Method and composition for producing acetyl-CoA derivatives

Abstract

Genetically engineered microbial strains and related bioprocesses for the production of products from acetyl-CoA. Specifically, the use of dynamically controlled synthetic metabolic valves to reduce the activity of specific enzymes results in increased product production in a two-step process.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of U.S. Provisional Patent Application No. 63 / 056,031, filed on July 24, 2020, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to metabolically engineered microorganisms, such as bacterial strains, and bioprocesses that utilize such strains. These strains provide dynamic control of metabolic pathways that result in the production of products from acetyl - CoA.

[0003] Sequence Listing This application includes a sequence listing that was electronically filed in ASCII format as 49186 - 48_ST25.txt, created on July 13, 2021, with a size of 26,740 bytes, the entire disclosure of which is incorporated herein by reference.

[0004] Research and Development Funded by the Federal Government The present invention was made with government support under NSF EAGER:#1445726, DARPA#HR 0011 - 14 - C - 0075, ONR YIP#N 00014 - 16 - 1 - 2558, DOE EERE Grant#EE 0007563. The government has certain rights in this invention.

Background Art

[0005] Biotechnology-based fermentation processes have made rapid progress in recent years due to technological developments in the fields of fermentation science and synthetic biology, as well as metabolism and enzyme engineering. However, improvements in rate, titer, and yield are often required to enable commercially competitive processes. Most metabolic engineering strategies aimed at improving these metrics rely on the overexpression of desired pathway enzymes and the deletion and / or downregulation of competing biochemical activities. Over the past few decades, stoichiometric models of metabolism have helped shift the field from manipulating gene expression levels to manipulating networks that can be designed to combine growth and product formation, and both can be optimized using selection.

[0006] The remaining limitation of these approaches is the metabolic boundary conditions required for cell proliferation. Dynamic metabolic control, and specifically two-step control, offers potential engineering strategies to overcome these limitations by switching to production states that can push the levels of metabolites and enzymes beyond the boundaries required for proliferation. Significant efforts have been made to develop tools for dynamic metabolic control, including control systems, metabolic valves, and modeling approaches. However, to date, previous studies have primarily focused on flux-dynamically redirecting pathways by switching "off" pathways that stoichiometrically compete with the desired pathway. [Overview of the project] [Means for solving the problem]

[0007] The inventors demonstrated that the increase in stationary-phase flux was due not to a decrease in competing metabolic pathways, but to a dynamic decrease in metabolites acting as feedback regulators of central metabolism. Using two-step dynamic metabolic control, the inventors describe the manipulation of feedback regulation in central metabolism and the improvement of biosynthesis in genetically modified microorganisms. Specifically, the inventors describe the effect of dynamic control on two central metabolic enzymes, citrate synthase and glucose-6-phosphate dehydrogenase, on stationary-phase flux. A decrease in citrate synthase levels leads to a decrease in α-ketoglutarate, an inhibitor of sugar transport, resulting in an increase in glucose uptake and glycolysis fluxes.

[0008] Other methods, features, and / or advantages will become apparent, or will become apparent, upon consideration of the following figures and detailed description. All such additional methods, features, and advantages are contained within this description and are intended to be protected by the attached claims.

[0009] The novel features of the present invention are described in detail in the claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are used, and to the accompanying drawings. [Brief explanation of the drawing]

[0010] [Figure 1] This figure shows a schematic diagram of the pCASCADE control plasmid construction scheme.

[0011] [Figure 2] This figure shows the pCASCADE construction scheme. (2A) Single sgRNA cloning, (2B) Double sgRNA.

[0012] [Figure 3A]This is a schematic diagram of a two-step dynamic control of feedback regulation of central metabolism that improves stationary-phase glucose uptake and acetyl-CoA flux dynamics. The metabolic valve (double triangle) dynamically reduces the levels of Zwf (glucose-6-phosphate dehydrogenase) and GltA (citrate synthase). The reduction in flux by the TCA cycle lowers αKG levels, mitigating feedback inhibition of PTS-dependent glucose uptake and improving glycolysis flux and pyruvate production. The reduction in flux, Zwf, lowers NADPH levels, activates SoxRS oxidative stress response regulation, increases the expression and activity of pyruvate ferredoxin oxireductase, and improves pyruvate oxidation and acetyl-CoA flux. [Figure 3B] This figure shows the time course of two-stage dynamic metabolic regulation during phosphate depletion. Biomass levels accumulate, consuming the limiting nutrient (in this case, inorganic phosphate), and depletion triggers a stationary production phase, while the levels of key enzymes are dynamically reduced by the synthetic metabolic valve (red) (c and d). The synthetic metabolic valve utilizes CRISPRi-based gene silencing and / or controlled proteolysis. [Figure 3C] This figure illustrates how a series of silencing guides can be used to silence multiple target genes of interest (GOIs). This involves the inducible expression of one or more guide RNAs, as well as the expression of a modified native cascade system lacking the cas3 nuclease. The gRNA / cascade complex binds to the target sequence in the promoter region and silences its transcription. [Figure 3D] This figure shows that when a C-terminal DAS+4 tag is added to the target enzyme (EOI) by chromosomal modification, it can be inductively degraded by the clpXP protease in the presence of an inducible sspB chaperone. [Figure 3E]This figure shows the dynamic regulation of protein levels in Escherichia coli (E. coli) using inducible proteolysis and CRISPRi silencing. As cells proliferate, phosphate is depleted, causing the cells to "turn off" mCherry and "turn on" GFPuv. The shaded area represents one standard deviation from the mean, and n=3. [Figure 3F] This figure shows the relative effects of individual and combined protein degradation and gene silencing on mCherry degradation. [Figure 3G] This figure shows the mCherry decay rate. [Figure 3H] This figure shows the dynamic regulation of the levels of central metabolic enzymes. [Figure 3I] This figure shows the dynamic regulation of the levels of key metabolic enzymes. The effects of silencing (pCASCADE) and proteolysis (DAS+4 tagging) on ​​protein levels were evaluated individually and in combination. (h) GltA (citrate synthase) and (i) Zwf (glucose-6-phosphate dehydrogenase). In all cases, chromosomal genes were tagged with C-terminal sfGFP. Protein levels were measured by ELISA 24 hours after induction by phosphate deficiency in microfermentation. Abbreviations are as follows: PTS for phosphotransferase transport system, PPP for pentose phosphate pathway, TCA for tricarboxylic acid, G6P for glucose-6-phosphate, 6-PGL for 6-phosphogluconolactone, 6PG for 6-phosphogluconic acid, PEP for phosphoenolpyruvate, Fd for ferredoxin, CoA for coenzyme A, OAA for oxaloacetate, and αKG for α-ketoglutarate.

[0013] [Figure 4A] The figure shows that the dynamic decrease in GltA reduces the αKG pool, mitigating αKG-mediated inhibition of PTS-dependent glucose uptake (specifically PtsI), and improving glucose uptake rate, glycolysis flux, and pyruvate production. [Figure 4B] This figure shows the effect of dynamic control of GltA and Zwf levels on pyruvate production in minimal medium microfermentation. [Figure 4C] Figure showing the effect of dynamic control of GltA and Zwf levels and dimethyl-αKG supplementation on the glucose uptake rate in microfermentation. [Figure 4D] Figure measuring pyruvate and biomass production for the control strain and the "G" valve strain. The biomass (gray) and pyruvate production (blue) of the control strain, and the biomass (black) and pyruvate production (green) of the "G" valve strain are plotted as a function of time. The dashed line represents the extrapolated growth by the defective sample.

[0014] [Figure 5A] Figure showing that a dynamic decrease in Zwf level activates the SoxRS regulon, increases the activity of pyruvate ferredoxin oxidoreductase (Pfo, ydbK), and improves acetyl-CoA flux and citramalate production. [Figure 5B] Figure showing the effect of dynamic control of GltA and Zwf levels on citramalate production in minimal medium microfermentation. Furthermore, proteolysis of Lpd (lpd-DAS+4, a subunit of the pyruvate dehydrogenase multienzyme complex) and deletion of ydbK were evaluated in the "GZ" valve background. [Figure 5C] Figure measuring citramalate and biomass production for the control strain (c) and the "GZ" valve strain (d). [Figure 5D] Figure measuring citramalate and biomass production for the control strain (c) and the "GZ" valve strain (d). (c) Two runs, gray and black biomass levels, green and blue citramalate titers. (d) Average of three runs, black biomass and green citramalate. The dashed line represents the extrapolated growth by the defective sample.

[0015] [Figure 6A] Figure measuring citramalate and biomass production for the control strain (a), the "G" valve strain (b), and the "GZ" valve strain (c) in fermentation targeting a biomass level of 10 g CDW / L. [Figure 6B] This figure shows the citramaric acid and biomass production measured for the control strain (a), the "G" valve strain (b), and the "GZ" valve strain (c) during fermentation targeting a biomass level of 10 g CDW / L. [Figure 6C] This figure shows the citramaric acid and biomass production measured for the control strain (a), the "G" valve strain (b), and the "GZ" valve strain (c) during fermentation targeting a biomass level of 10 g CDW / L. [Figure 6D] This figure shows the citramaric acid and biomass production measured in control strain (a), "G" valve strain (b), and "GZ" valve strain (c) during fermentation targeting a biomass level of 10 g CDW / L. Two runs are shown, with gray and black representing biomass levels and green and blue representing citramaric acid titers. (d) Citramaric acid production and biomass levels during fermentation targeting a biomass level of 25 g CDW. Average of three runs, with black representing biomass and green representing citramaric acid. The dashed line represents extrapolated growth due to defective samples.

[0016] [Figure 7A] This figure shows an overview of sugar uptake in the PTS-negative strain of Escherichia coli (E. coli). [Figure 7B] This figure shows the dynamic control of citrate synthase (GltA level) and pyruvate production in two-step microfermentation in DLF_00286 strain. [Figure 7C] This figure shows that glucose uptake is insensitive to dimethyl-αKG supplementation in the PTS(-) strain. [Figure 7D] This figure shows the measured pyruvate and biomass production of the DLF_00286 strain and its "G" valve derivative strain.

