Catalog #UFC8030) according to the manufacturer's protocol, then the buffer was replaced with reaction buffer (45 mM sodium phosphate, 10 mM MgCl ₂ , pH 7.6) to remove metabolites. Rinse 3 times. Samples were assayed in triplicate in 96-well plates by adding 100 μl of filtered cell extract per well containing 31.25 mM xylose, 0.5 mM NADPH and 1 ug/mL purified D-xylose reductase (see above). ). Absorbance at 340 nm was measured at 15 second intervals for 1.5 hours, and XylA activity was quantified using the slope of the linear region. The total protein concentration of each sample was determined by standard Bradford assay. Kinetic parameters are as follows: kcat: 13.56 ± 3.05 s ^-1 , Km: 35.12 ± 3.05 mM.

(UdhA) Quantification of soluble transhydrogenase

The activity of soluble transhydrogenase was quantified according to a previously published method (Chou, H.-H., Marx, CJ & Sauer, U. Transhydrogenase promotes the robustness and evolvability of E. coli deficient in NADPH production. PLoS Genet . Escherichia coli J. Biol. Chem. 279 , 6613-6619 (2004)). UdhA expression and cell lysis were performed using the same method as the above-mentioned XyrA expression. The cell lysate was centrifuged (4200 RPM, 4°C) for 15 minutes 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 soluble transhydrogenase. Cell lysates were diluted 1:5 with assay reaction buffer (50 mM Tris-HCl, 2 mM MgCl, pH 7.6) and transferred to Amicon Ultra centrifugal filters (10 kDa MWCO). Samples were centrifuged (4200 RPM, 4°C) for 30 minutes, this step was repeated 3 times to remove metabolites, and lysis buffer was replaced with assay buffer. After filtration, the protein concentration of the samples was quantified by standard Bradford assay.

Soluble transhydrogenase activity was then assayed at room temperature. Assay, cell lysate and reaction buffer were mixed in equal volumes of 1:1, in a final volume of 100 μl per well and final concentrations of 0.5 mM NADPH and 1 mM 3-acetylpyridine adenine dinucleotide (APAD ⁺ ), mixed in a black 96-well plate. was performed in Absorbance changes at 400 nm and 310 nm following APAD ⁺ reduction and NADPH oxidation, respectively, were simultaneously monitored at 30-second intervals for 30 minutes with a Spectramax Plus 384 microplate reader. The molar extinction coefficient of NADPH (3.04*10 ³ M ⁻¹ cm ⁻¹ ) was calculated using a standard curve. The slope of the straight line region measured using this molar extinction coefficient was converted into a change in concentration per minute. The specific activity (Unit/mg total protein) was obtained by dividing the concentration change per minute by the protein concentration.

FabI quantification

Quantification of FabI via a C-terminal GFP tag was performed using AbCam's GFP Quantification Kit (Cambridge, UK, Cat # ab171581) according to the manufacturer's instructions.

Quantification of xylose and xylitol

In the micro-fermentation, xylose and xylitol were quantified using commercially available bioassays from Megazyme (Wicklow, Ireland, Cat # K-XYLOSE and K-SORB) according to the manufacturer's instructions. Using the HPLC method coupled with a refractive index detector, both xylose and xylitol were quantified from the fermentation of the device. Briefly, a Rezex ROA-organic acid H ⁺ (8%) analytical HPLC column (Cat #: #00H-0138-K0, Phenomenex, Inc., Torrance, CA, 300 x 7.8 mme;) was used to separate xylose and xylitol. was used for 5 mM sulfuric acid was used as an isocratic mobile phase at a flow rate of 0.5 mL/min and 55°C. A Waters Acquity H-Class UPLC with integrated Waters 2414 refractive index (RI) detector (Waters Corp., Milford, MA. USA) was used for detection. The injection volume of samples and standards was 10 μl. Samples were diluted 20-fold with water to be in the linear range (0.01 to 20 g/L). MassLynx v4.1 software was used for all peak integration and concentration analysis.

NADPH pool quantification

NADPH pools were measured using the NADPH assay kit (AbCam, Cambridge, UK, Cat # ab186031) according to the manufacturer's instructions. Cultivation and phosphate depletion were performed as described above for XyrA expression (provided that no xyrA plasmid was present in the cells). Cells were lysed using the lysis buffer of the assay kit.

Building a metabolic model

Using the previously published reconstruction as a starting point, an in silico analysis was performed implementing a constraint-based (COBRA) model for E. coli developed using the COBRApy Python package. This carefully selected E. coli K-12 MG1655 reconstruction contains 2,719 metabolic reactions and 1,192 unique metabolites. This model was adjusted as follows. First, the omitted reactions and metabolites for xylitol production and efflux were added as shown in Table S5:

Table S5. Xylitol specific response added to metabolic model

designation 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 by passive diffusion xylt_e ↔ xylt_c XYLTt Flavodoxin Reductase (NADPH) 2.0 flxso_c + nadph_c ↔ 2.0 flxr_c + h_c + nadp_c FLDR2

All reactions, metabolite stoichiometry and identifiers were extracted from the BiGG Models database. The constructed model was verified for mass balance and metabolite compartment formulas by the COBRApy verification method. Upon reaching an appropriate balance, a proliferation model was constructed and analyzed. Specific evaluation conditions and biomass flux are shown in Table S6.

Table S6. Metabolic model validation using different carbon sources and oxygen levels (all flux values expressed in nmol/gDW*hr).

sole carbon source C.S Flux oxygen flux Biomass flux obtained from the model Previously published biomass flux* 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

* Figure derived from (Prasad, S., Singh, A. & Joshi, HC Ethanol as an alternative fuel from agricultural, industrial and urban residues. Resour. Conserv. Recycl. 50 , 1-39 (2007)).

Next, constraints were set on the model using experimental data obtained from xylitol micro-fermentation. Specific constraints are as follows: i) pyruvate dehydrogenase (PDH) and pyruvate-flavodoxin oxidoreductase ( ybdk ) set the pyruvate consumption rate to 10% and 90% of the total plus, respectively, and ii) setting ferredoxin/flavodoxin reductase to a reversible reaction, and iii) using xylose as the sole carbon source at an input flux of 10 mmol/gCDW*hr in minimal medium conditions. Finally, a set of specific xylitol-producing strains was constructed and evaluated in silico using Flux Balance Analysis (FBA) to determine the xylitol yield, cofactors and/or metabolites of interest, as well as produced and consumed fluxes. analyzed. Specific phenomena analyzed included decreased or increased activity of Zwf, FabI, GltA, XylA, PntAB and UdhA, as shown in Table S7. The following data were obtained for each event/condition: xylitol yield, NADPH production and consumptive g response fluxes, and Escher maps for central metabolism for flux distribution visualization. Finally, major changes in flux between the most relevant strains were analyzed.

