Methods and compositions for producing acetyl-coa-derived products

Abstract

A genetically engineered microbial strain for production of products from acetyl-CoA and related biological processes. In particular, a dynamically controlled synthetic metabolic valve is used to reduce the activity of certain enzymes, resulting in increased product yield in a two-stage process.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 056,031, filed July 24, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to metabolically engineered microorganisms, such as bacterial strains, and bioprocesses utilizing such strains. These strains provide dynamic control over metabolic pathways, enabling the production of products from acetyl-CoA.

[0004] sequence list

[0005] This application contains a sequence list created on July 13, 2021, which is 26,740 bytes in size and was submitted electronically in ASCII format as 49186-48_ST25.txt, the entire contents of which are incorporated herein by reference.

[0006] Federal government-funded research or development

[0007] This invention was made with the support of the governments of NSF EAGER: #1445726, DARPA# HR0011-14-C-0075, ONR YIP #N00014-16-1-2558, and DOE EERE grant #EE0007563. The governments hold certain rights to this invention. Background Technology

[0008] In recent years, biotechnology-based fermentation processes have made rapid progress due to technological advancements in fermentation science, synthetic biology, and metabolic and enzyme engineering. However, commercially competitive processes often require improvements in rate, titer, and yield. Most metabolic engineering strategies aimed at improving these metrics rely on the overexpression of desired pathway enzymes and the deletion and / or downregulation of competitive biochemical activities. Over the past few decades, stoichiometric models of metabolism have helped the field shift from manipulating gene expression levels to manipulating networks, which can now be designed to combine growth with product formation, and both can be optimized using selection.

[0009] An unresolved limitation of these methods is the metabolic boundary conditions required for cell growth. Dynamic metabolic control, particularly two-stage control, offers a potential design strategy to overcome these limitations by switching to a production state that allows metabolite and enzyme levels to exceed the growth-required boundaries. Significant efforts have been made to develop tools for dynamic metabolic control, including control systems, metabolic valves, and modeling methods. However, to date, previous work has primarily focused on dynamically redirecting fluxes by “off”ing pathways that stoichiometrically compete with the desired pathway. Summary of the Invention

[0010] We demonstrate that the increased stationary phase flux is attributable to a dynamic reduction in metabolites acting as feedback regulators of central metabolism, rather than a reduction in competing metabolic pathways. Employing two-stage dynamic metabolic control, we describe the manipulation of feedback regulation in central metabolism and the enhancement of biosynthesis in genetically modified microorganisms. Specifically, we describe the effects of dynamically controlling two central metabolic enzymes, citrate synthase and glucose-6-phosphate dehydrogenase, on stationary phase flux. Decreased citrate synthase levels lead to a reduction in the glucose transport inhibitor α-ketoglutarate, thereby increasing glucose uptake and glycolytic fluxes.

[0011] Other methods, features, and / or advantages will become apparent or will become apparent upon examination of the following drawings and detailed description. All such additional methods, features, and advantages are intended to be incorporated herein by reference and protected by the appended claims. Attached Figure Description

[0012] The novel features of the invention are particularly set forth in the claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description of illustrative embodiments using the principles of the invention, along with the accompanying drawings:

[0013] Figure 1 A schematic diagram of the pCASCADE-control plasmid construction scheme is shown.

[0014] Figure 2A-2B The pCASCADE construction scheme is shown. (2A) Single sgRNA clone; (2B) Double sgRNA.

[0015] Figure 3A-3I(3A) Schematic diagram of two-stage dynamic control of central metabolic feedback regulation improving glucose uptake and acetyl-CoA flux during the stationary phase. The metabolic valve (double triangle) dynamically reduces the levels of Zwf (glucose-6-phosphate dehydrogenase) and GltA (citrate synthase). The reduced flux through the TCA cycle lowers KG levels, thereby mitigating the feedback inhibition of PTS-dependent glucose uptake and improving glycolysis flux and pyruvate production. The reduced Zwf flux lowers NADPH levels, activates SoxRS oxidative stress regulation, and increases the expression and activity of pyruvate ferroreductase, improving pyruvate oxidation and acetyl-CoA flux. (3B) Time process of two-stage dynamic metabolic control after phosphate depletion. The accumulation of biomass and consumption of depleted nutrient triggers the entry into the stationary phase of production, which in turn dynamically reduces the levels of key enzymes under the anabolic valve (red) (3C and 3D). Synthetic valves for gene silencing and / or controlled proteolysis based on CRISPRi. (3C) Arrays of silencing guides can be used to silence multiple target genes (GOIs). This involves the induced expression of one or more guide RNAs and 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 transcription. (3D) C-terminal DAS+4 tags are added to target enzymes (EOIs) via chromosomal modification, which can be induced to degrade by the clpXP protease in the presence of an inducible sspB chaperone. (3E) Dynamic control of protein levels in *E. coli* using inducible proteolysis and CRISPRi silencing. As cells grow, phosphate is depleted, and cells “turn off” mCherry and “turn on” GFPuv. Shaded areas represent one standard deviation from the mean, n=3. (3F) The relative effects of proteolysis and gene silencing, used alone and in combination, on mCherry degradation; (3G) mCherry decay rates; (3H and 3I) Dynamic control of central metabolic enzyme levels. The effects of silencing (pCASCADE) and proteolysis (DAS+4 tag) on ​​protein levels, used alone and in combination, were evaluated: (3H) GltA (citrate synthase), (3I) 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 phosphate depletion induction via microfermentations.Abbreviations: PTS: Phosphotransferase transport system, PPP: Pentose phosphate pathway, TCA: Tricarboxylic acid, G6P: Glucose-6-phosphate, 6-PGL: 6-phosphogluconolactone, 6PG: 6-phosphogluconate, PEP: Phosphoenolpyruvate, Fd: Deferredoxin, CoA: Coenzyme A, OAA: Oxaloacetate, αKG: Alpha-ketoglutarate.

[0016] Figures 4A-4D :( Figure 4A The dynamic reduction of GltA decreased the KG pool and alleviated KG-mediated inhibition of PTS-dependent glucose uptake (especially Pts1), while increasing glucose uptake, glycolysis flux, and pyruvate production. Figure 4B The effect of dynamic control of GltA and Zwf levels on pyruvate production in basal medium microfermentation. Figure 4C The dynamic control of GltA and Zwf levels and the effect of dimethyl-KG supplementation on glucose uptake in microfermentation. Figure 4D Pyruvate and biomass production were measured for the control strain and the "G" valve strain. Biomass (grey) and pyruvate production (blue) for the control strain, and biomass (black) and pyruvate production (green) for the "G" valve strain, are plotted as a function of time. Dashed lines represent extrapolated growth due to missed samples.