[0017] [Figure 8A] This figure shows that the acetyl-CoA flux is dependent on Pfo(YdbK) activity. [Figure 8B]This figure shows the relative stationary ydbK enzyme activity as a function of the "G" valve and the "Z" valve. [Figure 8C] This figure shows the NADPH pool (gray bars) and ydbK expression levels (green bars) in the manipulated strain.

[0018] [Figure 9A] This figure shows that the acetyl-CoA flux can be improved independently of the "Z" valve, depending on soxS activation. [Figure 9B] Figure 9B) shows that the acetyl-CoA flux is dependent on soxS activation and can be improved independently of the "Z" valve. 9A) The strain was manipulated for hypophosphatemia induction of SoxS (independent of NADPH pool and SoxR activation). 9B) shows citramaric acid production in microfermentation in the PTS(+) strain manipulated with a combination of the "G" valve and hypophosphatemia-inducible soxS. [Modes for carrying out the invention]

[0019] This invention demonstrates the use of two-step dynamic metabolic regulation to manipulate feedback regulation in central metabolism and improve biosynthesis in genetically modified Escherichia coli (E. coli). Specifically, we report the effects of dynamic regulation on two central metabolic enzymes, citrate synthase and glucose-6-phosphate dehydrogenase, on stationary-phase fluxes. Firstly, a decrease in citrate synthase levels leads to a decrease in α-ketoglutarate, an inhibitor of sugar transport, resulting in an increase in glucose uptake and glycolysis fluxes. Reduced glucose-6-phosphate dehydrogenase activity activates the expression of SoxRS regulon and pyruvate ferredoxin oxireductase, which then leads to a significant increase in acetyl-CoA production. These two mechanisms result in improved stationary-phase production of citramarate, enabling a titer of 126 ± 7 g / L. These results identify pyruvate oxidation via ferredoxin oxireductase as a "central" metabolic pathway during the stationary phase and highlight the potential to improve flux by manipulating essential central regulatory mechanisms using two-step dynamic metabolic control.

[0020] definition As used herein and in the claims, the singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise. For example, a reference to "expression vector" includes a single expression vector as well as multiple expression vectors of the same (e.g., the same operon) or different; a reference to "microorganism" includes a single microorganism as well as multiple microorganisms, and so on.

[0021] As used herein, terms such as “heterogeneous DNA” and “heterogeneous nucleic acid sequence” refer to nucleic acid sequences in which at least one of the following is true: (a) the nucleic acid sequence is exogenous (i.e., naturally occurring) to a given host microorganism; (b) the sequence is naturally occurring in a given host microorganism but in an unnatural (e.g., larger than expected) quantity; or (c) the nucleic acid sequence contains two or more subsequences that are not found in nature in the same relation to each other. For example, with respect to example (c), recombinantly produced heterogeneous nucleic acid sequences have two or more sequences from unrelated genes arranged to create novel functional nucleic acids, such as non-natural promoters that drive gene expression.

[0022] As used herein, terms such as “synthetic metabolic valve” refer to the use of controlled proteolysis, gene silencing, or a combination of both proteolysis and gene silencing to alter metabolic flux.

[0023] The term “heterogeneous” is intended to include the term “exogenous,” as the latter is commonly used in the art. Referring to the genome of a host microorganism before the introduction of a heterogeneous nucleic acid sequence, the nucleic acid sequence encoding the enzyme is heterogeneous (whether or not the heterogeneous nucleic acid sequence is introduced into its genome). As used herein, chromosomes, as well as native and endogenous, refer to the genetic material of a host microorganism.

[0024] As used herein, the term “gene disruption” or its grammatical equivalents (including “disrupting enzyme function,” “disrupting enzyme function,” etc.) is intended to mean a genetic modification of a microorganism such that the encoded gene product has reduced polypeptide activity compared to the polypeptide activity in or derived from an unmodified microbial cell. Genetic modification may be, for example, by the deletion of an entire gene, the deletion or other modification of a regulatory sequence necessary for transcription or translation, the deletion of a portion of a gene resulting in a cleaved gene product (e.g., an enzyme), or by any of the various mutation strategies that reduce the activity of the encoded gene product (including making it undetectable). Disruption can broadly include the deletion of all or part of the nucleic acid sequence encoding an enzyme, and may also include, but are not limited to, other types of genetic modification, such as the introduction of a stop codon, frameshift mutation, introduction or removal of a portion of a gene, and introduction of a degradation signal. These genetic modifications affect mRNA transcription levels and / or stability and modify the promoter or repressor upstream of the enzyme-coding gene.

[0025] The biological production, micro-fermentation, or fermentation as used herein may be aerobic, microaerobic, or anaerobic.

[0026] Where a gene product, i.e., a genetic modification of an enzyme, is referred to herein, including in the claims, the genetic modification is understood to be of the gene product described, i.e., a gene or nucleic acid sequence containing a gene, such as a gene that normally codes for an enzyme.

[0027] As used herein, terms such as “metabolic flux” refer to metabolic changes that result in changes in product and / or byproduct formation, including the rate of production, potency, and yield of production from a given substrate.

[0028] Species and other phylogenetic identifications follow classifications known to those skilled in the art of microbiology.

[0029] Enzymes are listed herein with reference to their UniProt identification numbers, which are known to those skilled in the art. The UniProt database can be accessed at UniProt.org. Where a gene product, i.e., a genetic modification of an enzyme, is referred to herein, including in the claims, the genetic modification is understood to be of a nucleic acid sequence, such as a gene or a gene, that typically codes for the gene product, i.e., the enzyme.

[0030] Where the methods and processes described herein indicate specific events occurring in a particular order, those skilled in the art will recognize that the order of the specific steps can be changed, and such changes constitute modifications of the present invention. Furthermore, the specific steps may be performed sequentially, or simultaneously in parallel processing if possible.

[0031] The meanings of the abbreviations are as follows, and as is clear from their usage, "C" means Celsius or degrees Celsius or °C, DCW means dry cell weight, "s" means seconds, "min" means minutes, "h", "hr" or "hrs" means hours, "psi" means pounds per square inch, "nm" means nanometers, "d" means days, "μL", "uL", or "ul" means microliters, "mL" means milliliters, "L" means liters, "mm" means millimeters, "nm" means nanometers, "mM" means millimoles, "μmol" or "uMol" means micromoles, "g" means grams, "μg" or "ug" means micrograms, "ng" means nanograms, "PCR" means polymerase chain reaction, and "OD" means optical density. 600" means optical density measured at a photon wavelength of 600 nm, "kDa" means kilodalton, "g" means gravitational constant, "bp" means base pair, "kbp" means kilobase pair, "%w / v" means "weight / volume percent", "%v / v" means volume / volume percent, "IPTG" means isopropyl-μ-D-thiogalactopyranoidide, "aTc" means anhydrotetracycline, "RBS" means ribosome binding site, "rpm" means revolutions per minute, "HPLC" means high-performance liquid chromatography, and "GC" means gas chromatography.

[0032] I. Carbon source The biological culture medium used in the present invention with recombinant microorganisms must contain a carbon source or substrate suitable for both the growth and production stages. Suitable substrates may include, but are not limited to, glucose, or combinations of xylose, glucose, sucrose, xylose, mannose, arabinose, oil, carbon dioxide, carbon monoxide, methane, methanol, formaldehyde, or glycerol. All of the above carbon substrates and mixtures thereof are considered suitable as carbon sources for the present invention.

[0033] II. Microorganisms The features described and claimed herein may be provided in microorganisms selected from the list herein, or in other suitable microorganisms, including one or more natural, introduced, or enhanced biological production pathways for products. Thus, in some embodiments, the microorganism includes an endogenous product production pathway (which may be enhanced in some such embodiments), while in other embodiments, the microorganism does not include an endogenous product production pathway.

[0034] More specifically, based on the various criteria described herein, suitable microbial hosts for the biological production of chemical products may generally include, but are not limited to, the organisms described in the Common Methods section.

[0035] The host microorganisms or source microorganisms of any gene or protein described herein are the following list of microorganisms: Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces. The host microorganisms can be selected from Saccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces, and Pseudomonas. In some embodiments, the host microorganism is Escherichia coli.

[0036] III. Culture medium and culture conditions In addition to a suitable carbon source, such as one selected from among the types disclosed herein, the bio-producing medium must contain suitable minerals, salts, cofactors, buffers and other components known to those skilled in the art, suitable for promoting the growth of cultures and the bio-production of chemical products under the present invention.

[0037] Another aspect of the present invention relates to culture media and culture conditions comprising the genetically modified microorganisms of the present invention and optionally supplements.

[0038] Typically, cells are grown in a suitable culture medium at temperatures ranging from approximately 25°C to 40°C, and up to 70°C for thermophilic microorganisms. Suitable growth media are well-characterized and known in the art. The pH range suitable for biological production is pH 2.0 to pH 10.0, with pH 6.0 to pH 8.0 being a typical pH range for initial conditions. However, this does not mean that the actual culture conditions for a particular embodiment are limited by these pH ranges. Biological production can be carried out under aerobic, microaerophilic, or anaerobic conditions, with or without agitation.

[0039] IV. Biological Reactors and Systems Fermentation systems utilizing the methods and / or compositions according to the present invention are also within the scope of the present invention. Any recombinant microorganism described and / or referred to herein can be introduced into industrial bioproduction systems in which the microorganism converts a carbon source into a product in a commercially viable operation. The bioproduction system includes introducing such recombinant microorganisms into a bioreactor vessel using a carbon source substrate and a bioproduction medium suitable for growing the recombinant microorganisms, maintaining the bioproduction system for a suitable temperature range (and dissolved oxygen concentration range if the reaction is aerobic or microaerophilic) for a suitable time to obtain the desired conversion of a portion of the substrate molecules into selected chemical products. Bioproduction can be carried out under aerobic, microaerophilic or anaerobic conditions, with or without stirring. Industrial bioproduction systems and their operation are known to those skilled in the art of chemical engineering and bioprocess engineering.