Table S7. Building a strain model

individual valve valve combination strain valve silence strain valve silence WT -- Z-F zwf & fabI Z zwf Z-G zwf & gltA F fabI Z-X zwf & xylA G gltA Z-pAB zwf & pntAB X xylA Z-F-G zwf, fabI & gltA pAB pntAB Z-G-U zwf, gltA & udhA U udhA Z-F-pAB zwf, fabI & pntAB

Example 1: Characterization of XyrA xylose reductase

Referring now to FIG. 2(A), BL21, XyrA fermentation using a medium combination of SM10++ (growth medium) and SM10-No phos (fermentation medium) is described. After expression, post-production cells were lysed by freeze-thaw cycles. Then, using the His-tag for XyrA designed in the plasmid sequence, the xyrA protein was extracted through NN resin. In Fig. 2(B), the activity of xyrA using NADPH as a cofactor. Reaction rates were plotted as a function of xylose concentration. In this assay, NADPH was fixed at a constant initial level of 50 μM. Figure 2(C) Kinetic parameters of XyrA according to this experiment and other research sources as comparison. The kinetics of XyrA were measured using ²⁶ 50 μM NADPH in 50 mM sodium phosphate, pH 7.6 (containing 5 mM MgCl ₂ ). The assay results were determined by monitoring the absorbance of NADPH at 340 nm. Using the Edie-Hofsty equation, the parameters Vmax=22.6U, kcat=13.5s ⁻¹ and km=35.12mM were established, ie protein enzyme activity that could be used in a tank fermentation process was verified. If the Vmax is known, the minimum expression level required to reach the desired specific production rate can be established.

Example 2: Metabolic valve design for bio-production of xylitol

In the plateau phase of phosphate depletion, rationally designed strains were developed to optimize the production of xylitol from xylose using two-step dynamic metabolic control. As illustrated in FIG. 1 , this design involves overexpression of xylose reductase and dynamic reduction of xylose isomerase ( xylA ) activity to reduce xylose metabolism competing with xylitol production. For this purpose, the present inventors constructed strains and plasmids capable of achieving dynamic reduction of XylA activity and dynamic induction of xyrA upon phosphate depletion through gene silencing, proteolysis of XylA, or a combination thereof. See Tables 1 and 2 for plasmids and strains used. These strains were evaluated in a 96-well plate micro-fermentation published by Moreb et al., and the results are presented in FIG. 3 .

Example 3: Xylitol production using two-step dynamic control

Only a slight improvement in xylitol production was achieved upon dynamic modulation of XylA ("X") activity (FIG. 3), so we evaluated the potential impact of a larger scale valve set on xylitol production. We constructed a set of strains with valves in key metabolic pathways, Figure 4. These valves include citrate synthase, which controls flux through each of the tricarboxylic acid cycle, pentose phosphate pathway, fatty acid biosynthesis, and NADPH supply ( GltA-"G"), glucose-6-phosphate dehydrogenase (Zwf-"Z"), enoyl-ACP reductase (FabI-"F") and soluble transhydrogenase (udhA-"U") includes Strains with valve combinations of X, U, G, Z and F were constructed and xylitol production was evaluated. As described above, dynamic metabolic regulation was achieved by adding the C-terminal DAS+4 degron to the xylA, udhA, zwf, gltA and fabI genes and overexpressing a guide RNA capable of silencing its transcription. See Methods section for details on chromosome modification and plasmid construction.

The panel consisted of ~370 valve combinations of X, U, G, Z and F, and evaluated xylitol production at least in triplicates in a two-stage 96-well plate micro-fermentation. The results of these experiments are shown in Figures 5A and 5B. Xylitol titers ranged from -0 g/L-OD (600 nm) to -9.35 g/L-OD (600 nm). About -80% of the combinations of silencing and proteolysis outperformed the control strain with a titer of only 0.106 g/L-OD. A significant difference for specific xylitol production (xylitol (g/L)/unit OD600nm) between the valve strain and the control strain was confirmed by one-way ANOVA (F(414,851)=7.598, p<0.0001).α

A p-value heatmap was created using the P-value (FIG. 6), and only combinations with a p-value less than 0.05 were highlighted. Combinations that are not analyzed or have less than two consecutive replicates (due to poor cell proliferation) are not suitable for statistical analysis and are marked with gray dots. X-valve integration generally led to increased xylitol production, but surprisingly the two best xylitol-producing strains had neither an X-valve nor a U-valve (which should increase NADPH levels), but rather a combination of F, G, and Z valves. The best producing strain had the F and Z valve combination and was able to achieve a specific xylitol productivity of 9.35 g/L-OD600 nm. The performance of these gene combinations also had a synergistic effect compared to the F-valve or Z-valve alone. This was surprising since these two enzymes had no direct or predictable effect on xylitol biosynthesis, as can be seen in FIG. 4 .

Example 4: Xylitol production in a bioreactor device

Based on the micro-fermentation results (Figs. 5 and 6), we selected the "Z-FZ" valve strain (zwf "Z" silencing and fabI and zwf "FZ" proteolysis) with a titer of 9.35 g/L-OD600. and was selected as a control group for evaluation in the bioreactor device. Fermentation was carried out according to the literature of Menacho-Melgar et al., and as illustrated in FIG. 5 , phosphate was limited in the medium to induce phosphate depletion and xylitol production in a plateau phase. These fermentation results are shown in Figure 6 below. The Z-FZ strain (Fig. 6A) was able to produce up to 104+/-11.31 g/L of xylitol within 160 hours, whereas the control strain DLF_0025-EV (Fig. 6B) produced only ~3 g of xylitol within the same time period with xyrA expression. /L was induced.