[0017] Figures 5A-5D(5A) Dynamic reduction of Zwf levels activates the SoxRS regulator and increases the activity of pyruvate-ferrugin oxidoreductase (Pfo, ydbK), thereby improving acetyl-CoA flux and citrate production. (5B) Dynamic control of GltA and Zwf levels on citrate production in basal medium microfermentation. Furthermore, proteolytic degradation of Lpd (lpd-DAS+4, a subunit of the pyruvate dehydrogenase multienzyme complex) and deletion in ydbK were evaluated in a “GZ” valve background. (5C and 5D) Citrate and biomass yields were measured for control strain (c) and “GZ” valve strain (d). (5C) Duplicate runs, gray and black for biomass levels, green and blue for citrate titers. (5D) Average of triplicate runs, black for biomass and green for citrate. The dashed line represents extrapolation growth caused by missing samples.

[0018] Figures 6A-6D Cimicifuric acid and biomass yields were measured in control strain (6A), “G” valve strain (6B), and “GZ” valve strain (6C) in fermentation targeting a biomass level of 10 g CDW / L. Two runs were performed; gray and black represent biomass levels, and green and blue represent citric acid titers. (5D) Cimicifuric acid yield and biomass level in fermentation targeting a biomass level of 25 g CDW. The average of three runs was used; biomass is shown in black, and citric acid in green. Dashed lines indicate extrapolated growth due to sample loss.

[0019] Figures 7A-7D (7A) Overview of glucose uptake in PTS(-) strains of *Escherichia coli* (PTS minus strain). (7B) Pyruvate production in two-stage microfermentation by DLF_00286 strain and DLF_00286 strain with dynamic control of citrate synthase (GltA level). (7C) Glucose uptake is insensitive to dimethyl-αKG supplementation in PTS(-) strains. (7D) Measurement of pyruvate and biomass yield in strain DLF_00286 and its “G” valve derivative.

[0020] Figures 8A-8C (8A) Acetyl-CoA flux depends on Pfo (YdbK) activity. (8B) Stationary phase relative ydbK enzyme activity as a function of “G” and “Z” valves. (8C) NADPH library (grey bar) and ydbK expression level (green bar) in engineered strains.

[0021] Figures 9A-9BAcetyl-CoA flux depends on soxS activation and can be improved independently of the “Z” valve. (9A) strains were engineered for low-phosphate induction of soxS (independent of NADPH library and soxR activation). (9B) In the case of using the “G” valve and low-phosphate induction... soxS In the combined engineered PTS(+) strains, citric acid is produced by micro-fermentation. Detailed Implementation

[0022] This paper demonstrates the use of two-stage dynamic metabolic control to manipulate feedback regulation of central metabolism and improve biosynthesis in engineered *E. coli*. Specifically, we report the effects of dynamically controlling two central metabolic enzymes, citrate synthase and glucose-6-phosphate dehydrogenase, on stationary-phase flux. First, decreased citrate synthase levels reduced the glucose transport inhibitor α-ketoglutarate, thereby increasing glucose uptake and glycolysis flux. The reduced glucose-6-phosphate dehydrogenase activity activated the expression of the SoxRS regulator and pyruvate-ferrugin oxidoreductase, which in turn significantly increased acetyl-CoA production. These two mechanisms improved stationary-phase production of citrate, enabling a titer of 126 ± 7 g / L. These results identify pyruvate oxidation via pyruvate-ferrugin oxidoreductase as the “central” metabolic pathway in the stationary phase and highlight the potential to improve flux by manipulating fundamental central regulatory mechanisms using two-stage dynamic metabolic control.

[0023] definition

[0024] As used in the specification and claims, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” include plural objects. Thus, for example, reference to “expression vector” includes the same (e.g., the same operon) or different, a single expression vector and multiple expression vectors; reference to “microorganism” includes a single microorganism and multiple microorganisms, etc.

[0025] As used herein, the terms “heterologous DNA”, “heterologous nucleic acid sequence”, etc., refer to a nucleic acid sequence in which at least one of the following conditions is met: (a) the nucleic acid sequence is foreign to a given host microorganism (i.e., not naturally occurring); (b) the sequence may be naturally occurring in a given host microorganism, but in an anomalous quantity (e.g., greater than expected); or (c) the nucleic acid sequence contains two or more subsequences whose relationship differs from that found in nature. For example, with respect to instance (c), the recombinant heterologous nucleic acid sequence arranges two or more sequences from unrelated genes to produce a new functional nucleic acid, such as a non-natural promoter driving gene expression.

[0026] The term "synthetic metabolic valve" as used in this article refers to the use of controlled proteolysis, gene silencing, or a combination of proteolysis and gene silencing to alter metabolic flux.

[0027] The term "heterologous" is intended to include the term "exogenous," as the latter is commonly used in the art. For a host microbial genome prior to the introduction of a heterologous nucleic acid sequence, the nucleic acid sequence encoding the enzyme is heterologous (regardless of whether the heterologous nucleic acid sequence was introduced into the genome). As used herein, chromosomal and native, as well as endogenous, refer to the genetic material of the host microorganism.

[0028] As used herein, the term “gene disruption” or its grammatical equivalents (including “to disrupt enzymatic function”, “disruption of enzymatic function”, etc.) are intended to refer to a genetic modification of a microorganism that results in a reduced polypeptide activity of the encoded gene product compared to the polypeptide activity in or derived from an unmodified microbial cell. Genetic modifications can be, for example, the deletion of an entire gene, the deletion or other modification of regulatory sequences required for transcription or translation, the deletion of a portion of the gene resulting in a truncated gene product (e.g., an enzyme), or any of a variety of mutational strategies that reduce the activity of the encoded gene product, including to undetectable levels. Disruption can broadly include the deletion of all or part of the nucleic acid sequence encoding the enzyme, and also includes, but is not limited to, other types of genetic modifications such as the introduction of stop codons, frame shift mutations, the introduction or removal of gene parts, the introduction of degradation signals, genetic modifications that affect mRNA transcription levels and / or stability, and alterations to promoters or repressors upstream of the gene encoding the enzyme.

[0029] As used in this article, bioproduction, micro-fermentation, or fermentation can be aerobic, microaerobic, or anaerobic.

[0030] When this document (including the claims) refers to genetic modification of a gene product (i.e., an enzyme), it should be understood that genetic modification is a genetic modification of a nucleic acid sequence (e.g., or including a gene that typically encodes the gene product (i.e., an enzyme)).

[0031] As used herein, the term "metabolic flux" refers to metabolic changes that result in variations in the formation of products and / or byproducts, including productivity, production titers, and production yields from a given substrate.

[0032] Species and other phylogenic identifications are based on classifications known to technicians in the field of microbiology.

[0033] Enzymes are listed herein with reference to UniProt identification numbers well known to those skilled in the art, and the UniProt database can be accessed at UniProt.org. When this document (including the claims) refers to genetic modifications of a gene product (i.e., an enzyme), it should be understood that genetic modification is a genetic modification of a nucleic acid sequence (e.g., including or comprising a gene that typically encodes the gene product (i.e., an enzyme)).