[0040] The amount of product produced in a biological culture medium can generally be determined using many methods known in the art, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), or GC / mass spectrometry (MS).

[0041] V. Genetic modification, nucleotide sequence, and amino acid sequence Embodiments of the present invention may arise from the introduction of an expression vector into a host microorganism, the expression vector containing a nucleic acid sequence encoding an enzyme that is or is not typically found in the host microorganism.

[0042] The ability to genetically modify host cells is essential for creating any genetically modified (recombinant) microorganism. Modes of gene transfer technology may include electroporation, conjugation, transduction, or spontaneous transformation. A wide range of host-conjugating plasmids and drug resistance markers are available. Cloning vectors are tailored to the host organism based on the properties of the antibiotic resistance markers that can function in that host. Furthermore, as disclosed herein, genetically modified (recombinant) microorganisms may include modifications other than plasmid transfer, including modifications to their genomic DNA.

[0043] More generally, nucleic acid constructs can be prepared that contain isolated polynucleotides encoding an enzymatically active polypeptide, operably linked to one or more (several) control sequences that, under conditions compatible with the control sequences, direct the expression of a coding sequence in a microorganism such as Escherichia coli (E. coli). The isolated polynucleotides can be manipulated to result in polypeptide expression. Depending on the expression vector, manipulation of the polynucleotide sequence before insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well established in the art.

[0044] The regulatory sequence may be a suitable promoter sequence, a nucleotide sequence recognized by the host cell for the expression of the polynucleotide encoding the polypeptide of the present invention. The promoter sequence may include a transcriptional regulatory sequence that mediates polypeptide expression. The promoter may be any nucleotide sequence that exhibits transcriptional activity in a preferred host cell, including mutant promoters, cleaved promoters, and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to the host cell. Techniques for modifying and utilizing recombinant DNA promoter sequences are well established in the art.

[0045] In various embodiments of the present invention, genetic manipulation may include operations aimed at regulating enzymes, and therefore their final activity, or altering the enzymatic activity of enzymes identified in any of the respective pathways. Such genetic modifications may target transcription, translation, and post-translational modifications that result in changes in enzyme activity and / or selectivity under selected culture conditions. Genetic manipulation of nucleic acid sequences may include increasing the copy number and / or using variants of enzymes associated with product production. Specific methods and approaches for achieving such genetic modifications are known to those skilled in the art.

[0046] In various embodiments, microorganisms may contain one or more gene deletions to function more efficiently. For example, in Escherichia coli (E. coli), genes encoding 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 transcription bimodifier (arcA), and iclR transcription regulator (iclR) can be disrupted, including by deletion. Such gene disruption, including deletion, is not intended to be limiting and can be carried out in various combinations in various embodiments. Gene deletion can be achieved by a number of strategies known in the art, as well as methods for incorporating foreign DNA into host chromosomes.

[0047] In various embodiments, to function more efficiently, microorganisms may include one or more synthetic metabolic valves composed of enzymes targeting controlled proteolysis, expression silencing, or a combination of both controlled proteolysis and expression silencing. For example, a single enzyme encoded by one gene in Escherichia coli (E. coli) or a combination of multiple enzymes encoded by multiple genes can be designed as a synthetic metabolic valve to alter metabolism and improve product formation. Representative genes in Escherichia coli (E. coli) include, but are not limited to, fabI, zwf, gltA, ppc, udhA, lpd, sucD, aceA, pfkA, lon, rpoS, pykA, pykF, tktA, or tktB. It is understood that methods for identifying homologs of these genes and / or other genes in further microbial species are known to those skilled in the art.

[0048] All nucleic acids and amino acid sequences provided herein include conservatively modified variants of these sequences, which are understood to be within the scope of the present invention in various embodiments. Functionally equivalent nucleic acids and amino acid sequences (functional variants) may include conservatively modified variants, as well as more broadly variable sequences that are well within the skill of those skilled in the art, and microorganisms containing these, and as well as methods and systems containing such sequences and / or microorganisms, are within the scope of various embodiments of the present invention.

[0049] Accordingly, as described in the various sections above, some compositions, methods, and systems of the present invention include providing genetically modified microorganisms that include both a synthetic metabolic valve for redistributing flux and a production pathway for producing a desired product from a central intermediate.

[0050] Aspects of the present invention also relate to providing a number of genetic modifications for improving the overall effectiveness of microorganisms in converting a selected carbon source into a selected product. Specific combinations for increasing specific productivity, volume productivity, potency, and yield, beyond more basic combinations of genetic modifications, are shown in examples, etc.

[0051] In addition to the gene modifications described above, various embodiments also provide gene modifications that include synthetic metabolic valves to increase or decrease the pool and availability of cofactors such as NADPH and / or NADH that can be consumed in the production of the product.

[0052] VI. Synthetic metabolic valve

[0053] The use of synthetic metabolic valves allows for a simpler modeling of metabolic fluxes and physiological requirements during the production phase, transforming proliferating cells into stationary-phase biocatalysts. These synthetic metabolic valves can be used in multi-step fermentation processes to switch off essential genes and redirect carbon, electron, and energy fluxes towards product formation. One or more of the following—1) transcription gene silencing or repression techniques, 2) inducible and selective enzymatic degradation, and 3) nutrient restriction to induce a stationary or non-dividing cell state—provide the described synthetic valves. SMVs can be generalized to any pathway and microbial host. These synthetic metabolic valves enable novel, rapid metabolic engineering strategies useful for the production of renewable chemicals and fuels, as well as any product that can be produced by whole-cell catalysis.

[0054] In particular, the present invention describes the construction of a synthetic metabolic valve comprising one or more or a combination of controlled gene silencing and controlled proteolysis. It will be understood that those skilled in the art are aware of several methods for gene silencing and controlled proteolysis.

[0055] VI.A Gene Silencing In particular, the present invention describes the use of controlled gene silencing to provide control of metabolic fluxes in controlled multi-step fermentation processes. There are several methods known in the art for controlled gene silencing, including but not limited to mRNA silencing or RNA interference, transcriptional repressor-mediated silencing, and CRISPR interference. Methods and mechanisms for RNA interference are taught by Agrawal et al. "RNA Interference: Biology, Mechanism, and Applications" Microbiology and Molecular Biology Reviews, December 2003;67(4)p657-685.DOI:10.1128 / MMBR.67.657-685.2003. Methods and mechanisms for CRISPR interference are taught by Qi et al., "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression," Cell February 2013;152(5)p1173-1183.DOI:10.1016 / j.cell.2013.02.022. Furthermore, methodologies and mechanisms for CRISPR interference using a native Escherichia coli (E. coli) CASCADE system are taught by Luo et al., "Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression," NAR.October 2014;DOI:10.1093. Numerous other transcriptional repressor systems are well known in the art and can be used to turn off gene expression.

[0056] VI.B Regulated Proteolysis In particular, the present invention describes the use of controlled protein degradation or proteolysis to provide control of metabolic flux in controlled multi-step fermentation processes. There are several methods known in the art for controlled proteolysis, including, but not limited to, controlled targeting of proteins for targeted protein cleavage by specific proteases and degradation by specific peptide tags. A system for the use of *E. coli* clpXP protease for controlled proteolysis is taught by McGinness et al. "Engineering controllable protein degradation" Mol Cell. June 2006;22(5)p701-707. This method relies on the addition of a specific C-terminal peptide tag, such as a DAS4 (or DAS+4) tag. Proteins with this tag are not degraded by the clpXP protease until the specificity-enhancing chaperone sspB is expressed. sspB induces degradation of DAS4-tagged proteins by the clpXP protease. For numerous other sites, specific protease systems are known in the art. Proteins can be engineered to contain a specific target site for a given protease and then cleaved after controlled expression of the protease. In some embodiments, cleavage can be expected to result in protein inactivation or degradation. For example, Schmidt et al. ("ClpS is the recognition component for Escherichia coli substrates of the N-end rule degradation pathway" Molecular Microbiology March 2009.72(2),506-517.doi:10.1111) teach that an N-terminal sequence can be added to a protein of interest to provide clpS-dependent clpAP degradation. Furthermore, this sequence can be further masked by an additional N-terminal sequence that can be controllly cleaved by ULP hydrolase, for example.This enables controlled N-terminal degradation dependent on hydrolase expression. Therefore, it is possible to tag proteins for controlled proteolysis at either the N-terminus or the C-terminus. The choice between using an N-terminal or C-terminal tag largely depends on which tag affects protein function before controlled degradation initiation.

[0057] This invention describes the use of controlled protein degradation or proteolysis in Escherichia coli (E. coli) to provide control of metabolic fluxes in a controlled multi-step fermentation process. Several methods for controlled proteolysis in other microbial hosts, including a wide range of Gram-negative and Gram-positive bacteria, yeasts, and even archaea, are known in the art. In particular, systems for controlled proteolysis can be transferred from native microbial hosts and used in non-native 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, teaches the expression and use of the E. coli (E. coli) clpXP protease in yeast (Saccharomyces cerevisiae). Such approaches can be used to transfer methods for synthetic metabolic valves to any genetically manageable host.

[0058] Control of the VI.C Synthetic Metabolism Valve In particular, the present invention describes the use of synthetic metabolic valves for controlling metabolic flux in multi-stage fermentation processes. There are numerous methods known in the art for inducing expression that can be used in the transitions between stages in multi-stage fermentation. These include, but are not limited to, artificial chemical inducers, including tetracycline, anhydrotetracycline, lactose, IPTG (isopropyl-β-D-1-thiogalactopyranoside), arabinose, raffinose, and tryptophan. Systems that link the use of these known inducers to the control of gene expression silencing and / or controlled proteolysis can be incorporated into genetically modified microbial systems to control the transition between the growth phase and the production phase in multi-stage fermentation processes.

[0059] Furthermore, it may be desirable to control the transition between growth and production in multi-stage fermentation by depleting one or more restriction nutrients consumed during growth. Restriction nutrients may include, but are not limited to, phosphates, nitrogen, sulfur, and magnesium. Using natural gene expression systems that respond to these nutrient restrictions, gene expression silencing and / or controlled proteolysis can be operatively linked to the transition between growth and production phases in the multi-stage fermentation process.