Example 5: Improvement of NADPH flux and xylitol biosynthesis

Most previous studies of producing xylitol from xylose have been based on bioconversion requiring an additional sugar (usually glucose) as an electron donor (Albuquerque, TL de, da Silva, IJ, de Macedo, GR & Rocha Cirino , PC, Chin , JW & Ingram, LO Engineering Escherichia coli for xylitol production from glucose- xylose mixtures. Bioeng . _ Eng. 31 , 112-122 (2015)). Upon oxidation of glucose (by-product gluconic acid production), NADPH is produced, which is then utilized 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. Appl. Biochem. Biotechnol. 187 , 1143-1157 (2019)). Although this process provides high xylitol titers and good yields when considering xylose, it is quite inefficient since it requires glucose at the same molar level as xylose. More broadly, improving NADPH availability or flux in cell-based biotransformation as well as synthesis of various metabolites has long been a challenge in metabolic engineering.

We have utilized two-step dynamic metabolic control (DMC) to improve NADPH flux and xylitol production using xylose as the sole feedstock (Burg, JM, Cooper, CB, Ye, Z., Reed, BR & Moreb, EA Large-scale bioprocess competitiveness: the potential of dynamic metabolic control in two-stage fermentations. Current opinion in (2016)). Dynamic control of metabolism has become a common approach in metabolic engineering and is used to produce a variety of products ranging from 3-hydroxypropionic acid to myo-inositol and many other products. The present inventors recently published a plan to extend dynamic metabolic control to a two-step bioprocess, producing products in a plateau phase when metabolic productivity phosphate is depleted. This approach is based on the combined use of controlled proteolysis and gene silencing using degron tags and CRISPR interference, respectively. Importantly, in these early studies, we demonstrated that improvements in metabolic flux due to dynamic metabolic regulation could be the result of reduced levels of pivotal metabolites that are feedback regulators of other key metabolic pathways. Specifically, the inventors recently found that reducing glucose-6-phosphate dehydrogenase levels activates SoxRS regulon to increase the expression and activity of pyruvate ferredoxin/flavodoxin oxidoreductase (Pfo). , proved. Pfo induces an improvement in acetyl-CoA production in the plateau phase (see FIG. 11(A)). In this study, we describe the results of evaluating combinations of synthetic metabolic valves in producing xylitol from xylose. First, increasing Pfo activity not only improves acetyl-CoA flux but also induces NADPH production. NADPH is produced from reduced flavodoxin/ferredoxin by the action of NADPH-dependent flavodoxin/ferredoxin reductase (Fpr). We also identified a key regulatory mechanism regulating NADPH flux, namely inhibition of membrane-bound transhydrogenase (PntAB) by fatty acid metabolites. We mitigate the inhibition of PntAB by dynamically perturbing fatty acid biosynthesis. This mechanism works synergistically with Pfo activation, greatly increasing NADPH flux and xylitol production. We compare this “modulation” approach to a more intuitive stoichiometric strategy that dynamically lowers the levels of key enzymes that compete with xylitol production. Importantly, the improved NADPH flux is partly the result of a reduced NADPH pool. Reducing the NADPH pool induces changes in expression and activity that result in increased NADPH flux, presumably an evolved regulatory mechanism to restore setpoint NADPH levels. These results are a reminder that pool and flux are not equivalent, nor are they necessarily correlated.

Stoichiometric strategy

We initially rationally designed strains to optimize xylitol production from xylose using a two-step dynamic metabolic control, based on lowering the level of a key competitive pathway. As illustrated in FIG. 1 , this design involves dynamic reduction of xylose isomerase ( xylA ) and soluble transhydrogenase ( udhA ) activities. These modifications were designed to reduce xylose metabolism, which competes with xylitol production, and increase NADPH supply. NADPH can be consumed by soluble transhydrogenases. To this end, the present inventors constructed strains and plasmids capable of dynamically reducing XylA and UdhA levels upon phosphate depletion. See Appendix Table S1 for the strains and plasmids used in this experiment. As mentioned above and previously published, the dynamic reduction of activity can be achieved by adding a C-terminal DAS+4 degron tag to the chromosomal xylA and udhA genes, as well as overexpression of the guide RNA for the purpose of silencing expression. achieved by The effect of these modifications on enzyme levels in two-stage culture is shown in Figures 9A-B. In the case of XylA, proteolysis reduces activity by -60%. Surprisingly, silencing gRNAs actually led to increased levels of XylA activity. Its underlying mechanism is currently unknown and requires further research. Upon combination of silencing and proteolysis, no additional activity reduction was achieved when compared to proteolysis alone. In the case of UdhA, activity decreased by -30% upon proteolysis, whereas silencing had no detectable effect on the level of activity with or without proteolysis.

Combinations of proteolysis and silencing for XylA or "X valve", and proteolysis alone for UdhA, "U valve" were evaluated for xylitol production. Specifically, strains were engineered to contain these metabolic valves, as well as to overexpress xylose reductase ( xyrA from Aspergillus nira) and evaluated in a two-stage minimal medium micro-fermentation as published by Moreb et al. . The results are shown in Figures 10A-C. In addition, a confirmatory analysis for XyrA kinetics was performed, and the results are presented in Appendix Figure 11. The combination of modifications achieved a 16-fold increase in xylitol production compared to the control.