[0034] Where the methods and steps described herein instruct specific events to occur in a particular order, those skilled in the art will recognize that the order of specific steps can be modified and such modifications are consistent with variations of the invention. Furthermore, certain steps may be performed simultaneously in parallel processes, as well as sequentially, where possible.

[0035] The abbreviations have the following meanings: "C" represents Celsius temperature or °C, which is clearly visible in their usage; DCW represents cell dry weight; "s" represents second; "min" represents minute; "h", "hr", or "hrs" represents hour; "psi" represents pounds per square inch; "nm" represents nanometer; "d" represents day; "µL", "uL", or "ul" represents microliter; "mL" represents milliliter; "L" represents liter; "mm" represents millimeter; "nm" represents nanometer; "mM" represents millimole; "µM" or "uM" represents micromolar; "M" represents mole; "mmol" represents millimole; "µmol" or "uMol" represents micromolar; "g" represents gram; "µg" or "ug" refers to microgram; "ng" refers to nanogram; "PCR" refers to polymerase chain reaction; "OD" refers to optical density; "OD600" refers to optical density measured at a photon wavelength of 600 nm; "kDa" represents kilodaltons; and "g" represents the gravitational constant. "constant", "bp" represents base pair(s), "kbp" represents kilobase pair(s), "% w / v" represents weight / volume percentage, "%v / v" represents volume / volume percentage, "IPTG" represents isopropyl-µ-D-thiogalactopyranoiside, "aTc" represents anhydrotetracycline, "RBS" represents ribosome binding site, "rpm" represents revolutions per minute, "HPLC" represents high performance liquid chromatography, and "GC" represents gas chromatography.

[0036] I. Carbon Source

[0037] In this invention, the bio-production media used with recombinant microorganisms must contain a carbon source or substrate suitable for 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-mentioned carbon substrates and mixtures thereof are contemplated as suitable carbon sources for use in this invention.

[0038] II. Microorganisms

[0039] Features as described and claimed herein may be provided in a selection of microorganisms listed herein or another suitable microorganism, which also include one or more natural, introduced, or enhanced product bioproduction pathways. 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.

[0040] More specifically, based on the various criteria described herein, suitable microbial hosts for the bioproduction of chemical products may generally include, but are not limited to, the organisms described in the Common Methods Section.

[0041] The host or source microorganism for any gene or protein described herein may be selected from the following list of microorganisms: *Citrobacter*, *Enterobacter*, *Clostridium*, *Klebsiella*, *Aerobacter*, *Lactobacillus*, *Aspergillus*, *Saccharomyces*, *Schizosaccharomyces*, and *Zygosaccharomyces*. The genera include Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces, and Pseudomonas. In some respects, the host microorganism is Escherichia coli.

[0042] III. Culture medium and culture conditions

[0043] In addition to a suitable carbon source (e.g., selected from one of the types disclosed herein), the bio-production media must also contain suitable minerals, salts, cofactors, buffers, and other components known to those skilled in the art as suitable for the cultivation and growth promotion of the chemical products of this invention.

[0044] Another aspect of the invention relates to culture media and culture conditions comprising the genetically modified microorganism of the invention and optional supplements.

[0045] Typically, cells are grown in suitable culture media at temperatures ranging from about 25°C to about 40°C, and up to 70°C for thermophilic microorganisms. Suitable growth media are characterized and known in the art. The suitable pH range for bioproduction is between pH 2.0 and pH 10.0, with pH 6.0 to pH 8.0 being the typical pH range for initial conditions. However, the actual culture conditions of a particular embodiment are not intended to be limited to these pH ranges. Bioproduction can be carried out with or without stirring, under aerobic, microaerobic, or anaerobic conditions.

[0046] IV. Bio-production Reactors and Systems

[0047] Fermentation systems using the methods and / or compositions according to the invention are also within the scope of this invention. Any recombinant microorganisms described and / or mentioned herein can be introduced into industrial bioproduction systems, wherein the microorganisms convert a carbon source into a product in a commercially viable operation. A bioproduction system comprises introducing such recombinant microorganisms into a bioreactor vessel having a carbon source substrate and a bioproduction culture medium suitable for the growth of the recombinant microorganisms, and maintaining the bioproduction system within a suitable temperature range (and a dissolved oxygen concentration range (if the reaction is aerobic or microaerophilic)) for an appropriate time to achieve the desired partial conversion of substrate molecules into a selected chemical product. Bioproduction can be carried out with or without stirring, under aerobic, microaerophilic, or anaerobic conditions. Industrial bioproduction systems and their operation are well known to those skilled in the art of chemical engineering and bioprocess engineering.

[0048] The amount of product produced in a bioproduction culture medium can typically be determined using a variety of methods known in the art, such as high performance liquid chromatography (HPLC), gas chromatography (GC), or GC / mass spectrometry (MS).

[0049] V. Genetic modifications, nucleotide sequence and amino acid sequence

[0050] Embodiments of the present invention can be obtained by introducing an expression vector into a host microorganism, wherein the expression vector contains a nucleic acid sequence encoding an enzyme that may or may not be present in the host microorganism.

[0051] The ability to genetically modify host cells is essential for the production of any genetically modified (recombinant) microorganism. Gene transfer technology can be performed via electroporation, conjugation, transduction, or natural transformation. A wide range of host-conjugating plasmids and antibiotic resistance markers can be used. Cloning vectors are tailored to the host organism based on the nature of the antibiotic resistance markers that can function within the host. Furthermore, as disclosed herein, genetically modified (recombinant) microorganisms can contain modifications beyond those introduced via plasmids, including modifications to their genomic DNA.

[0052] More generally, nucleic acid constructs can be prepared comprising isolated polynucleotides encoding enzymatically active polypeptides, which are operatively linked to one or more (several) control sequences that direct the expression of the coding sequence in microorganisms (e.g., *E. coli*) under conditions compatible with the control sequences. The isolated polynucleotides can be manipulated to provide polypeptide expression. Depending on the expression vector, manipulation of the polynucleotide sequence may be required or necessary prior to insertion into the vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well-established in the field.

[0053] The control sequence can be a suitable promoter sequence, i.e., a nucleotide sequence of a polynucleotide that is recognized by the host cell to express the polypeptide encoding the present invention. The promoter sequence can contain a transcriptional control sequence mediating polypeptide expression. The promoter can be any nucleotide sequence exhibiting transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell. Techniques for modifying and utilizing recombinant DNA promoter sequences are well-established in the art.

[0054] For various embodiments of the present invention, genetic manipulations can include operations aimed at altering the regulation of an enzyme and thus its final activity or the activity of an enzyme identified in any corresponding pathway. Such genetic modifications can be directed at transcriptional, translational, and post-translational modifications that result in altered enzyme activity and / or selectivity under selected culture conditions. Genetic manipulation of nucleic acid sequences can increase copy number and / or include the use of mutants of enzymes associated with product production. Specific methods and approaches for achieving such genetic modifications are well known to those skilled in the art.