[0060] The scope of the present invention includes genetically modified microorganisms that can produce products at specific rates selected from rates greater than 0.05 g / gDCW-hr, greater than 0.08 g / gDCW-hr, greater than 0.1 g / gDCW-hr, greater than 0.13 g / gDCW-hr, greater than 0.15 g / gDCW-hr, greater than 0.175 g / gDCW-hr, greater than 0.2 g / gDCW-hr, greater than 0.25 g / gDCW-hr, greater than 0.3 g / gDCW-hr, greater than 0.35 g / gDCW-hr, greater than 0.4 g / gDCW-hr, greater than 0.45 g / gDCW-hr, or greater than 0.5 g / gDCW-hr.

[0061] In various embodiments, the present invention comprises a culture system comprising a carbon source in an aqueous medium and a genetically modified microorganism described in any one of the claims herein, wherein the genetically modified microorganism is present in an amount selected from more than 0.05 g DCW / L, more than 0.1 g DCW / L, more than 1 g DCW / L, more than 5 g DCW / L, more than 100,000 L, or more than 200,000 L, such as when the volume of the aqueous medium is selected from more than 5 mL, more than 100 mL, more than 0.5 L, more than 1 L, more than 2 L, more than 10 L, more than 250 L, more than 1,000 L, more than 10,000 L, more than 50,000 L, more than 100,000 L, or more than 200,000 L, such as when the volume of the aqueous medium is more than 250 L and is contained in a steel container.

[0062] Summary of the Invention In one embodiment, a genetically modified microorganism usable in a biofermentation process is provided, the microorganism comprising a production pathway including at least one enzyme for producing a product from an acetyl-CoA precursor. The microorganism is induced into a stationary phase or non-dividing cell state under conditions that deplete restriction nutrients from the growth medium in which the genetically modified microorganism is growing. In this stationary phase, pyruvate flavodoxin / ferredoxin oxireductase enzyme activity is increased in the genetically modified microorganism under aerobic or partially aerobic conditions during the stationary phase or non-dividing cell state to produce an acetyl-CoA pool, and further sugar uptake is enhanced in the genetically modified microorganism compared to a non-genetically modified microorganism.

[0063] In one embodiment, a genetically modified microorganism includes a conditionally triggered synthetic metabolic valve that silences the gene expression of citrate synthase (gltA) and / or glucose-6-phosphate dehydrogenase (zwf) genes, or a conditionally triggered synthetic metabolic valve that enables selective proteolysis of the citrate synthase (gltA) and / or glucose-6-phosphate dehydrogenase (zwf) enzymes, the synthetic metabolic valve of the microorganism being conditionally triggered during a stationary phase or non-dividing cell state.

[0064] In one embodiment, the genetically modified microorganism includes deletions of the endogenous poxB and pflB genes.

[0065] In one embodiment, the increased activity of the pyruvate flavodoxin / ferredoxin oxireductase enzyme in genetically modified microorganisms is attributed to the overexpression of the gene encoding pyruvate ferredoxin oxireductase during the stationary phase or non-dividing cell state.

[0066]

[0067] In one embodiment, the pyruvate flavodoxin / ferredoxin oxireductase enzyme activity is increased in a genetically modified microorganism, the pyruvate flavodoxin / ferredoxin oxireductase enzyme is encoded by the ydbK gene, and the genetically modified microorganism is an Enterobacter microorganism.

[0068] In one embodiment, the increased pyruvate flavodoxin / ferredoxin oxireductase enzyme activity in genetically modified microorganisms is attributed to the induction of oxidative soxRS regulon during the stationary phase or non-dividing cell state.

[0069] In one embodiment, the increased pyruvate flavodoxin / ferredoxin oxireductase enzyme activity in genetically modified microorganisms is a result of a decrease in NADPH levels within the genetically modified microorganisms during the stationary phase or non-dividing cell state.

[0070] In one embodiment, the activity of at least one sugar transporter in a genetically modified microorganism is increased to enhance sugar uptake.

[0071] In one embodiment, the activity of at least one sugar transporter in a genetically modified microorganism results in increased sugar transporter activity within the genetically modified microorganism as a result of constitutive expression of the sugar transporter gene.

[0072] In one embodiment, the activity of at least one sugar transporter in a genetically modified microorganism is a result of conditional overexpression during the stationary phase or non-dividing cell state.

[0073] In one embodiment, the sugar transporter of a genetically modified microorganism is encoded by a pts gene.

[0074] In one embodiment, the genetically modified microorganism is an Enterobacter microorganism. In another embodiment, the microorganism is an E. coli microorganism.

[0075] In one embodiment, the genetically modified microorganism contains citramalate synthase as an enzyme in the production pathway.

[0076] In one embodiment, a bioprocess for producing protein products from genetically modified microorganisms is provided. The bioprocess includes, in the first step, growing the genetically modified microorganisms in a culture medium, and in the second step, inducing a stationary phase or non-dividing cell state when the restriction nutrients from the growth medium are depleted. The bioprocess, consisting of genetically modified microorganisms in the stationary phase or non-dividing cell state, produces products at a rate of 30 g / L or more.

[0077] In another embodiment, the bioprocess includes the activity of the pyruvate flavodoxin / ferredoxin oxireductase enzyme caused by overexpression of a gene encoding active pyruvate ferredoxin oxireductase, induction of an oxidative soxRS regulon, a decrease in NADPH levels, a decrease in glucose-6-phosphate dehydrogenase levels by a synthetic metabolic valve targeting gene silencing of the zwf gene or selective proteolysis of the glucose-6-phosphate dehydrogenase enzyme, a valve activated in the stationary phase or non-dividing cell state, or a combination thereof.

[0078] In one embodiment, the bioprocess involves increased activity of at least one sugar transporter.

[0079] In one embodiment, the bioprocess yields a citramaric acid product, the enzyme in the production pathway includes citramaric acid synthase, and the bioprocess produces 100 g / L or more of citramaric acid. In one embodiment, the citramaric acid synthase enzyme is encoded by the cimA3.7 gene.

[0080] In one embodiment, the genetically modified microorganism for the bioprocess comprises a plasmid containing a citramarate synthase gene operably linked to a low-phosphate-inducible promoter.

[0081] In one embodiment, the bioprocess includes the use of genetically modified microorganisms containing deletions of endogenous poxB and pflB genes.

[0082] The disclosed embodiments are not limiting.

[0083] While various embodiments of the present invention have been shown and described herein, it is emphasized that such embodiments are provided only as examples. In these various embodiments, numerous modifications, changes, and substitutions can be made without departing from the present invention as herein. Specifically, for any reason whatsoever, any grouping of compounds, nucleic acid sequences, polypeptides containing specific proteins including functional enzymes, metabolic enzymes or intermediates, elements or other compositions, or concentrations, described herein in lists, tables or other groups (such as metabolic enzymes shown in Figures 3A, 4A, and 5A), or otherwise presented, unless otherwise explicitly stated, each such grouping is intended to provide a basis for various subset embodiments and to help identify them, and the broadest subset embodiment is intended to include all subsets of such grouping by excluding one or more members (or subsets) of the grouping described, respectively. Furthermore, where any range is described herein, unless otherwise explicitly stated, that range includes all values ​​within it and all subranges within it.

[0084] More generally, conventional molecular biology, cell biology, microbiology, and recombinant DNA techniques can be used within the scope of the art of those skilled in the art, as disclosed, discussed, examples, and embodiments herein. Such techniques are fully described in the literature. For example, see Sambrook and Russell, "Molecular Cloning: A Laboratory Manual," Third Edition 2001 (volumes 1-3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Animal Cell Culture, R. Freshney, ed., 1986. These published resources are incorporated herein by reference.

[0085] The following publicly available resources are incorporated herein by reference as useful in conjunction with the invention described herein, for example, with respect to methods for industrially bio-producing chemical products from sugar sources and industrial systems that can be used to achieve such conversions (for example, with respect to the design of bioreactors, Biochemical Engineering Fundamentals, 2 nd See Chapter 9, pages 533-657, Ed. JEBailey and DFOllis, McGraw Hill, New York, 1986, for example, regarding process and separation technology analysis, Unit Operations of Chemical Engineering, 5 th Ed., WLMcCabe et al., McGraw Hill, New York 1993, for example, regarding the teaching of separation techniques, Equilibrium Staged Separations, PCWankat, Prentice Hall, Englewood Cliffs, NJ USA, 1988).

[0086] All publications, patents, and patent applications referenced herein, including International Application US2015 / 035306 filed on 11 June 2015 and International Application US2018 / 019040 filed on 21 February 2018, are incorporated herein in full by reference.

[0087] example The examples provided herein are not intended to be limiting and are merely a selection of examples. All reagents are commercially available unless otherwise specified. Species and other phylogenetic identifications follow classifications known to those skilled in the art of microbiology, molecular biology, and biochemistry.

[0088] General method

[0089] Culture medium and reagents

[0090] Unless otherwise specified, all materials and reagents were purchased from Sigma (St. Louis, Missouri). Lennox LB preparations were used for routine strain and plasmid growth and construction. FGM1, FGM30, and SM10++ seed media were prepared as previously described by Menacho-Melgar et al. (doi:10.1101 / 820787). SM10++ and SM10 phosphate-free media were prepared as previously described by Moreb et al. (doi:10.1021 / acssynbio.0c00182). FGM3 medium used in biolector studies is detailed in the supplementary materials. The antibiotic concentrations used were as follows: kanamycin 35 μg / mL, chloramphenicol 35 μg / mL, zeosin 100 μg / mL, blasticidine 100 μg / mL, spectinomycin 25 μg / mL, and tetracycline 5 μg / mL.

[0091] FGM3 medium / concentrated medium solution

[0092] Mix 30g of (NH4)2SO4 and 1.5g of citric acid with water while stirring, and adjust the pH to 7.5 with NaOH to obtain 10-fold concentrated ammonium citrate 30 salt (1L). Autoclave and store at room temperature (RT).