regulatory strategy

To investigate the impact of the regulatory strategy, the inventors next sought to evaluate the potential impact of a larger set of valves on xylitol production, as illustrated in Figure 12A. We have identified citrate synthase (GltA), 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. ) to establish a set of strains with valves. We present the construction of metabolic valves in GltA (“G valve”) and Zwf (“Z valve”), consisting of proteolytic denaturation (DAS+4 tag), gene silencing (zwf promoter or gltAp2 promoter), or both. have done In the case of FabI, we constructed new strains and plasmids to evaluate the two-step dynamic regulation at the level of FabI. To this end, superfolder GFP was added to the C-terminus of the fabI allele so that the protein level could be quantified by ELISA, as reported by Li et al. Unfortunately, unexpectedly, when the fabI expression silencing plasmid was evaluated, guide RNA trophosphacer loss was observed (FIG. 13A-D), and as a result, we were unable to reliably obtain fabI silencing results. FabI proteolysis reduced FabI levels by -75% (FIG. 14), resulting in only proteolytic denaturation referred to as "F valve". Strains with combinations of "X", "U", "G", "Z" and "F" valves were constructed and again evaluated for xylitol production in minimal medium micro-fermentation. The results are presented in Figures 12A-D. Surprisingly, the best xylitol producers did not have "X" or "U" valves, but rather a combination of "F" and "Z" valves. Xylitol production in the "FZ" valve strain had a synergistic effect compared to the "F" and "Z" valves alone (FIG. 12B). This was a surprising result in that these two enzymes had no direct or predictable effect on xylitol biosynthesis, as can be seen in FIG. 12A. The inventors recently published that the "Z" valve achieves an increase in acetyl-CoA flux by inducing activation of Pfo (encoded by the ydbK gene). With the increased flux through Pfo, we hypothesized that NADPH could be generated from reduced ferredoxin/flavodoxin via ferredoxin reductase (Fpr). As shown in Figure 15, using deletions in ydbK and fpr , it was confirmed that this pathway, specifically Fpr, contributes in part to the increased NADPH flux and xylitol production observed in the actual "FZ" valve strain. This is, as far as the present inventors are aware, the first verification that Fpr is reversible in vivo. This reverse flux through Fpr may be dependent on the low NADPH pool (as described below). The synergistic effect of the "F" valve was somewhat unexpected. However, increased NADPH flux due to dynamic regulation of FabI (enoyl-ACP reductase) may be due to reduced levels of fatty acid metabolites, particularly acyl-CoA (and potentially its precursor acyl-ACP). Fatty acyl-CoA is a competitive inhibitor of the membrane-bound transhydrogenase encoded by the pntAB gene (FIG. 12A). In particular, palmitoyl-CoA has a reported Ki of 1-5 μM. Modulation of FabI levels and/or activity has previously been shown to reduce the acyl-ACP pool and consequently alleviate feedback inhibition of acetyl-CoA carboxylase and malonyl-CoA synthesis. To the best of our knowledge, this is the first study to demonstrate the importance of these metabolites in regulating NADPH flux. Although previous reports have demonstrated PntAB inhibition by acyl-CoAs (derived from fatty acid biosynthesis in minimal medium), it remains possible that acyl-ACPs also act as inhibitors, so further studies are needed to verify this hypothesis. do.

Next, we investigated several additional modifications at the top of the “FZ” valve that could potentially affect xylitol production (FIGS. 15B-D). Specifically, we investigated the addition of “G” and “U” valves and overexpression of pntAB. A plasmid based on overexpression of the pntAB gene (using a promoter inducible with low phosphate concentration) achieved significant improvement in xylitol production (FIG. 15B). In contrast, when the "G" or "U" valve was added to the "FZ" combination, xylitol synthesis did not increase, but xylitol production was significantly reduced (FIG. 15C-D). This suggests that citrate synthase (GltA) activity, and flux through the TAC cycle, are required for optimal NADPH flux.

Using the results of these experiments, we were able to estimate boundary conditions for several intracellular fluxes. For example, from Figure 15B, we can estimate that flux through the Pfo/Fpr pathway accounts for up to -55% of NADPH/xylitol production. As a result, the present inventors were able to construct a stoichiometric metabolic model comparing the optimal growth period and the xylitol production period, as illustrated in FIGS. 16A-B. Importantly, this model demonstrates that actual TCA flux is important for xylitol production (FIG. 17A-B) and that a 4-fold increase in PntAB activity is required to account for increased NADPH flux and xylitol production in addition to flux through the Pfo/Fpr pathway. that it verifies. The model predicts xylose ~0.864 g/g as the total xylitol maximum yield at this metabolic state, which is consistent with the yield measured in feed batch fermentation as described below.

Produced in a bioreactor unit

Next, we compared the xylitol production in the bioreactor device using the "FZ" valve strain with and without pntAB overexpression to the control strain. Minimal medium feed batch fermentation is described by Menacho-Melgar et al., in which the medium contains sufficient batch phosphate to support induction of xylitol biosynthesis at the target biomass level (˜25 g CDW/L) prior to phosphate depletion and stationary phase. performed as is done. The results of this study are presented in FIG. 18 . In the control strain of the present invention (DLF_Z0025), when xyrA was expressed, xylitol was only several grams per L (Fig. 18A), but titers of over 100 g/L were achieved with 160 hours of production when integrated with the "FZ" valve (Fig. 18B). Additional overexpression of pntAB (FIG. 18C) resulted in peak titers of over 200 g/L (185-204 g/L) over 170 hours. In this duplicate fermentation, the average yield of total xylitol (stationary phase) was 0.935 +/- 0.011 g/g xylose.

NADPH flux improvement does not correlate with NADPH pool.

Finally, we measured NADPH levels in our set of engineered strains. The results are presented in Figure 19A. Interestingly, there was no correlation between specific xylitol production and NADPH pools. In this case, the three strains with the highest NADPH pool levels were the control strain and strains that dynamically modulated enoyl-ACP levels ("F" valve) or soluble transhydrogenase ("U" valve) levels. Addition of a "Z" valve (reducing the level of glucose-6-phosphate dehydrogenase) reduced the NADPH pool, but increased the NADPH flux. Deletion of ydbK and/or fpr also led to a decrease in NADPH, and overexpression of pntAB increased xylitol production rate and flux, but did not improve the NADPH pool in the “FZ” background.

When using a two-step dynamic regulation, an overall metabolic state leading to enhanced NADPH flux and xylitol production was established. As far as the present inventors are aware, this is the highest titer and xylitol production yield so far in engineered E. coli , especially in a strain using xylose as the sole carbon source. In addition, the production plateau established with this modification can be extended to at least 170 hours. Although these studies have focused on xylitol production, the identification of the "F" and "Z" valves that influence NADPH fluxes have made possible other NADPH-dependent processes, including more complex pathways, as well as routine NADPH-dependent biotransformation. can be an easy way to The influence of FabI activity and fatty acid metabolite pool on transhydrogenase activity is consistent with previous biochemical studies and likely evolved to balance NADPH supply with demand for fatty acid synthesis. Unfortunately, this feedback control mechanism has largely disappeared from metabolic engineering studies in E. coli in the past decades, but it is still a powerful way to improve NADPH flux. The unexpected combination of "F" and "Z" valves is contrary to standard thinking about NADPH fluxes, as Zwf is often considered one of the major sources of NADPH in cells, and reducing Zwf activity is intended to increase NADPH supply. It didn't rank high on the list of changes.