[0055] In various implementation schemes, microorganisms may contain one or more gene deletions in order to function more effectively. For example, in *E. coli*, it can be disrupted, including deletions, of enzymes 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-specific enhancing factor (SSPB), ATP-dependent Lon protease (Ion), outer membrane protease (OMPT), arcA transcriptional dual regulator (arcA), and iclR transcriptional regulator (iclR). Genes that act as regulators (iclRs). Such gene disruption, including deletion, is not intended to be limiting and can be implemented in various combinations across a variety of embodiments. Gene deletions can be achieved through many strategies well known in the art, such as methods of incorporating exogenous DNA into the host chromosome.

[0056] In various implementations, to function more effectively, the microorganism may contain one or more anabolic valves, which consist of enzymes targeting controlled proteolysis, expression silencing, or a combination of controlled proteolysis and expression silencing. For example, an enzyme encoded by a single gene or a combination of multiple enzymes encoded by numerous genes in *E. coli* can be designed as anabolic valves to alter metabolism and improve product formation. Representative genes in *E. coli* may include, but are not limited to, the following: fabI, zwf, gltA, ppc, udhA, lpd, sucD, aceA, pfkA, lon, rpoS, pykA, pykF, tktA or tktB It should be understood that those skilled in the art are well aware of how to identify homologs of these genes and / or other genes in other microbial species.

[0057] For all nucleic acid and amino acid sequences provided herein, it should be understood that conserved variations of these sequences are included and are within the scope of the various embodiments of the present invention. Functionally equivalent nucleic acid and amino acid sequences (functional variants), including conserved variations within the skill of those skilled in the art and more broadly varied sequences, as well as microorganisms comprising these functionally equivalent nucleic acid and amino acid sequences, and methods and systems comprising such sequences and / or microorganisms, are also within the scope of the various embodiments of the present invention.

[0058] Therefore, as described in the different sections above, some compositions, methods, and systems of the present invention include providing genetically modified microorganisms that comprise a combination of a production pathway for preparing desired products from a central intermediate and a synergistic metabolic valve for redistributing flux.

[0059] The invention also relates to providing a variety of genetic modifications to improve the overall efficiency of microorganisms in converting selected carbon sources into selected products. For example, examples show that, relative to more basic combinations of genetic modifications, specific combinations significantly improve specific productivity, volumetric productivity, titer, and yield.

[0060] In addition to the genetic modifications described above, various embodiments also provide genetic modifications including anabolic valves to increase or decrease the pool and availability of cofactors such as NADPH and / or NADH that may be consumed in the production of the product.

[0061] VI. Anabolism Valve

[0062] Using anabolic valves allows for simpler models of metabolic flux and physiological requirements during the production phase, transforming growing cells into stationary biocatalysts. These valves can be used to shut down essential genes and redirect carbon, electron, and energy fluxes to product formation in multi-stage fermentation processes. One or more of the following provide anabolic valves: 1) transcriptional gene silencing or repression techniques combined with 2) inducible and selective enzymatic degradation and 3) nutrient restriction to induce a stable or non-dividing cellular state. SMVs can be extended to any pathway and microbial host. These anabolic valves enable novel rapid metabolic engineering strategies for the production of renewable chemicals and fuels, as well as any product that can be produced via whole-cell catalysis.

[0063] In particular, this invention describes the construction of an anabolic valve comprising one or more, or a combination thereof, controlled gene silencing and controlled proteolysis. It should be understood that those skilled in the art are familiar with several methods for gene silencing and controlled proteolysis.

[0064] VI. Gene Silencing

[0065] In particular, this invention describes the use of controlled gene silencing to control metabolic flux in a controlled multistage fermentation process. Several methods for controlling gene silencing are known in the art, including but not limited to mRNA silencing or RNA interference, silencing via transcriptional repressors, and CRISPR interference. 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 teaches methods and mechanisms of RNA interference. 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, taught methods and mechanisms for CRISPR interference. Furthermore, Luo et al., “Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression” NAR. October 2014; DOI: 10.1093, taught methods and mechanisms for CRISPR interference using the native E. coli CASCADE system. In addition, many transcriptional repressor systems are well-known in the field and can be used to shut down gene expression.

[0066] VI. B-controlled proteolysis

[0067] In particular, this invention describes the use of controlled protein degradation or proteolysis to control metabolic flux in a controlled multistage fermentation process. Several methods known in the art for controlling protein degradation exist, including but not limited to targeted protein cleavage via specific proteases and controlled protein-targeted degradation via specific peptide tags. A system using the *E. coli* clpXP protease to control protein degradation is taught in 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 expression of the specifically enhanced molecular chaperone sspB is specifically enhanced. sspB induces the clpXP protease to degrade proteins tagged with DAS4. In addition, many site-specific protease systems are well known in the art. Proteins can be programmed to contain specific target sites of a given protease and then cleaved after controlled expression of the protease. In some embodiments, cleavage is 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) taught that N-terminal sequences can be added to proteins of interest to provide clpS-dependent clpAP degradation. Furthermore, this sequence can be further masked by other N-terminal sequences that can be controlled to cleave (e.g., by ULP hydrolases). This allows for controlled N-terminal rule degradation dependent on hydrolase expression. Therefore, proteins can be tagged with either the N-terminus or the C-terminus to control protein hydrolysis. The preference for using N-terminal versus C-terminal tags largely depends on whether either tag affects protein function before controlled degradation begins.

[0068] This invention describes the use of controlled protein degradation or proteolysis to control metabolic flux in a controlled, multi-stage fermentation process in *E. coli*. Several methods are known in the art for controlling protein degradation in other microbial hosts, including a wide range of Gram-negative and Gram-positive bacteria, yeasts, and even archaea. In particular, systems for controlled proteolysis can be transferred from natural microbial hosts and used in non-natural hosts. For example, Grilly et al.'s "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* clpXP protease in *Saccharomyces cerevisiae*. Such methods can be used to transfer the methodology of anabolic valves to any genetically manipulated host.

[0069] VI. C-anabolism valve control

[0070] In particular, this invention describes the use of anabolic valves to control metabolic flux in multi-stage fermentation processes. Numerous methods of inducing expression known in the art can be used for transitions between stages in multi-stage fermentation. These include, but are not limited to, artificial chemical inducers, including tetracycline, anhydrous tetracycline, lactose, IPTG (isopropyl-β-D-1-thiogalactopyranoside), arabinose, raffinose, tryptophan, and many other substances. Systems linking the use of these well-known inducers with the control of gene expression silencing and / or controlled proteolysis can be integrated into genetically modified microbial systems to control transitions between growth and production stages in multi-stage fermentation processes.

[0071] Furthermore, it may be necessary to control the transition between growth and production in multi-stage fermentation by depleting one or more limiting nutrients consumed during growth. Limiting nutrients include, but are not limited to, phosphates, nitrogen, sulfur, and magnesium. Natural gene expression systems responsive to these nutrient limitations can be used to operatively link the control of gene expression silencing and / or controlled proteolysis to the transition between growth and production phases in a multi-stage fermentation process.