[0093] Mix 90g of (NH4)2SO4 and 2.5g of citric acid with water while stirring, and adjust the pH to 7.5 with NaOH to obtain 10-fold concentrated ammonium citrate 90 salt (1L). Autoclave and store at room temperature.

[0094] 1M potassium 3-(N-morpholino)propanesulfonic acid (MOPS), adjusted to pH 7.4 with KOH. Sterilize by filtration (0.2 μm) and store at room temperature.

[0095] Prepare a 0.5M potassium phosphate buffer, pH 6.8, by mixing 248.5 mL of 1.0 M K2HPO4 and 251.5 mL of 1.0 M KH2PO4 and adjusting the final volume to 1000 mL with ultrapure water. Filter sterilize (0.2 μm) and store at room temperature.

[0096] 2M MgSO4 and 10mM CaSO4 solutions. Sterilize by filtration (0.2μm) and store at room temperature.

[0097] A 50 g / L solution of thiamine-HCl. Sterilize by filtration (0.2 μm) and store at 4°C.

[0098] A 500 g / L glucose solution, dissolved by heating and stirring. Cool, filter sterilize (0.2 μm), and store at room temperature.

[0099] 500x Trace Metal Stock: Prepare a micronutrient solution in 1000 mL of water containing 10 mL of concentrated H2SO4. The components are: 0.6 g CoSO4·7H2O, 5.0 g CuSO4·5H2O, 0.6 g ZnSO4·7H2O, 0.2 g Na2MoO4·2H2O, 0.1 g H3BO3, and 0.3 g MnSO4·H2O. Sterilize by filtration (0.2 μm) and store at room temperature in a dark place.

[0100] Prepare a fresh aqueous solution of 40 mM ferric sulfate heptahydrate and filter it (0.2 μm) each time before preparing the culture medium.

[0101] Culture medium components: Prepare the final working medium by aseptically mixing the stock solutions according to the table below in the order listed to minimize precipitation, and then filter sterilize (using a 0.2 μm filter).

[0102] [Table 1]

[0103] Modified stock

[0104] [Table 2]

[0105] [Table 3]

[0106] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7]

[0107] Strains and plasmids

[0108] Plasmid and strain information is shown in Tables 2-4. Sequences of oligonucleotides and synthetic linear DNA (Gblocks™) were obtained from Integrated DNA Technologies (IDT, Coralville, Iowa). Deletions were constructed by tet-sacB-based selection and reverse selection. C-terminal DAS+4 tags (with or without superfolder GFP tags) were added to chromosomal genes by direct insertion, and selection was performed by insertion of the 3' antibiotic resistance cassette of the gene. All strains were confirmed by PCR and agarose gel electrophoresis, and sequenced using adjacent pairs of oligonucleotides (Eton Biosciences or Genewiz) for the entire region. Recombinant plasmid pSIM5 and tet-sacB selection / reverse selection marker cassettes were obtained from Donald Court (NCI, redrecombineering.ncifcrf.gov / court-lab.html). Strain BW25113 was obtained from the Yale Genetic Stock Center (CGSC: cgsc.biology.yale.edu). Strain DLF_R002 was constructed as previously reported by Menacho-Melgar et al. (doi: 10.1101 / 820787). Strain DLFZ_0025 was constructed from DLF_R002 by first deleting the native sspB gene (using tet-sacB-based selection and reverse selection). Subsequently, the cas3 gene was deleted and replaced with a hypophosphate-inducible sspB (using the ugpB gene promoter) allele and a constitutive promoter to drive Cascade operon expression (again using tet-sacB-based selection and reverse selection). The C-terminal DAS+4 tag modification (with or without a superfolder GFP tag) was added to the chromosomes of DLF_Z0025 and its derivatives by incorporation, and selection was made by incorporation of the gene's antibiotic resistance cassette 3'.

[0109] Plasmids pCDF-ev (Addgene#89596), pHCKan-yibDp-GFPuv (Addgene#127078), and pHCKan-yibDp-cimA3.7 (Addgene#134595) were constructed as previously reported (doi:10.1101 / 820787). Plasmids pCDF-mCherry1 (Addgene#87144) and pCDF-mCherry1 (Addgene#87145) were constructed from pCDF-ev by PCR and Gibson assembly, along with synthetic DNA encoding an mCherry open reading frame with or without a C-terminal DAS+4 degron tag, using the potent synthetic constitutive proD promoter previously reported by Davis et al.

[0110] Gene silencing guides and guide arrays were expressed from a series of pCASCADE plasmids. A pCASCADE control plasmid was prepared by replacing the pTet promoter in pcrRNA.Tet (acquired from C. Beisel) with an isolated hypophosphate-inducible ugpB promoter. To design the CASCADE guide array, the CASCADE PAM site near the -35 or -10 box of the promoter of interest was identified, the 30 bp 3' end of the PAM site was selected as the guide sequence, and cloned into the pCASCADE plasmid using Q5 site-directed mutagenesis (NEB, Massachusetts) according to the manufacturer's protocol, with the addition of 5% v / v DMSO to the Q5 PCR reaction as a modification. The PCR cycle was as follows: amplification followed an initial denaturation step of 30 seconds at 98°C, followed by 25 cycles of 10 seconds at 98°C, 30 seconds at 72°C, and 1.5 minutes at 72°C (extension rate was 30 seconds / kb), followed by a final extension of 2 minutes at 72°C. Two μL of PCR mixture was used in a 10 μL KLD reaction (NEB, Massachusetts), which was allowed to proceed at room temperature for 1 hour. Subsequently, one μL of the KLD mixture was used for electroporation. The pCASCADE guide array plasmid (pCASCADE-G2Z) was prepared by sequentially amplifying the complementary half of each smaller guide plasmid by PCR, followed by DNA assembly as shown in the table. The primers and gRNA sequences used for pCASCADE assembly are provided in Supplementary Table 5 below. Furthermore, all strains containing the gRNA plasmid were routinely examined, and gRNA stability was evaluated by PCR as described below.

[0111] [Table 5]

[0112] bioLector research

[0113] A single colony from each strain was inoculated into 5 mL of LB containing the appropriate antibiotic and cultured at 37°C and 220 rpm for 9 hours, or until OD 600 reached >2. 500 μL of the culture was inoculated into 10 mL of SM10 medium containing the appropriate antibiotic and cultured in a square shaking flask (CAT#: 25-212, Genesee Scientific, Inc., San Diego, California) at 37°C and 220 rpm for 16 hours. Cells were pelletized by centrifugation, and the culture density was normalized to OD600=5 using FGM3 medium. Growth and fluorescence measurements were obtained using a Biolector (m2p labs, Besweiler, Germany) with a high-mass-transfer FlowerPlate (catalog number: MTP-48-B, m2p-labs, Germany). 40 μL of the OD-normalized culture was inoculated into 760 μL of FGM3 medium containing the appropriate antibiotic. The Biolector settings were as follows: RFP increase = 100, GFP increase = 20, biomass increase = 20, shaking speed = 1300 rpm, temperature = 37°C, humidity = 85%. Each strain was analyzed in three consecutive samples.

[0114] ELISA

[0115] Protein quantification via C-terminal GFP tag was performed using AbCam's GFP quantification kit (Cambridge, UK, product number ab171581) according to the manufacturer's instructions. Briefly, samples were obtained from micro-fermentation as described above. Cells were harvested 24 hours after phosphate depletion, washed with water, and lysed in the provided extraction buffer.

[0116] Guide RNA stability testing

[0117] The stability of the guide RNA array was confirmed by colony PCR using the following two primers, gRNA-for:5'-GGGAGACCACAACGG-3' (SEQ ID NO: 25) and gRNA-rev:5'-CGCAGTCGAACGACCG-3' (SEQ ID NO: 26), with 10 μL of PCR reaction mixture consisting of 5 μL of 2X EconoTaq Master mix (Lucigen), 1 μL of each primer (10 μM), and 3 μL of dH2O. After initial denaturation at 98°C for 2 minutes, 35 cycles were performed at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, followed by final extension at 72°C for 5 minutes. The PCR reaction mixture was then run on an agarose gel, and the band size was compared with a control PCR reaction using purified plasmid DNA as a template. When the guide array size was smaller than expected, loss of guide protospacers occurred, showing loss of one or more protospacers.

[0118] fermentation

[0119] Minimal medium microfermentation was performed as previously reported (doi:10.1021 / acssynbio.0c00182). For microfermentation using paraquat induction, paraquat was added 1 hour before phosphate depletion and subsequently removed during the cell washing step used to deplete phosphate in the medium. 1L fermentation in an instrumented bioreactor was also performed as previously reported, with slight modifications to the glucose supply profile, which is a function of the strain and process. In general, the supply was increased to allow for excess residual glucose to ensure that the production rate was not limited by the supply. The glucose supply was as follows: For 10g CDW / L fermentation, the starting batch glucose concentration was 25g / L. When the cells entered metaphase of the exponential growth phase, a constant concentrated sterile filtered glucose supply (500g / L) was added to the tank at 1.5g / h. For 25g CDW / L fermentation, the starting batch glucose concentration was 25g / L. When the cells entered metaphase of the exponential growth phase, a concentrated, sterile, filtered glucose feed (500 g / L) was added to the tank at an initial rate of 9 g / hr. This rate was then increased exponentially, doubling every 1.083 hours (65 minutes) until a total of 40 g of glucose had been added, after which the feed rate was maintained at 1.75 g / hr.

[0120] Production of isotope-labeled metabolites.

[0121] C 13 Pyruvate (CLM-1082-PK) and C 13 D-glucose (U-13C6, 99%) was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, Massachusetts). Isotope-labeled citramarate was produced using the DLF_Z0044 strain expressing cimA3.7 in a two-step minimal medium shaking flask study mimicking microfermentation. Briefly, 20 mL of culture in SM10++ medium was inoculated with a strain that had been grown overnight at 37°C in a 250 mL baffled Erlenmeyer shaking flask with shaking at 150 rpm. After 16 hours of growth, cells were harvested by centrifugation, washed, and glucose was added to C. 13The cells were resuspended in 20 mL of SM10 minimal medium (phosphate-free) replaced with labeled glucose. The cultures were grown at 37°C for 25 hours with shaking at 150 rpm, after which the cells were removed by centrifugation, and the used medium was sterilized before being used as an internal standard.