To elucidate the reason for the lack of association between the NAPHA pool and our results, the inventors developed a conceptual model as illustrated in FIG. 1 . The "Z" valve causes a decrease in the NADPH pool that activates SoxRS regulon, which is sensitive to oxidants and NADPH levels. SoxRS activation leads to increased expression of Pfo, which is required to maintain high rates of pyruvate oxidation, and NADPH is produced through Fpr. Uniquely, this experiment identified previously unpublished pathways for NADPH production using Pfo and Fpr, and confirmed that Fpr catalyzes a reversible reaction in vivo. Pfo expression is required for pyruvate oxidation and sugar consumption as well as NADH production through the TAC cycle. Increased TCA flux produces excess NADH required as a substrate for PntAB for maximal NADPH flux. Disruption of the TCA cycle ("G" valve, Figure 15B) halts NADH production and acetyl-CoA consumption, resulting in a large reduction in NADPH flux. The increased NADPH level due to the "F" valve is significant in light of the results discussed and is due to increased activity of the membrane-bound transhydrogenase, PntAB. Decreased soluble transhydrogenase (UdhA, FIG. 15D) levels lead to increased NADPH levels ( FIG. 19 ), which presumably reduces SoxRS activation and Pfo expression. Briefly, the metabolic network responds by increasing sugar consumption and NADPH fluxes to compensate for the reduced NADPH and acyl-CoA pools. If the "set" NADPH pool is restored or continued sugar catabolism ceases, the continued NADPH flux ceases.

Finally, the metabolic states leading to NADPH flux enhancement and xylitol production will be difficult to identify and/or manipulate in proliferation-linked processes, as they are based on feedback inhibition manipulations due to central metabolites. These central metabolic regulatory circuits have evolved to balance fluxes to optimize proliferation and to respond adaptively to environmental and physiological perturbations. Dynamic metabolic regulation, particularly two-step dynamic metabolic regulation, is particularly suited to manipulating levels of key metabolites without affecting cell proliferation or survival. This approach may lead to the discovery and manipulation of central regulatory mechanisms, which will likely enhance metabolic flux and prompt future metabolic engineering strategies.