[0072] Within the scope of this invention are genetically modified microorganisms, wherein the microorganisms are capable of producing products at a specific rate selected from greater than 0.05 g / gDCW-hour, 0.08 g / gDCW-hour, greater than 0.1 g / gDCW-hour, greater than 0.13 g / gDCW-hour, greater than 0.15 g / gDCW-hour, greater than 0.175 g / gDCW-hour, greater than 0.2 g / gDCW-hour, greater than 0.25 g / gDCW-hour, greater than 0.3 g / gDCW-hour, greater than 0.35 g / gDCW-hour, greater than 0.4 g / gDCW-hour, greater than 0.45 g / gDCW-hour, or greater than 0.5 g / gDCW-hour.

[0073] In various embodiments, the present invention includes a culture system comprising a carbon source in an aqueous culture medium and a genetically modified microorganism according to any one of the claims herein, wherein, for example, when the volume of the aqueous culture medium is selected from greater than 5 mL, greater than 100 mL, greater than 0.5 L, greater than 1 L, greater than 2 L, greater than 10 L, greater than 250 L, greater than 1000 L, greater than 10,000 L, greater than 50,000 L, greater than 100,000 L, or greater than 200,000 L, for example, when the volume of the aqueous culture medium is greater than 250 L and contained in a steel container, the genetically modified organism is present in an amount selected from greater than 0.05 gDCW / L, 0.1 gDCW / L, greater than 1 gDCW / L, greater than 5 gDCW / L, greater than 10 gDCW / L, greater than 15 gDCW / L, or greater than 20 gDCW / L.

[0074] Overview of the invention

[0075] On the one hand, genetically modified microorganisms are provided for use in biofermentation processes, comprising a production pathway for the production of products from acetyl-CoA precursors containing at least one enzyme. Under conditions of depletion of limiting nutrients from the growth medium in which the genetically modified microorganisms are grown, the microorganisms are induced into a stationary phase or a non-dividing cell state. During this stationary phase, pyruvate-flavin oxidoreductase / ferredoxin oxidoreductase activity increases in the genetically modified microorganisms under aerobic or partially aerobic conditions to produce an acetyl-CoA library; and compared to non-genetically modified microorganisms, the genetically modified microorganisms exhibit further enhanced sugar uptake.

[0076] On the one hand, genetically modified microorganisms include conditionally triggered anabolic valves that silence the expression of citrate synthase (gltA) and / or glucose-6-phosphate dehydrogenase (zwf) genes; or conditionally triggered anabolic valves that selectively proteolytically hydrolyze citrate synthase (gltA) and / or glucose-6-phosphate dehydrogenase (zwf) enzymes, and the microbial anabolic valves are conditionally triggered during the stationary phase or non-dividing cell state.

[0077] On the one hand, genetically modified microorganisms include the deletion of endogenous poxB and pflB genes.

[0078] On the one hand, the increased pyruvate-flavin oxidoreductase activity in genetically modified microorganisms is due to the overexpression of genes encoding pyruvate oxidoreductase during the stationary phase or non-dividing cell state.

[0079] On the one hand, the genetically modified microorganisms exhibit increased pyruvate-flavin oxidoreductase / ferroreductase activity, which is encoded by the ydbK gene, and the genetically modified microorganisms are Enterobacter species.

[0080] On the one hand, the increased pyruvate-flavin oxidoreductase / ferroreductase activity in genetically modified microorganisms is due to the induction of the oxidative soxRS regulator during the stationary phase or non-dividing cell state.

[0081] On the one hand, the activity of pyruvate-flavin oxidoreductase / ferreductin oxidoreductase is increased in genetically modified microorganisms. The increased activity of pyruvate-ferreductin oxidoreductase is due to the decrease in NADPH levels in genetically modified microorganisms during the stationary phase or non-dividing cell state.

[0082] On the one hand, the activity of at least one sugar transporter in genetically modified microorganisms increases the activity of at least one sugar transporter to enhance sugar uptake.

[0083] On the one hand, the activity of at least one sugar transporter in genetically modified microorganisms is a result of constitutive expression of sugar transporter genes leading to increased sugar transporter activity within the genetically modified microorganisms.

[0084] On the one hand, the activity of at least one sugar transporter protein in genetically modified microorganisms is the result of conditional overexpression during the stationary phase or non-dividing cell state.

[0085] On the one hand, the sugar transport proteins of genetically modified microorganisms are... pts Gene encoding.

[0086] On one hand, the genetically modified microorganisms are Enterobacteriaceae. On the other hand, the microorganisms are Escherichia coli.

[0087] On the one hand, genetically modified microorganisms, including citrate synthase, are used as enzymes in the production process.

[0088] On one hand, a bioprocess is provided for the production of protein products from genetically modified microorganisms. The bioprocess includes: a first stage in which the genetically modified microorganisms are grown in a culture medium; and a second stage in which a stationary or non-dividing cell state is induced after the limiting nutrients from the growth medium have been depleted. In the bioprocess, the genetically modified microorganisms in the stationary or non-dividing cell state produce the product at a rate of 30 g / L or higher.

[0089] In another aspect, the bioprocess includes: the activity of pyruvate-flavin oxidoreductase / ferreductase is caused by: overexpression of the gene encoding active pyruvate ferreductase, induction of the oxidative soxRS regulator, reduction of NADPH levels, reduction of glucose-6-phosphate dehydrogenase levels using an anabolic valve, or a combination thereof, said anabolic valve being designed to silence the gene. zwf A gene or selective protein hydrolyzing glucose-6-phosphate dehydrogenase, the valve of which is activated during the stationary phase or non-dividing cell state.

[0090] On the one hand, the activity of at least one sugar transporter increases in bioprocessing.

[0091] On the one hand, the bioprocess yields citrate, and the enzymes involved in the production pathway include citrate synthase, with the bioprocess producing citrate at a concentration of 100 g / L or greater. On the other hand, citrate synthase is encoded by the cimA3.7 gene.

[0092] On the one hand, the genetically modified microorganisms used in bioprocessing contain plasmids containing a citrate synthase gene operatively linked to a low-phosphate-inducible promoter.

[0093] On the one hand, bioprocesses include the use of endogenous... poxB Genes and pflB Genetically modified microorganisms with missing genes.

[0094] The publicly disclosed implementation plan is non-restrictive.

[0095] While various embodiments of the invention have been shown and described herein, it is emphasized that these embodiments are provided by way of example only. Many variations, alterations, and substitutions can be made without departing from the various embodiments of the invention. Specifically, for whatever reason, any grouping of compounds, nucleic acid sequences, polypeptides (including specific proteins comprising functional enzymes, metabolic pathway enzymes, or intermediates), elements, or other components, or concentrations stated or presented herein in lists, tables, or tables, or other groupings (e.g., Figure 3A , 4A The metabolic pathway enzymes shown in 5A, unless otherwise explicitly stated, are intended to provide a basis for identifying various subset implementations, which in their broadest extent include every subset of such groups by excluding one or more members (or subsets) of each of the said groups. Furthermore, when any range is described herein, unless otherwise explicitly stated, the range includes all values ​​therein and all subranges therein.