[0122] Analysis method

[0123] Cell dry weight: The OD / cell dry weight correlation coefficient (1 OD (600 nm) = 0.35 g CDW / L) determined by Menacho-Melgar et al. was used in this study.

[0124] Determination of glucose and organic acids: Two methods were used to determine glucose and organic acids. First, a UPLC-RI method was developed for the simultaneous determination of glucose, citramalic acid, acetic acid, pyruvate, citraconic acid, citric acid, and other organic acids including lactic acid, succinic acid, fumaric acid, malic acid, and mevalonate. Chromatographic separation was performed at 55°C using a Rezex Fast Acid Analysis HPLC column (100 × 7.8 mm, particle size 9 μm, CAT#:#1250100, Bio-Rad Laboratories, Inc., Hercules, California). 5 mM sulfuric acid was used as the constant composition eluent, and the flow rate was X mL / min. The sample injection volume was 10 μL. Next, quantification was performed at 65°C using a Bio-Rad Fast Acid Analysis HPLC column (100 × 7.8 mm, particle size 9 μm, CAT#:#1250100, Bio-Rad Laboratories, Inc., Hercules, California). 10 mM sulfuric acid was used as the eluent, and the constant composition flow rate was 0.3 mL / min. In both methods, the sample injection volume was 10 μL, and chromatography and detection were performed using a Waters 2414 Refractive Index (RI) detector (Waters Corp., Milford, Massachusetts, USA) integrated with a Waters Acquity H-Class UPLC. The sample was diluted as needed to reach a precise linear range. Dilution was performed using ultrapure water.

[0125] Quantitative determination of organic acids by RapidFire-qTOF-MS: Micro-fermentation samples (and confirmation subsets of samples from bioreactors) were centrifuged to remove cells. The broth was diluted 100-fold with water to a final volume of 20 μL. To this, either 10 mg / L of C13 pyruvate was added, or 2 μL of broth containing C13-labeled citramaric acid was added. The final sample was injected into a HILIC (H1 type or equivalent H6) RapidFire® cartridge (Agilent Technologies, Santa Clara, California). After aspiration for 600 ms, the injector was filled into the cartridge containing 95% hexane and 5% isopropanol over 3000 ms at a flow rate of 1.0 mL / min. After filling, the cartridge was washed with isopropanol for 2000 ms at a flow rate of 1.0 mL / min. Elution was performed for 8000 ms at a flow rate of 1.0 mL / min using 50% methanol containing 50% water / 0.2% acetic acid and 0.5 μM (NH4)3PO4. Column equilibration was performed for 4000 ms. qTOF was adjusted to a mass range of 50–250 m / z in fragile ion, negative ESI mode. Detection settings were: dry gas flow rate 13 L / min at 250 C, sheath gas flow rate 12 L / min at 400 C, nebulizer pressure 35 psi, fragmenter voltage 100 V, skimmer voltage 65 V, nozzle voltage 2000 V, and capillary voltage 3500 V. The acquisition rate was 1 spectrum / second.

[0126] Example 1: Gene silencing array and pathway expression construct pCASCADE guide array-based gene silencing

[0127] The design and structure of the CASCADE guide and guide array are shown in Figures 1 and 2 below. The pCASCADE control plasmid was prepared by replacing the pTet promoter in pcrRNA.Tet with an isolated hypophosphate-inducible ugpB promoter, as shown in Figure 1. Two promoters were responsible for regulating the gltA gene, and sgRNAs were designed for both promoters. During the exponential growth phase, gapA mRNA was mainly initiated by the highly efficient gapA P1 promoter and remained high during the stationary phase compared to the other three gapA promoters; therefore, four promoters were involved in regulating the gapA gene, and an sgRNA was designed for the first promoter. Multiple promoters upstream of the lpd gene are involved in lpd regulation (ecocyc.org / gene?orgid=ECOLI&id=EG 10543#tab=showAll), and therefore, it was not possible to design a unique and effective sgRNA for lpd alone. The promoter sequences for fabI, udhA, and zwf were obtained from the EcoCyc database (ecocyc.org). To design the CASCADE guide array, the CASCADE PAM site near the -35 or -10 box of the desired promoter was identified, the 30 bp at the 3' end of the PAM site was selected as the guide sequence, and cloned into pCASCADE plasmids using Q5 site-directed mutagenesis (NEB, Massachusetts) according to the manufacturer's protocol, with the addition of 5% v / v DMSO to the Q5 PCR reaction as a modification. The pCASCADE-control vector was used as a template. pCASCADE plasmids with two or more guide arrays are described below and prepared as shown in Figure 2. The pCASCADE guide array plasmids were prepared by sequentially amplifying complementary halves of each smaller guide plasmid by PCR, followed by DNA assembly. Tables 6 and 7 list the sgRNA guide sequences and the primers used to construct them. All pCASCADE silencing plasmids are listed in the table below and are available from Addgene.

[0128] [Table 6]

[0129] [Table 7]

[0130] Example 2: Dynamic control at the protein level

[0131] Plasmids expressing fluorescent proteins and silencing guides were transformed into the corresponding host strains listed in Table 2. The strains were evaluated in triplicate using the m2p-labs Biolector®, which simultaneously measures fluorescence including GFPuv, mCherry levels, and biomass levels. The results are shown in Figure 5.

[0132] [Table 8]

[0133] The OD600 reading is corrected using the following formula, where OD600 refers to the offline measurement. * t0 indicates the Biolector biomass reading, t0 indicates the starting point, and tf indicates the ending point.

[0134]

number

[0135] Example 3: The effect of dynamic regulation of two central metabolic pathways, TCA and PPP, on flux induced by glycolysis and pyruvate oxidation.

[0136] As shown in Figure 3A, the dynamic control of two central metabolic pathways (the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway (PPP)) to the flux mediated by glycolysis and pyruvate oxidation is desirable. We have achieved this by constructing a synthetic metabolic valve that dynamically reduces the levels of the first committed step in each pathway, namely citrate synthase (GltA, "G", encoded by the gltA gene) and glucose-6-phosphate dehydrogenase (Zwf, "Z", encoded by the zwf gene). We demonstrate that dynamic control of these two enzymes improves stationary-phase production of pyruvate and citramarate and is applicable to the production of numerous products requiring pyruvate and / or acetyl-CoA.

[0137] The inventors first developed a control system capable of dynamically reducing protein levels in a two-step process, as shown in Figures 3B–3D. The valve can include controlled proteolysis or CRISPRi / cascade-based gene silencing, or both proteolysis and silencing combined to reduce levels of critical metabolic enzymes. Induction is carried out using phosphate depletion as an environmental trigger. A naturally occurring E. coli (E. coli) IE-type cascade / CRISPR system is used for gene silencing (Figures 3Ci–iii). Targeted proteolysis is carried out by linking the expression of the chaperone SspB to phosphate depletion. When induced, SspB binds to the C-terminal DAS+4 peptide tag on any target protein, causing degradation by the E. coli (E. coli) ClpXP protease (Figure 3D). Using the engineered strain, protein levels can be controlled in a two-step process, as demonstrated in Figure 1E, as exemplified by turning GFP "on" and constitutively expressing mCherry "off". In this case, the combination of gene silencing and proteolysis yields the maximum proteolysis rate (Figures 3F-3G), but the effects of each approach and specific decay rate vary depending on the target gene / enzyme and its specific innate turnover rate and expression level.

[0138] To dynamically reduce GltA and Zwf levels (Figures 3H-3I), strains were engineered using chromosomal modifications that added a C-terminal DAS+4 degron tag to these genes. Furthermore, the inventors engineered several strains to have a C-terminal superfolder GFP tag behind each gene, with or without the C-terminal degron tag. Plasmids expressing gRNA were designed to suppress expression from the gltAp2 promoter and zwf promoter. Using these strains and plasmids, dynamic regulation of enzyme levels was monitored by tracking GFP via an ELISA assay in a two-step minimal medium microfermentation, as reported by Moreb et al. ELISA was used because the protein levels in the engineered strains were too low to use GFP fluorescence directly as a reporter. In the case of GltA proteolysis and silencing, GltA levels decreased by 70% and 85%, respectively, and by 90% in combination. In the case of Zwf, in all cases of proteolysis, silencing, and combination, protein levels were below the quantification limit of the inventors' assay.

[0139] The effect of the combination of "G" and "Z" valves on metabolic flux was measured in minimal medium microfermentation carried out without heterologous production pathways. Since the strains used had deletions in the major pathways leading to acetic acid production (poxB and pta-ackA), pyruvate synthesis was first evaluated as a measure of metabolic flux via glycolysis (Figure 4). The "G" valve had the greatest effect on pyruvate production, and no detectable product was measured in the control strain without SMV. The improvement in pyruvate production could be attributed to either a stoichiometric effect where some of the flux normally entering the TCA cycle is redistributed to overflow metabolites, or a more overall increase in glucose uptake rate enabling greater overflow metabolism and pyruvate synthesis. To evaluate these two options, the effect of the "G" valve on glucose uptake rate was measured. The results shown in Figure 4C indicate that the increase in pyruvate production is primarily attributable to the increase in uptake rate rather than the redistribution of basal flux.