SEQUENCE LISTING <110> DUKE UNIVERSITY <120> METHODS AND COMPOSITIONS FOR THE PRODUCTION OF XYLITOL FROM XYLOSE UTILIZING DYNAMIC METABOLIC CONTROL <130> 49186-46 <150> 63/004,740 <151> 2020-04-03 <150> 63/056,08 <151> 2020-07-24 <160> 31 <170> PatentIn version 3.5 <210> 1 <211> 1487 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 1 gatacgatgg cactggcgct gaaaattgca gcgcgcatga ttgaagatgg cgagctggat 60 aaacgcatcg cgcagcgtta ttccggctgg aatagcgaat tgggccagca aatcctgaaa 120 ggccaaatgt cactggcaga tttagccaaa tatgctcagg aacatcattt gtctccggtg 180 catcagagtg gtcgccagga acaactggaa aatctggtaa accattatct gttcgacaaa 240 gcggccaacg atgaaaacta ttctgaaaac tatgcggatg cgtcttaatg ataaggaccg 300 tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa 360 ctaaaccatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 420 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 480 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 540 acgccccgaa gaacgttt tc caatgatgag cacttttaaa gttctgctat gtggcgcggt 600 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 660 tgacttggtt gagtactcac cagtcacaga aaagcatctc acggatggca tgacagtaag 720 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctggc 780 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 840 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 900 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 960 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggatcact 1020 tctgcgctcg gccctcccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 1080 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gcatcgtagt 1140 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 1200 aggtgcctca ctgattaagc attggtagta agtagggata acagggtaat cggctaactg 1260 tgcagtccgt tggcccggtt atcggtagcg ataccgggca tttttttaag gaacgatcga 1320 tatgtatatc gggatagatc ttggcacctc gggcgtaaaa gttattttgc tcaacgagca 1380 gggtgaggtg gttgctgcgc aaacggaaaa gctgaccgtt tcgcgcccgc atccactctg 1440 gtcggaacaa gacccggaac agtggtggca ggcaactgat cgcgcaa 1487 <210> 2 <211> 970 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 2 ctattgaaga tgtgggtaac tctgcggcat tcctgtgctc cgatctctct gccggtatct 60 ccggtgaagt ggtccacgtt gacggcggtt tcagcattgc tgcaatgaac gaactcgaac 120 tgaaagcggc caacgatgaa aactattctg aaaactatgc ggatgcgtct taataggaag 180 ttcctattct ctagaaagta taggaacttc cgaatccatg tgggagttta ttcttgacac 240 agatatttat gatataataa ctgagtaagc ttaacataag gaggaaaaac atatgttacg 300 cagcagcaac gatgttacgc agcagggcag tcgccctaaa acaaagttag gtggctcaag 360 tatgggcatc attcgcacat gtaggctcgg ccctgaccaa gtcaaatcca tgcgggctgc 420 tcttgatctt ttcggtcgtg agttcggaga cgtagccacc tactcccaac atcagccgga 480 ctccgattac ctcgggaact tgctccgtag taagacattc atcgcgcttg ctgccttcga 540 ccaagaagcg gttgttggcg ctctcgcggc ttacgttctg cccaagtttg agcagccgcg 600 tagtgagatc tatatctatg atctcgcagt ctccggcgag caccggaggc agggcattgc 660 caccgcgctc atcaatctcc tcaagcatga ggccaactgcg cttgg ctt atgtgatcta 720 cgtgcaagca gattacggtg acgatcccgc agtggctctc tatacaaagt tgggcatacg 780 ggaagaagtg atgcactttg atatcgaccc aagtaccgcc acctaagaag ttcctattct 840 ctagaaagta taggaacttc cgttctgttg gtaaagatgg gcggcgttct gccgcccgtt 900 atctctgtta tacctttctg atatttgtta tcgccgatcc gtctttctcc ccttcccgcc 960 ttgcgtcagg 970 <210> 3 <211> 869 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 3 gtattccgtc ttccatgttc accgtcattt tcgcaatggc acgtaccgtt ggctggatcg 60 cccactggag cgaaatgcac agtgacggta tgaagattgc ccgtccgcgt cagctgtata 120 caggatatga aaaacgcgac tttaaaagcg atatcaagcg tgcggccaac gatgaaaact 180 attctgaaaa ctatgcggat gcgtcttaat agttgacaat taatcatcgg catagtatat 240 cggcatagta taatacgact cactatagga gggccatcat ggccaagttg accagtgccg 300 ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg 360 ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc 420 tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg 480 tgcgcggggcct ggacgagccgagggtg ggacgagccgagggt cgtgtccacg aacttccggg 540 acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc 600 tgcgcgaccc ggccggcaac tgcgtgcact ttgtggcaga ggagcaggac tgaggataag 660 taatggttga ttgctaagtt gtaaatattt taacccgccg ttcatatggc gggttgattt 720 ttatatgcct aaacacaaaa aattgtaaaa ataaaatcca ttaacagacc tatatagata 780 tttaaaaaga atagaacagc tcaaattatc agcaacccaa tactttcaat taaaaacttc 840 atggtagtcg catttataac cctatgaaa 869 <210> 4 <211> 852 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 4 tctgggtatt cactgctttg gcgagcgcgc tgccgaaatt attcatatcg gtcaggcgat 60 tatggaacag aaaggtggcg gcaacactat tgagtacttc gtcaacacca cctttaacta 120 cccgacgatg gcggaagcct atcgggtagc tgcgttaaac ggtttaaacc gcctgtttgc 180 ggccaacgat gaaaactatt ctgaaaacta tgcggatgcg tcttaatagt tgacaattaa 240 tcatcggcat agtatatcgg catagtataa tacgactcac tataggaggg ccatcatgaa 300 gaccttcaac atctctcagc aggatctgga gctggtggag gtcgccactg agaagatcac 360 catgctctat gaggacaaca agcaccatgt cggggcggcc atcaggacca agactgggga 420 gatcatctct gct gtccaca ttgaggccta cattggcagg gtcactgtct gtgctgaagc 480 cattgccatt gggtctgctg tgagcaacgg gcagaaggac tttgacacca ttgtggctgt 540 caggcacccc tactctgatg aggtggacag atccatcagg gtggtcagcc cctgtggcat 600 gtgcagagag ctcatctctg actatgctcc tgactgcttt gtgctcattg agatgaatgg 660 caagctggtc aaaaccacca ttgaggaact catccccctc aagtacacca ggaactaaag 720 taaaacttta tcgaaatggc catccattct tgcgcggatg gcctctgcca gctgctcata 780 gcggctgcgc agcggtgagc caggacgata aaccaggcca atagtgcggc gtggttccgg 840 cttaatgcac gg 852 <210> 5 <211> 898 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 5 gaagtggaag aagcctggaa atgggtagac tccattactg aggcgtgggc gatggacaat 60 gatgcgccga aaccgtatca ggccggaacc tggggacccg ttgcctcggt ggcgatgatt 120 acccgtgatg gtcgttcctg gaatgagttt gaggcggcca acgatgaaaa ctattctgaa 180 aactatgcgg atgcgtctta atagttgaca attaatcatc ggcatagtat atcggcatag 240 tataatacga ctcactatag gagggccatc atgaagacct tcaacatctc tcagcaggat 300 ctggagctgg tggaggtcgc cactgagaag atcaccatgc tctatgagga caacaagcac 360 cat gtcgggg cggccatcag gaccaagact ggggagatca tctctgctgt ccacattgag 420 gcctacattg gcagggtcac tgtctgtgct gaagccattg ccattgggtc tgctgtgagc 480 aacgggcaga aggactttga caccattgtg gctgtcaggc acccctactc tgatgaggtg 540 gacagatcca tcagggtggt cagcccctgt ggcatgtgca gagagctcat ctctgactat 600 gctcctgact gctttgtgct cattgagatg aatggcaagc tggtcaaaac caccattgag 660 gaactcatcc ccctcaagta caccaggaac taaagtaata tctgcgctta tcctttatgg 720 ttattttacc ggtaacatga tcttgcgcag attgtagaac aatttttaca ctttcaggcc 780 tcgtgcggat tcacccacga ggcttttttt attacactga ctgaaacgtt tttgccctat 840 gagctccggt tacaggcgtt tcagtcataa atcctctgaa tgaaacgcgt tgtgaatc 898 <210> 6 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 6 tcgagttccc cgcgccagcg gggataaacc gaaaagcata taatgcgtaa aagttatgaa 