[0096] Furthermore, and more generally, conventional molecular biology, cell biology, microbiology, and recombinant DNA techniques within the scope of this art can be employed based on the disclosures, discussions, examples, and implementations herein. These techniques are well explained in the literature. See, for example, Sambrook and Russell, “Molecular Cloning: A Laboratory Manual,” Third Edition 2001 (volumes 1-3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Animal Cell Culture, RI Freshney, ed., 1986. These published resources are incorporated herein by reference.

[0097] The following disclosed resources are incorporated herein by reference for use in conjunction with the inventions described herein, for example, methods for the industrial bioproduction of chemical products from sugar sources, and industrial systems that can be used to achieve such conversions (Biochemical Engineering Fundamentals, 2nd ed., JE Bailey and DF Ollis, McGraw Hill, New York, 1986, e.g., Chapter 9, pp. 533-657, for biological reactor design; Unit Operations of Chemical Engineering, 5th ed., WL McCabe et al., McGraw Hill, New York, 1993, e.g., for process and separation technologies analyses; Equilibrium Staged Separations, PC Wankat, Prentice Hall, Englewood Cliffs, NJ USA, 1988, e.g., for separation technologies teachings).

[0098] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety, including PCT / US2015 / 035306 filed June 11, 2015 and PCT / US2018 / 019040 filed February 21, 2018.

[0099] Example

[0100] The examples provided herein are for illustrative purposes only and are not intended to be limiting. All reagents are commercially available unless otherwise stated. Species and other phylogenetic identification are performed according to classifications known to those skilled in the art of microbiology, molecular biology, and biochemistry.

[0101] Common methods

[0102] Culture media and reagents

[0103] Unless otherwise noted, all materials and reagents were purchased from Sigma (St. Louis, MO). LuriaBroth Lennox formulations were used for the propagation and construction of standard strains and plasmids. FGM1, FGM30, and SM10++ seed media were prepared according to Menacho-Melgar et al. (doi: 10.1101 / 820787) as previously described. SM10++ and SM10 phosphate-free media were prepared according to Moreb et al. (doi: 10.1021 / acssynbio.0c00182) as previously described. The FGM3 medium used in the microbioreactor studies is described in detail in the Supplementary Materials. The working antibiotic concentrations are as follows: kanamycin: 35 µg / mL, chloramphenicol: 35 µg / mL, zeocin: 100 µg / mL, blasticidin: 100 µg / mL, spectinomycin: 25 µg / mL, and tetracycline: 5 µg / mL.

[0104] FGM 3 medium / stock medium:

[0105] 10X concentrated ammonium citrate 30 salt (1L) was prepared by mixing 30 g of (NH4)2SO4 and 1.5 g of citric acid in water and stirring, adjusting the pH to 7.5 with NaOH. It was then autoclaved and stored at room temperature (RT).

[0106] 10X concentrated ammonium citrate 90 salt (1L) was prepared by mixing 90 g of (NH4)2SO4 and 2.5 g of citric acid in water and stirring, adjusting the pH to 7.5 with NaOH. It was then autoclaved and stored at RT.

[0107] 1 M 3-(N-morpholino)propanesulfonate (MOPS), pH adjusted to 7.4 with KOH. Sterilize by filtration (0.2 µm) and store at room temperature (RT).

[0108] 0.5M potassium phosphate buffer, pH 6.8, was prepared by mixing 248.5 mL of 1.0 M K₂HPO₄ and 251.5 mL of 1.0 M KH₂PO₄, and adjusting to a final volume of 1000 mL with ultrapure water. The solution was then filtered sterilized (0.2 µm) and stored at RT.

[0109] 2M MgSO4 and 10 mM CaSO4 solutions. Sterilize by filtration (0.2 µm) and store at room temperature (RT).

[0110] 50 g / L thiamine-HCl solution. Sterilize by filtration (0.2 µm) and store at 4°C.

[0111] Dissolve in 500 g / L glucose solution by heating and stirring. Cool, filter sterilize (0.2 µm), and store at room temperature (RT).

[0112] 500X Trace Metal Stock Solution: A solution of micronutrients was prepared in 1000 mL of water containing 10 mL concentrated H₂SO₄, 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. The solution was filtered and sterilized (0.2 µm) and stored at room temperature protected from light.

[0113] Prepare a fresh 40 mM ferric sulfate heptahydrate solution in water, and filter sterilize (0.2 µm) each time before preparing the culture medium.

[0114] Culture medium composition: Aseptically mix the stock solutions in the order written in the table below to reduce precipitation, and then filter sterilize (using a 0.2 µm filter) to prepare the final working culture medium.

[0115] Table 1: FGM3 medium, pH 6.8:

[0116]

[0117] Modified Strains

[0118] Table 2: List of strains with chromosome modifications.

[0119]

[0120] Table 3: Oligonucleotides used in strain construction.

[0121]

[0122] Table 4: Synthetic DNA used for strain construction.

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] strains and plasmids

[0131] Plasmid and strain information is shown in Tables 2 through 4. Oligonucleotide and synthetic linear DNA (Gblocks™) sequences were obtained from Integrated DNA Technologies (IDT, Coralville, Iowa (IA)). Deletions were constructed using tet-sacB-based selection and anti-selection. C-terminal DAS+4 tags (with or without superfolded GFP tags) were added directly to the chromosomal gene and selected using the 3' antibiotic resistance cassette of the integrated gene. All strains were confirmed by PCR, agarose gel electrophoresis, and sequencing using paired oligonucleotides (flanking the entire region) (Eton Biosciences or Genewiz). The recombinant engineered plasmid pSIM5 and the tet-sacB selection / anti-selection cassette were gifts 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). This was achieved by first deleting the natural... sspB Genes (using tet-sacB-based selection and anti-selection) were constructed from DLF_R002 strain DLFZ_0025. Subsequently, cas3 The gene was deleted and replaced with low phosphate-inducible sspB (using ugpBGene promoter alleles and constitutive promoters driving Cascade operon expression (again using tet-sacB-based selection and anti-selection). C-terminal DAS+4 tag modifications (with or without a hyperfolded GFP tag) are added to the chromosome of DLF_Z0025 and its derivatives via direct integration, and selection is performed via integration of the gene's antibiotic resistance cassette 3'.

[0132] The construction of plasmids, pCDF-ev (Addgene #89596), pHCKan-yibDp-GFPuv (Addgene #127078), and pHCKan-yibDp-cimA3.7 (Addgene #134595) was as described previously (doi: 10.1101 / 820787). Using mCherry open reading frames with or without C-terminal DAS+4 degron tags and the strongly synthetic constitutive proD promoter previously reported by Davis et al., plasmids pCDF-mCherry1 (Addgene #87144) and pCDF-mCherry 1 (Addgene #87145) were constructed from pCDF-ev by PCR and Gibson assembly.