[0140] Therefore, the increase in glucose uptake by the "G" valve may be due to the direct regulatory effect of a metabolite produced by the TCA cycle, namely α-ketoglutarate (αKG). αKG, a precursor of glutamate, has several important regulatory roles, including the regulation of glucose transport by direct inhibition of enzyme I of the PTS-dependent glucose transporter (Figure 3). This feedback regulation is a way of coordinating glucose uptake with nitrogen assimilation (glutamate synthesis). We performed supplementation experiments by adding 20 mM dimethyl-αKG (DM-αKG) to microfermentation at the start of production. We used DM-αKG instead of αKG because it has been shown to cross the membrane more effectively, and added it to the intracellular αKG pool after hydrolysis. As seen in Figure 4, DM-αKG inhibited glucose uptake in control cells as well as in strains with a valve that reduces GltA levels. In summary, these results support the dynamic decrease in GltA levels and subsequent decrease in the αKG pool, as they are primarily related to the improvement of glucose uptake rate and pyruvate biosynthesis. Next, we evaluated pyruvate production in an instrumented bioreactor. As previously reported by Menacho-Melgar et al., we performed minimal medium-feed batch fermentation in which phosphate concentration limits biomass levels and induces the expression of silencing gRNA and SspB chaperone once consumed. Figure 4D shows the results of comparing the control host strain with the strain with dynamic control over GltA levels. Minimal pyruvate temporarily accumulated in the control strain, but with dynamic control, the maximum titer exceeded approximately 30 g / L.

[0141] To evaluate the effect of dynamic regulation on the acetyl-CoA flux, the inventors utilized a citramaric acid synthase that produces 1 mole of citramaric acid from 1 mole of pyruvate and 1 mole of acetyl-CoA. Citramaric acid is a precursor to the industrial chemicals itaconic acid and methyl methacrylate, and is an intermediate in branched-chain amino acid biosynthesis. To produce citramaric acid, the inventors used a hypophosphate-inducible plasmid expressing a previously reported feedback-resistant mutant citramaric acid synthase (cimA3.7). This plasmid was introduced into a set of "G" and "Z" valve strains, which were then evaluated for citramaric acid production in two-step microfermentation (Figure 5). The best-producing strains possessed both the "G" and "Z" valves.

[0142] In the case of pyruvate, the "Z" valve did not have a significant impact on production (Figure 4B). Citramarate and pyruvate are similar products in that they are both oxidized and do not require redox cofactors (such as NADPH) for biosynthesis. A key difference between the two products is that citramarate requires an additional precursor, namely acetyl-CoA. The "Z-valve"-dependent improvement in citramarate production may depend on the improvement in acetyl-CoA production in strains with reduced Zwf activity. This suggests that either Zwf levels or downstream metabolite levels have a negative regulatory effect on stationary-phase acetyl-CoA synthesis. It is important to note that the strains used for pyruvate and citramarate production have deletions in poxB and pflB (which can lead to acetyl-CoA synthesis), and it was initially assumed that all acetyl-CoA fluxes are mediated through well-characterized pyruvate dehydrogenase (PDH) multienzyme complexes. Unexpectedly, proteolysis of Lpd (a subunit of PDH) did not affect citramalate production. Based on this, we investigated the possibility of an alternative major pathway for acetyl-CoA production in stationary-phase culture, namely pyruvate flavodoxin / ferredoxin oxireductase (Pfo) encoded by the ydbK gene.

[0143] As shown in Figure 3, Pfo(ydbK) may be partially involved in stationary-phase acetyl-CoA synthesis, and due to its role in the oxidative stress response, this activity was regulated by a PPP intermediate, which is also known to be involved in the response to oxidative stress. To test this hypothesis, we constructed ydbK deletions in citramarate strains containing both the "G" and "Z" valves and measured citramarate production. As seen in Figure 5B, ydbK deletion significantly reduced citramarate synthesis, confirming the role of Pfo in the acetyl-CoA flux. Since Pfo has been shown to be induced during oxidative stress via the SoxRS regulon (which is also regulated by the NADPH pool), its expression may be due to changes in NADPH levels caused by decreased Zwf activity.

[0144] Finally, the inventors evaluated citramaric acid-producing strains in a bioreactor equipped with instruments. The control strain yielded a reasonable citramaric acid titer (approximately 40 g / L), but the introduction of SMV improved production. The combined "GZ" valve strain had the highest citramaric acid production, reaching a titer of approximately 100 g / L. This process was then enhanced by increasing the biomass level from approximately 10 g CDW / L to approximately 25 g CDW / L, resulting in a titer of 126 + / - 7 g / L. This process is shown in Figure 5C. The overall process yield was 0.74–0.77 g citramaric acid / g glucose, with yields during the production stage approaching 0.80–0.82 g citramaric acid / g glucose. The theoretical yield of citramaric acid from glucose is 1 mole / mol or 0.817 g / g.

[0145] Previous studies utilizing dynamic regulation have been primarily informed by stoichiometric frameworks, where pathways are switched "on" and "off" to reduce stoichiometrically competing fluxes for the desired product, i.e., pathway redirection. For example, Venayak et al., based partly on stoichiometric modeling, highlighted the importance of GltA / CS as a candidate central valve for dynamic metabolic regulation. However, these studies and models overlook the importance of the regulatory role of downstream metabolites such as αKG. This study demonstrates that increased fluxes due to dysregulation of feedback control can have a significant impact on production, regardless of stoichiometric or competing pathway minimization. In particular, the increase in acetyl-CoA flux when Zwf activity is reduced was unexpected.

[0146] This is the first report of the interaction between minimum Zwf levels, SoxRS activation, and Pfo activity in the stationary phase. Furthermore, the magnitude of the metabolic flux mediated by Pfo is unexpected. Pfo, an enzyme containing an iron-sulfur cluster, was well expressed under both aerobic and anaerobic conditions, but was rapidly inactivated by molecular oxygen in vitro, and consequently, previous findings suggested that it was unlikely to support these types of fluxes. These data suggest that the Pfo pathway can act as a central metabolic pathway under certain conditions and can maintain high levels of activity even aerobic in vivo. A deeper understanding of this could lead to alternative strategies (independent of reduced Zwf levels) to optimize the flux through this pathway, such as pathway overexpression and / or enzyme engineering.

[0147] Example 4: Stationary-phase glucose uptake and pyruvate synthesis are insensitive to α-ketoglutarate levels in the PTS-negative strain of Escherichia coli (E. coli).

[0148] Referring to Figure 7, 7A) Overview of glucose uptake in PTS-negative strains of Escherichia coli (E. coli). Strain DLF_00286 (genotype 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, ΔptsG:glk, proDp-galP) has a mutation in the ptsG gene that eliminates PTS-dependent glucose uptake. Glucose uptake is restored by overexpression of galP galactose permyase (which can also transport glucose) and glucose-activating glucokinase (glk). Figure 7B) Pyruvate production in two-step microfermentation in DLF_00286 strain and DLF_00286 strain under dynamic control of citrate synthase (GltA level). Station-phase pyruvate synthesis is improved in LF_00286 strain compared to the PTS(+) control (DLF_0025). Dynamic control of citrate synthase (gltA level) does not improve pyruvate synthesis in the DLF_00286 host background. Figure 7C) Glucose uptake is insensitive to dimethyl-αKG supplementation in the PTS(-) strain. Station-phase pyruvate synthesis is improved in LF_00286 strain compared to the PTS(+) control (DLF_0025). Dynamic control of citrate synthase (gltA level) does not improve pyruvate synthesis in the DLF_00286 host background. Figure 7D) Pyruvate and biomass production were measured for DLF_00286 strain and its "G" valve derivative strain. This figure plots the biomass (gray) and pyruvate production (blue) of the control strain, and the biomass (black) and pyruvate production (green) of the "G" valve strain as a function of time.

[0149] Example 5: Acetyl-CoA flux depends on Pfo(YdbK) activity.

[0150] Referring to Figure 8A, Lpd proteolysis (lpd-DAS+4, a subunit of the pyruvate dehydrogenase multienzyme complex) and ydbK deletion were evaluated against a "GZ" valve background. Figure 8B shows the relative station-phase ydbK enzyme activity as a function of the "G" valve and "Z" valve. YdbK activity was measured in crude lysates using pyruvate and CoA as substrates and methyl viologen as the electron acceptor. Figure 8C shows the NADPH pool (gray bars) and ydbK expression levels (green bars) in the engineered strains. Superfolder GFP (sfGFP) reporter expression is driven by the ydbK promoter.

[0151] Example 6: Acetyl-CoA flux can be improved independently of the "Z" valve, depending on soxS activation.

[0152] Referring here to Figure 9A, the strain was engineered for hypophosphatemia induction of SoxS (independent of NADPH pool and SoxR activation). This was achieved by manipulating an extra copy of SoxS on the chromosome induced by the hypophosphatemia-inducible yibD gene promoter. Figure 9B shows citramarate production in microfermentation in a PTS(+) strain engineered with a combination of the "G" valve and hypophosphatemia-inducible soxS. Importantly, deletion of ydbK in strains with soxS induction still reduces the citramarate flux.

[0153] More generally, the present invention highlights the potential to manipulate known and unknown feedback regulatory mechanisms to improve in vivo enzyme activity and metabolic flux. This approach can open up numerous novel engineering strategies, leading to significant improvements in production rate, titer, and yield. Furthermore, these results confirm the metabolic capacity of stationary-phase cultures. Dynamic metabolic control in two-stage culture is particularly well-suited for implementing these strategies. Simply overexpressing key enzymes does not bypass innate regulation, and complete removal of central metabolic enzymes and / or metabolites often results in growth defects or strains that need to evolve compensatory metabolic changes to meet growth requirements. In contrast, altering the levels of central regulatory metabolites in the stationary phase allows for the rearrangement of regulatory networks and metabolic fluxes without this constraint.