60 gttcgagttc cccgcgccag cggggataaa ccg 93 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 7 ccggatgagc attcatcagg cgggcaag 28 <210> 8 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> S ynthetic <400> 8 cggtttatcc ccgctggcgc ggggaactcg aacttcataa cttttac 47 <210> 9 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 9 tattgaccaa ttcattcggg acagttatta gttcggagttc c0ggataccacca 6 > 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 10 ccggatgagc attcatcagg cgggcaag 28 <210> 11 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 11 cggtttatcc ccgctggcgc ggggaactcg aactaataac tgtc 44 <210> 12 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 12 ttaccattct gttgctttta tgtataagaa tgtataagaa tcccgagttcccagc 61 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 13 ccggatgagc attcatcagg cgggcaag 28 <210> 14 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 14 cggtttatcc ccgctggcgc ggggaactcg attcttatac ataaaagc 48 <210> 15 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 15 ctcgtaaaa g cagtacagtg caccgtaaga tcgagttccc cgcgccagcg gggataaacc 60 g 61 <210> 16 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 16 ccggatgagc attcatcagg cgggcaag 28 <210> 17 <211> 49 <211> 212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 17 cggtttatcc ccgctggcgc ggggaactcg atcttacggt gcactgtac 49 <210> 18 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> Synthetic < 400> 18 ggagtgccca atattacgac atcatccatc tcgagttccc cgcgccagcg gggataaacc 60 g 61 <210> 19 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 19 ccagcgggga taaaccggga gtgcc4 caata <20 ttac 211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 20 cttgcccgcc tgatgaatgc tcatccgg 28 <210> 21 <211> 1655 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 21 aaagacttcc gcaaaatgct ggctcattgc gaagccgtta ccccgattcg ccgtaccgtt 60 actattgaag atgtgggtaa ctctgcggca ttcctgtgct ccgatctctc tgccggtatc 120 tccggtgaag tggtccacgt tgacggcggt ttcagcattg ctgcaatgaa cgaactcgaa 180 ctgaaagggg gttcaggcgg gtcgggtggc gtgagcaagg gcgaggagct gttcaccggg 240 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgcgc 300 ggcgagggcg agggcgatgc caccaacggc aagctgaccc tgaagttcat ctgcaccacc 360 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc 420 ttcagccgct accccgacca catgaagcgc cacgacttct tcaagtccgc catgcccgaa 480 ggctacgtcc aggagcgcac catcagcttc aaggacgacg gcacctacaa gacccgcgcc 540 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 600 aaggaggacg gcaacatcct ggggcacaag ctggagtaca acttcaacag ccacaacgtc 660 tatatcaccg ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac 720 gtggaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 780 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgtgct gagcaaagac 840 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 900 cacggcatgg acgagctgta caagtaatga cgaatccatg tgggagttta ttcttgacac 960 agatatttat gatataataa ctgagtaagc ttaacataag gaggaaaaac atatgttgc g 1020 tagctctaac gatgtgacgc aacaaggttc gcgtccaaag acaaaattgg gaggcagtag 1080 catggggatc attcgcactt gtcgcctggg gccagaccag gtgaagtcaa tgcgtgcggc 1140 tctggactta ttcgggcgcg aatttggaga tgtagccact tactcacagc accaaccgga 1200 cagtgattac ttggggaatt tacttcgcag taaaactttt atcgctttgg ccgctttcga 1260 ccaggaggct gtagtaggtg cgttggcagc ctatgttctt cctaaattcg agcaaccgcg 1320 tagcgaaatt tacatctatg atcttgcagt ctccggcgaa catcgccgtc aggggatcgc 1380 cacagcttta atcaaccttt tgaagcatga ggctaatgca cttggagcgt acgtgattta 1440 tgtgcaggct gactacggtg atgatcctgc agtcgctctg tacaccaaac tgggtatccg 1500 cgaggaggtc atgcactttg atattgaccc gtcgacggct acctaagttc tgttggtaaa 1560 gatgggcggc gttctgccgc ccgttatctc tgttatacct ttctgatatt tgttatcgcc 1620 gatccgtctt tctccccttc ccgccttgcg tcagg 1655 <210> 22 <211> 1730 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 22 aaagacttcc gcaaaatgct ggctcattgc gaagccgtta ccccgattcg ccgtaccgtt 60 actattgaag atgtgggtaa ctctgcggca ttcctgtgct ccgatctctc tgccggtatc 120 tccggtgaag tg gtccacgt tgacggcggt ttcagcattg ctgcaatgaa cgaactcgaa 180 ctgaaagggg gttcaggcgg gtcgggtggc gtgagcaagg gcgaggagct gttcaccggg 240 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgcgc 300 ggcgagggcg agggcgatgc caccaacggc aagctgaccc tgaagttcat ctgcaccacc 360 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc 420 ttcagccgct accccgacca catgaagcgc cacgacttct tcaagtccgc catgcccgaa 480 ggctacgtcc aggagcgcac catcagcttc aaggacgacg gcacctacaa gacccgcgcc 540 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 600 aaggaggacg gcaacatcct ggggcacaag ctggagtaca acttcaacag ccacaacgtc 660 tatatcaccg ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac 720 gtggaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 780 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgtgct gagcaaagac 840 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 900 cacggcatgg acgagctgta caagggtggg ggtgggagcg gcggcggtgg ctccgcggcc 960 aacgatgaaa actattctga aaactatgcggatgcgtctt aatgacgaat ccatgtggga 1020 gtttattctt gacacagata tttatgatat aataactgag taagcttaac ataaggagga 1080 aaaacatatg ttgcgtagct ctaacgatgt gacgcaacaa ggttcgcgtc caaagacaaa 1140 attgggaggc agtagcatgg ggatcattcg cacttgtcgc ctggggccag accaggtgaa 1200 gtcaatgcgt gcggctctgg acttattcgg gcgcgaattt ggagatgtag ccacttactc 1260 acagcaccaa ccggacagtg attacttggg gaatttactt cgcagtaaaa cttttatcgc 1320 tttggccgct ttcgaccagg aggctgtagt aggtgcgttg gcagcctatg ttcttcctaa 1380 attcgagcaa ccgcgtagcg aaatttacat ctatgatctt gcagtctccg gcgaacatcg 1440 ccgtcagggg atcgccacag ctttaatcaa ccttttgaag catgaggcta atgcacttgg 1500 agcgtacgtg atttatgtgc aggctgacta cggtgatgat cctgcagtcg ctctgtacac 1560 caaactgggt atccgcgagg aggtcatgca ctttgatatt gacccgtcga cggctaccta 1620 agttctgttg gtaaagatgg gcggcgttct gccgcccgtt atctctgtta tacctttctg 1680 atatttgtta tcgccgatcc gtctttctcc ccttcccgcc ttgcgtcagg 1730 <210> 23 <211> 19 <212> DNA < 213> Artificial Sequence <220> <223> Synthetic <400> 23 gcaaaatgct ggctcattg 19 <210> 24 <21 1> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 24 gcgatgaatg tcttactacg ga 22 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 25 tatcatcctg aaagcgatgg 20 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 26 actgaagccc agacgatc 18 <210> 27 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 27 ctgctggaaa ccatgcg 17 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 28 caaaagagat tctgggtatt cact 24 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> < 223> Synthetic <400> 29 gagcatggtg atcttctcag t 21 <210> 30 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 30 agatggcgag ctggata 17 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic<400> 31 agtactcaac caagtcattc tg 22