[0133] Gene silencing guides and guide arrays were expressed by a series of pCASCADE plasmids. The pCASCADE control plasmids were prepared by exchanging the pTet promoter (a gift from C. Beisel) in pcrRNA.Tet with an insulated, low-phosphate-inducible ugpB promoter. To design the CASCADE guide arrays, the CASCADE PAM site near the -35 or -10 box of the target promoter was identified. Following the manufacturer's experimental protocol (with the following adjustment: 5% v / v DMSO was added to the Q5 PCR reaction), the 30 bp from the 3' end of the PAM site was selected as the guide sequence and cloned into the pCASCADE plasmid using Q5 directed mutations (NEB, MA). The PCR cycles were as follows: amplification involved an initial denaturation step at 98°C for 30 seconds, followed by cycles of 10 seconds at 98°C, 30 seconds at 72°C, and 1.5 minutes at 72°C (extension rate of 30 seconds / kb), for 25 cycles, followed by a final extension at 72°C for 2 minutes. 2 μL of the PCR mixture was used for a 10 μL KLD reaction (NEB, MA), which was carried out at room temperature for 1 hour, after which 1 μL of the KLD mixture was used for electroporation. The pCASCADE guide array plasmid (pCASCADE-G2Z) was prepared by sequentially amplifying complementary halves of each smaller guide plasmid via PCR, followed by subsequent DNA assembly, as shown in the table. Primers used for pCASCADE assembly and gRNA sequencing are provided in Supplementary Table 5 below. Furthermore, all strains containing the gRNA plasmid underwent routine validation to evaluate gRNA stability by PCR, as described below.

[0134] Table 5: List of sgRNA guide sequences and primers used to construct them. Spacers are indicated in italics.

[0135]

[0136] Microbioreactor studies

[0137] Single colonies of each strain were inoculated into 5 mL LB medium containing appropriate antibiotics and cultured at 37°C and 220 rpm for 9 hours or until OD600 reached >2. 500 μL of culture was inoculated into 10 mL SM10 medium containing appropriate antibiotics and cultured at 37°C and 220 rpm in a square shake flask (CAT#: 25-212, Genesee Scientific, Inc., San Diego, CA) for 16 hours. Cells were pelleted by centrifugation, and the culture density was normalized to OD600=5 using FGM3 medium. Growth and fluorescence measurements were obtained using a high-quality transfer FlowerPlate (CAT#: MTP-48-B, m2p-labs, Germany) in a microbioreactor (m2p labs, Baesweiler, Germany). 40 μL of OD-normalized culture was inoculated into 760 μL of FGM3 medium containing appropriate antibiotics. The microbioreactor was set up as follows: RFP gain = 100, GFP gain = 20, biomass gain = 20, shaking speed = 1300 rpm, temperature = 37 °C, humidity = 85%. Each strain was analyzed in triplicate.

[0138] ELISA

[0139] According to the manufacturer's instructions, protein quantification was performed using the GFP quantitative kit from AbCam (Cambridge, UK, product #ab171581) via a C-terminal GFP tag. In short, the sample was obtained from microfermentation as described above. Cells were harvested 24 hours after phosphate depletion, washed with water, and lysed with the provided extraction buffer.

[0140] Guide RNA stability test

[0141] The stability of the guide RNA array was confirmed by colony PCR using 2X EconoTaq Master mix (Lucigen) in a 10 µL PCR reaction with the following two primers: gRNA-forward (for): 5'-GGGAGACCACAACGG-3' (SEQ ID NO: 25) and gRNA-reverse (rev): 5'-CGCAGTCGAACGACCG-3' (SEQ ID NO: 26). The 10 µL PCR reaction consisted of: 5 µL of 2X EconoTaq Master mix (Lucigen), 1 µL of each primer (10 µM), and 3 µL of dH2O. Initial denaturation was performed at 98 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min. PCR reactions were then performed on agarose gels, and band sizes were compared with control PCR reactions using purified plasmid DNA as a template. When the guide array size was smaller than expected, guide protospacer loss occurred, indicating the loss of one or more protospacer sequences.

[0142] Fermentation

[0143] Microfermentation of basal medium was performed as previously described (doi: 10.1021 / acssynbio.0c00182). For microfermentation induced by paraquat, paraquat was added 1 hour before phosphate depletion and subsequently removed in a cell washing step to deplete phosphate in the medium. 1 L fermentation in an instrumented bioreactor was also performed as previously reported, with slight modifications to the glucose feeding profiles as a function of strain and process. Typically, the feed was increased to ensure a glucose surplus, thus ensuring productivity was not limited by the feed. Glucose feeding was as follows: For 10 g CDW / L fermentation, the starting batch glucose concentration was 25 g / L. When cells entered mid-exponential growth, concentrated sterile filtered glucose (500 g / L) was continuously added to the tanks at a rate of 1.5 g / h. For 25 g CDW / L fermentation, the starting batch glucose concentration was 25 g / L. When the cells entered mid-exponential growth, concentrated sterile filtered glucose (500 g / L) was added to the tank at an initial rate of 9 g / h. This rate was then increased exponentially, doubling every 1.083 hours (65 minutes) until 40 g of total glucose was added, after which the feed was maintained at 1.75 g / h.

[0144] Production of isotope-labeled metabolites.

[0145] C 13 Pyruvic acid (CLM-1082-PK) and C 13 D-glucose (U-13C6, 99%) was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). Isotopically labeled cimic acid was produced in two-stage shake flask studies using the cimA3.7-expressing strain DLF_Z0044, mimicking microfermentation. Briefly, 20 mL of culture in SM10++ medium was inoculated with the strain grown overnight in baffled 250 mL Erlenmyer shake flasks at 37°C and 150 rpm. After 16 hours of growth, cells were collected by centrifugation, washed, and resuspended in 20 mL of SM10 basal medium (phosphate-free), in which glucose was converted to C10 basal medium. 13Labeled glucose was used as a substitute. Cultures were grown at 37°C with shaking at 150 rpm for 25 hours, after which cells were removed by centrifugation, and the used culture medium was filtered sterilized before being used as an internal standard.

[0146] Analytical methods

[0147] Cell dry weight: This work uses the OD / cell dry weight correlation coefficient (1 OD (600 nm) = 0.35 gCDW / L, determined by Menacho-Melgar et al.).