[0154] As described above, this application is illustrated by the description of embodiments, and the embodiments are described in considerable detail, but it is not intended to limit, or in any way restrict, the scope of the appended claims to such detail. Additional advantages and modifications will be readily apparent to those skilled in the art who are interested in this application. Accordingly, this application, in its broader embodiments, is not limited to the specific details and exemplary examples shown. Such details and examples can be deviated from without departing from the spirit or scope of the general concept of the invention. [Sequence Listing Free Text]

[0155] Sequence Listings 1-40 <223> synthesis

Claims

1. A genetically modified Escherichia coli (E. coli) microorganism, A production pathway for citramaric acid production, including citramaric acid synthase. Conditional induction of SoxS by depleting restriction nutrients from the growth medium in which the genetically modified Escherichia coli (E. coli) microorganisms are growing, Conditional silencing of the gene expression of the glucose-6-phosphate dehydrogenase (ZWF) gene; or Conditional selective proteolysis of glucose-6-phosphate dehydrogenase (ZWF) enzyme; The genetically modified Escherichia coli (E. coli) microorganism undergoes conditional silencing or selective proteolysis during the stationary phase or non-dividing cell state. Under conditions that deplete restriction nutrients from the growth medium in which the genetically modified Escherichia coli (E. coli) microorganisms are growing, a stationary phase or non-dividing cell state is induced. The pyruvate flavodoxin / ferredoxin oxireductase enzyme activity is overexpressed in the genetically modified Escherichia coli (E. coli) microorganisms under aerobic or partially aerobic conditions during the stationary phase or non-dividing cell state when restriction nutrients are depleted from the growth medium in which the genetically modified Escherichia coli (E. coli) microorganisms are growing during the stationary phase or non-dividing cell state, and produces an acetyl-CoA pool. Sugar uptake is enhanced in the genetically modified Escherichia coli (E. coli) microorganisms compared to non-genetically modified microorganisms. Genetically modified Escherichia coli (E. coli) microorganisms.

2. The genetically modified Escherichia coli (E. coli) microorganism according to claim 1, wherein the genetically modified Escherichia coli (E. coli) microorganism comprises a deletion of the endogenous poxB and pflB genes.

3. The genetically modified Escherichia coli (E. coli) microorganism according to claim 1, wherein the pyruvate flavodoxin / ferredoxin oxireductase enzyme is encoded by the ydbK gene.

4. The genetically modified Escherichia coli (E. coli) microorganism according to claim 1, wherein the increase in pyruvate flavodoxin / ferredoxin oxireductase enzyme activity is due to the induction of oxidative soxRS regulon during the stationary phase or non-dividing cell state.

5. The genetically modified Escherichia coli (E. coli) microorganism according to claim 1, wherein the increase in pyruvate flavodoxin / ferredoxin oxireductase enzyme activity is a result of a decrease in NADPH levels within the genetically modified Escherichia coli (E. coli) microorganism during the stationary phase or non-dividing cell state.

6. A genetically modified Escherichia coli (E. coli) microorganism according to claim 1, wherein the activity of at least one sugar transporter is increased during the stationary phase or non-dividing cell state, thereby enhancing sugar uptake.

7. The genetically modified Escherichia coli (E. coli) microorganism according to claim 1, wherein constitutive expression of sugar transporter genes results in increased sugar transporter activity within the genetically modified Escherichia coli (E. coli) microorganism.

8. The genetically modified Escherichia coli (E. coli) microorganism according to claim 6, wherein the sugar transporter is conditionally overexpressed during the stationary phase or non-dividing cell state.

9. The genetically modified Escherichia coli (E. coli) microorganism according to claim 6, wherein the sugar transporter is encoded by the pts gene.

10. A genetically modified Escherichia coli (E. coli) microorganism according to claim 1, wherein gene expression silencing is induced by CRISPR interference.

11. A bioprocess for producing a product from a genetically modified Escherichia coli (E. coli) microorganism according to claim 1, In the first step, the genetically modified Escherichia coli (E. coli) microorganism is grown in a culture medium, In the second stage, when restriction nutrients from the growth medium are depleted, the stationary phase or non-dividing cell state is induced. Includes, A bioprocess in which the genetically modified Escherichia coli (E. coli) microorganisms in the stationary phase or non-dividing cell state produce a product at a rate of 30 g / L or more.

12. The bioprocess according to claim 11, wherein the increased activity of the pyruvate flavodoxin / ferredoxin oxireductase enzyme is caused by overexpression of a gene encoding active pyruvate ferredoxin oxireductase, induction of oxidative soxRS regulon, a decrease in NADPH levels, gene silencing of the zwf gene or a decrease in glucose-6-phosphate dehydrogenase levels accompanied by selective proteolysis of the glucose-6-phosphate dehydrogenase enzyme, silencing or selective proteolysis of the conditional gene expression activated in the stationary phase or non-dividing cell state, or a combination thereof.

13. The bioprocess according to claim 11, wherein the activity of at least one sugar transporter is increased.

14. The bioprocess according to claim 11, wherein the product is citramaric acid, the enzyme in the production pathway includes citramaric acid synthase, and the bioprocess produces 100 g / L or more of citramaric acid.

15. The bioprocess according to claim 11, wherein the citramalate synthase is encoded by the cimA3.7 gene.

16. The bioprocess according to claim 11, wherein the genetically modified Escherichia coli (E. coli) microorganism comprises a plasmid containing a citramarate synthase gene operably linked to a hypophosphate-inducible promoter.

17. The bioprocess according to claim 11, wherein the genetically modified Escherichia coli (E. coli) microorganism comprises deletions of endogenous poxB and pflB genes.

18. A genetically modified Escherichia coli (E. coli) microorganism, A production pathway comprising at least one enzyme for producing a product from acetyl-CoA, Conditional silencing of the gene expression of the glucose-6-phosphate dehydrogenase (ZWF) gene, Conditional selective proteolysis of glucose-6-phosphate dehydrogenase (ZWF) enzyme and This includes deletions of endogenous poxB and pflB genes, The genetically modified Escherichia coli (E. coli) microorganism undergoes conditional silencing or selective proteolysis during the stationary phase or non-dividing cell state. Under conditions that deplete restriction nutrients from the growth medium in which the genetically modified Escherichia coli (E. coli) microorganisms are growing, a stationary phase or non-dividing cell state is achieved. The pyruvate flavodoxin / ferredoxin oxireductase enzyme activity increases in the genetically modified Escherichia coli (E. coli) microorganism under aerobic or partially aerobic conditions during the stationary phase or non-dividing cell state, producing an acetyl-CoA pool. Sugar uptake is enhanced in the genetically modified Escherichia coli (E. coli) microorganisms compared to non-genetically modified microorganisms. A genetically modified Escherichia coli (E. coli) microorganism in which the increase in pyruvate flavodoxin / ferredoxin oxireductase enzyme activity is due to the overexpression of the gene encoding pyruvate ferredoxin oxireductase during the stationary phase or non-dividing cell state.

19. The genetically modified Escherichia coli (E. coli) microorganism according to claim 18, wherein the increase in pyruvate flavodoxin / ferredoxin oxireductase enzyme activity is due to the overexpression of the gene encoding pyruvate ferredoxin oxireductase during the stationary phase or non-dividing cell state.

20. The genetically modified Escherichia coli (E. coli) microorganism according to claim 18, wherein the pyruvate flavodoxin / ferredoxin oxireductase enzyme is encoded by the ydbK gene.

21. The genetically modified Escherichia coli (E. coli) microorganism according to claim 18, wherein the increase in pyruvate flavodoxin / ferredoxin oxireductase enzyme activity is due to the induction of oxidative soxRS regulon during the stationary phase or non-dividing cell state.

22. The genetically modified Escherichia coli (E. coli) microorganism according to claim 18, wherein the increase in pyruvate flavodoxin / ferredoxin oxireductase enzyme activity is a result of a decrease in NADPH levels within the genetically modified Escherichia coli (E. coli) microorganism during the stationary phase or non-dividing cell state.

23. The genetically modified Escherichia coli (E. coli) microorganism according to claim 18, wherein the activity of at least one sugar transporter is increased, thereby enhancing sugar uptake.

24. The genetically modified Escherichia coli (E. coli) microorganism according to claim 18, wherein constitutive expression of sugar transporter genes results in increased sugar transporter activity within the genetically modified Escherichia coli (E. coli) microorganism.

25. The genetically modified Escherichia coli (E. coli) microorganism according to claim 18, wherein sugar uptake is enhanced by conditional overexpression of a sugar transporter during the stationary phase or non-dividing cell state.

26. The genetically modified Escherichia coli (E. coli) microorganism according to claim 25, wherein the sugar transporter is encoded by the pts gene.

27. The genetically modified Escherichia coli (E. coli) microorganism according to claim 18, wherein the enzyme in the production pathway is citramalate synthase.

28. The genetically modified Escherichia coli (E. coli) microorganism according to claim 18, wherein the product is pyruvate.

29. The genetically modified Escherichia coli (E. coli) microorganism according to claim 18, wherein CRISPR interference results in silencing of gene expression.

30. A bioprocess for producing a product from a genetically modified Escherichia coli (E. coli) microorganism according to claim 18, In the first step, the genetically modified Escherichia coli (E. coli) microorganism is grown in a culture medium, In the second stage, when restriction nutrients from the growth medium are depleted, the stationary phase or non-dividing cell state is induced. Includes, A bioprocess in which the genetically modified Escherichia coli (E. coli) microorganisms in the stationary phase or non-dividing cell state produce a product at a rate of 30 g / L or more.

31. The bioprocess according to claim 30, wherein the increased activity of the pyruvate flavodoxin / ferredoxin oxireductase enzyme is caused by overexpression of a gene encoding active pyruvate ferredoxin oxireductase, induction of oxidative soxRS regulon, a decrease in NADPH levels, gene silencing of the zwf gene or a decrease in glucose-6-phosphate dehydrogenase levels accompanied by selective proteolysis of the glucose-6-phosphate dehydrogenase enzyme, silencing or selective proteolysis of the conditional gene expression activated in the stationary phase or non-dividing cell state, or a combination thereof.

32. The bioprocess according to claim 30, wherein the activity of at least one sugar transporter is increased.

33. The bioprocess according to claim 30, wherein the product is citramaric acid, the enzyme in the production pathway includes citramaric acid synthase, and the bioprocess produces 100 g / L or more of citramaric acid.

34. The bioprocess according to claim 30, wherein the enzyme in the production pathway is citramarate synthase, and the citramarate synthase is encoded by the cimA3.7 gene.

35. The bioprocess according to claim 30, wherein the genetically modified Escherichia coli (E. coli) microorganism comprises a plasmid containing a citramarate synthase gene operably linked to a hypophosphate-inducible promoter.

Images