Claims (31)

As a genetically modified microorganism for producing xylitol from xylose,
inducible modification of the expression of xylose reductase, and
Including an inducible synthetic metabolic valve,
the valve,
A gene expression-silenced synthetic metabolic valve characterized by gene expression silencing of one or more genes encoding one or more enzymes, or
An enzymatically modified synthetic metabolic valve characterized by inducing enzymatic denaturation of one or more enzymes, or
combination of these
Including, genetically modified microorganisms. The genetically modified microorganism according to claim 1, wherein the xylose reductase is a NADPH-dependent xylose reductase. The genetically modified microorganism according to claim 1, wherein the xylose reductase is the xyrA gene of Aspergillus niger ( A. niger ). The genetically modified microorganism of claim 1, wherein the genetically modified microorganism produces xylitol from a xylose feedstock. The genetically modified microorganism according to claim 1, wherein the gene-silenced synthetic metabolic valve or the enzymatically modified synthetic metabolic valve directs regulation of a gene encoding xylose isomerase or a xylose isomerase enzyme. The method of claim 1, wherein the gene-silent synthetic metabolic valve or the enzymatically modified synthetic metabolic valve is a gene encoding glucose-6-phosphate dehydrogenase ( zwf ) or glucose-6-phosphate dehydrogenase ( zwf ) genetically modified microorganisms that direct control over enzymes. The genetically modified microorganism according to claim 1, wherein the gene-silenced synthetic metabolic valve or the enzymatically modified synthetic metabolic valve directs regulation of two or more genes, including:
a gene encoding glucose-6-phosphate dehydrogenase ( zwf ) or a glucose-6-phosphate dehydrogenase ( zwf ) enzyme; and
A gene encoding xylose isomerase or a xylose isomerase enzyme. The genetically modified microorganism of claim 1, wherein the gene-silenced synthetic metabolic valve or the enzymatically modified synthetic metabolic valve directs regulation of two or more genes, including:
a gene encoding glucose-6-phosphate dehydrogenase ( zwf ) or a glucose-6-phosphate dehydrogenase ( zwf ) enzyme; and
A gene encoding enoyl-ACP reductase ( fabI ) or an enoyl-ACP reductase ( fabI ) enzyme. The method according to claim 1, wherein the gene-silenced synthetic metabolic valve or the enzymatically modified synthetic metabolic valve is silencing the gene encoding glucose-6-phosphate dehydrogenase ( zwf ); and a genetically modified microorganism consisting of enzymatic modification of glucose-6-phosphate dehydrogenase ( zwf ) and enoyl-ACP reductase ( fabI ) enzymes. The genetic modification according to claim 1, wherein the expression of the xylose reductase, the gene expression-silencing synthetic metabolic valve and the enzymatically modified synthetic metabolic valve are induced under conditions of a transition phase of a multi-step biofermentation process. microorganisms. The genetically modified microorganism of claim 1 , wherein the induction is caused by nutrient depletion. The genetically modified microorganism of claim 1 , wherein the induction is caused by phosphate depletion. The genetically modified microorganism of claim 1 , further comprising a chromosomal defect. The method of claim 1 , wherein the silencing of gene expression comprises CRISPR interference, and the genetically modified microorganism also expresses a cascade guide array, wherein the array targets multiple genes for simultaneous silencing of multiple genes. A genetically modified microorganism comprising two or more genes each encoding a specific small guide RNA. The genetically modified microorganism according to claim 1, wherein the microorganism produces xylitol at a production titer higher than 20 g/L in about 24 hours in a biofermentation process. A multi-step fermentation bioprocess for producing xylitol from genetically modified microorganisms,
(a) expression modification of xylose reductase; and a synthetic metabolic valve; and
(b) growing the genetically modified microorganism in a medium containing a xylose feedstock;
(c) transitioning from the proliferative phase to the xylitol producing phase by inducing the synthetic metabolic valve(s) to slow or stop the growth of the microorganism; and
(d) producing xylitol;
Gene expression-silencing synthetic metabolic valve characterized by silencing of gene expression of one or more genes encoding one or more enzymes, or enzymatically modified synthetic metabolism, characterized in that the synthetic metabolic valve induces enzymatic denaturation of one or more enzymes a valve, or a combination thereof;
A multi-step fermentation bioprocess in which one or more enzymes in each synthetic metabolic valve are the same or different. The method of claim 16, wherein the gene-silent synthetic metabolic valve of the genetically modified microorganism or the enzymatically modified synthetic metabolic valve is a gene encoding glucose-6-phosphate dehydrogenase ( zwf ) or glucose-6-phosphate dehydrogenase hydrogenase ( zwf ) enzyme; gene encoding enoyl-ACP reductase ( fabI ) or enoyl-ACP reductase ( fabI ) enzyme; and a gene encoding xylose isomerase or a xylose isomerase enzyme. 17. The multi-stage fermentation bioprocess of claim 16, wherein the bioprocess produces xylitol at a production titer greater than 20 g/L in about 24 hours in a biofermentation process. 17. The multi-stage fermentation bioprocess according to claim 16, wherein the transition state is caused by phosphate depletion of the culture medium. 17. The multi-stage fermentation bioprocess of claim 16, wherein the genetically modified microorganism further comprises a chromosomal defect. As a genetically modified microorganism for producing xylitol,
reducing the inducibility of xylose isomerase by the microorganism; comprising an inducible decrease in glucose-6-phosphate dehydrogenase activity;
A microorganism, wherein the microorganism produces xylitol from feedstock xylose upon induction. 22. The genetically modified microorganism of claim 21, wherein the microorganism is an E. coli microorganism. 22. The genetically modified microorganism of claim 21, wherein the induction occurs by nutrient depletion. 22. The genetically modified microorganism of claim 21, wherein the induction occurs by phosphate depletion. A multi-step fermentation bioprocess for producing xylitol from the genetically modified microorganism of claim 21,
(a) providing a genetically modified microorganism;
(b) growing the genetically modified microorganism in a medium containing a xylose feedstock;
(c) transitioning from the proliferative phase to the xylitol producing phase by inducing the synthetic metabolic valve(s) to slow or stop the growth of the microorganism; and
(d) a multi-step fermentation bioprocess comprising producing xylitol. As a genetically modified microorganism for producing xylitol,
Reducing the inducibility of xylose reductase by the microorganism; an inducible decrease in glucose-6-phosphate dehydrogenase activity; Including inducible reduction of enoyl-ACP reductase,
A genetically modified microorganism, wherein the microorganism produces xylitol from feedstock xylose upon induction. 27. The genetically modified microorganism of claim 26, wherein the microorganism is an E. coli microorganism. 27. The genetically modified microorganism of claim 26, wherein the induction occurs by nutrient depletion. 27. The genetically modified microorganism of claim 26, wherein the induction occurs by phosphate depletion. A multi-step fermentation bioprocess for producing xylitol from the genetically modified microorganism of claim 26,
(a) providing a genetically modified microorganism;
(b) growing the genetically modified microorganism in a medium containing a xylose feedstock;
(c) transitioning from the proliferative phase to the xylitol producing phase by inducing the synthetic metabolic valve(s) to slow or stop the growth of the microorganism; and
A multi-step fermentation bioprocess comprising inducing the expression of xylose reductase to (d) produce xylitol. As a genetically modified microorganism,
the activity of membrane-bound transhydrogenase is increased; the activity of pyruvate ferredoxin oxidoreductase is increased; NADPH-dependent ferredoxin reductase activity is increased,
A genetically modified microorganism, wherein the microorganism produces one or more chemical products that require NADPH for biosynthesis.

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