[0148] Quantitative analysis of glucose and organic acids: Two methods were used for the quantification of glucose and organic acids. First, a UPLC-RI method was developed for the simultaneous quantification of glucose, citric acid, acetic acid, pyruvic acid, citraconate, citric acid, and other organic acids (including lactic acid, succinic acid, fumaric acid, malic acid, and mevalonic acid). Chromatographic separation was performed at 55 °C using a Rezex Fast Acid Analysis HPLC column (100 × 7.8 mm, 9 μm particle size; CAT#: #1250100, Bio-Rad Laboratories, Inc., Hercules, CA). 5 mM sulfuric acid was used as the isoclinic eluent at a flow rate of X mL / min. The sample injection volume was 10 µL. Next, quantification was performed using a Bio-Rad Fast Acid Analysis HPLC column (100 × 7.8 mm, 9 μm particle size; CAT#: #1250100, Bio-Rad Laboratories, Inc., Hercules, CA) at 65 °C. 10 mM sulfuric acid was used as the eluent, and the isocratic flow rate was 0.3 mL / min. In both methods, the injection volume was 10 µL. Chromatographic analysis and detection were performed using a Waters Acquity H-Class UPLC with an integrated Waters 2414 Refractive Index (RI) detector (Waters Corp., Milford, MIT, USA). Samples were diluted to the accurate linear range as needed. Diluents were performed using ultrapure water.

[0149] Quantification of organic acids using RapidFire-qTOF-MS Centrifuge the microfermentation sample (and the confirmatory subset from the bioreactor) to remove cells. Dilute the broth medium 100-fold in water to a final volume of 20 μL. Add C13 pyruvate to a final concentration of 10 mg / L or add 2 μL of broth medium containing C13-labeled citrate. Inject the final sample into a HILIC (HI type or equivalent H6) RapidFire™ cartridge (Agilent Technologies, Santa Clara, CA). After aspiration for 600 ms, load the injection solution onto a cartridge containing 95% hexane and 5% isopropanol at a flow rate of 1.0 mL / min for 3000 ms. After loading, wash the cartridge with isopropanol at a flow rate of 1.0 mL / min for 2000 ms. Elution was performed using 50% water / 50% methanol containing 0.2% acetic acid and 0.5 μM (NH4)3PO4 at a flow rate of 1.0 mL / min for 8000 ms. Column equilibration was performed for 4000 ms. qTOF was tuned in the fragile ion and negative ESI modes within a mass range of 50–250 m / z. The detection setup was as follows: dry gas: 250 °C, flow rate 13 L / min; sheath gas: 400 °C, flow rate 12 L / min; nebulizer pressure: 35 psi; fragmenter voltage: 100 V; skater voltage: 65 V; nozzle voltage: 2000 V; capillary voltage: 3500 V. The acquisition rate was 1 spectra / second.

[0150] Example 1: Gene Silencing Arrays and Pathway Expression Constructs

[0151] Gene silencing based on pCASCADE guide array

[0152] The design and construction of the CASCADE wizard sequence and wizard array are as follows: Figure 1 and Figure 2A-2B As shown. The pCASCADE-control plasmid was prepared by exchanging the pTet promoter in pcrRNA.Tet with an insulating, low-phosphate-inducible ugpB promoter, as shown. Figure 1As shown. Two promoters regulate the gltA gene, and sgRNAs were designed for both promoters. Four promoters regulate the gapA gene, and sgRNAs were designed for the first promoter because, during the exponential growth phase, gapA mRNA is primarily initiated at the highly efficient gapA P1 promoter and remains at a high level during the stationary phase compared to the other three gapA promoters. Multiple promoters upstream of the lpd gene are involved in lpd regulation (ecocyc.org / gene?orgid=ECOLI&id=EG10543#tab=showAll), therefore it is not possible to design unique and effective sgRNAs specifically for lpd. The promoter sequences for fabI, udhA, and zwf were obtained from the EcoCyc database (ecocyc.org). To design CASCADE guide arrays, the CASCADE PAM site near the -35 or -10 box of the target promoter was identified. Following the manufacturer's experimental protocol (with the following adjustment: 5% v / v DMSO was added to the Q5 PCR reaction), the 30 bp 3' end of the PAM site was selected as the guide sequence and cloned into the pCASCADE plasmid using Q5 directed mutagenesis (NEB, MA). The pCASCADE control vector was used as a template. pCASCADE plasmids with two or more guide arrays were prepared as described below and... Figure 2A-2B As shown. pCASCADE guide array plasmids are prepared by sequentially amplifying the complementary halves of each smaller guide plasmid via PCR, followed by subsequent 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.

[0153] Table 6: List of guide sequence sgRNAs and primers used to construct them. Spacers are shown in italics.

[0154]

[0155] Table 7: List of plasmids used in this study.

[0156]

[0157] Example 2: Dynamic control of protein levels

[0158] Plasmids expressing fluorescent proteins and silencing guide sequences were transformed into the corresponding host strains listed in Table 2. The strains were evaluated in triplicate in m2p-labs Biolector™, with fluorescence, including GFPuv and mCherry levels, and biomass levels, measured simultaneously. Results are presented in... Figures 5A-5D The information is provided in the text.

[0159] Table 8: Strains used for dynamic control of protein levels

[0160]

[0161] Use the following formula to correct the OD600 reading, where OD600 refers to the offline measurement result. Refers to Biolector biomass readings. t0 Indicates the starting point. tf Indicates the end point.

[0162]

[0163] Example 3: The effect of dynamic control of two central metabolic pathways, TCA and PPP, on fluxes through glycolysis and pyruvate oxidation.

[0164] like Figure 3A As shown, we sought to obtain the dynamic control of the effects of two central metabolic pathways (the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway (PPP)) on the fluxes via glycolysis and pyruvate oxidation. We achieved this by creating an anabolic valve to dynamically reduce the levels of the first critical step in each pathway: citrate synthase (GltA, “G”, encoded by the gltA gene) and glucose-6-phosphate dehydrogenase (Zwf, “Z”, encoded by the zwf gene). We demonstrated that dynamic control of these two enzymes increased the stationary-phase yields of pyruvate and citrate, and is applicable to the production of numerous products requiring pyruvate and / or acetyl-CoA.

[0165] We first developed a control system capable of dynamically reducing protein levels in two-stage processes, such as... Figure 3B-3D As shown. The valve may include controlled proteolysis or CRISPRi / Cascade-based gene silencing, or a combination of proteolysis and silencing, to reduce the levels of key metabolic enzymes. Induction is implemented using phosphate depletion as an environmental trigger. The native E. coli IE type Cascade / CRISPR system is used for gene silencing ( Figure 3C (i-iii) Targeted proteolysis is achieved by linking the expression of the molecular chaperone SspB to phosphate deprivation. When induced, SspB binds to the C-terminal DAS+4 peptide tag on any target protein and induces degradation by the *E. coli* ClpXP protease. Figure 3D Using engineered strains, such as Figure 1As shown in E, protein levels can be controlled in two phases, such as “turning on” GFP and “turning off” constitutively expressed mCherry. Although in this case, the combination of gene silencing and proteolysis results in the highest protein degradation rate (…). Figure 3F-3G However, the effects and specific decay rates of each pathway will depend on the target gene / enzyme and its specific natural turnover rate and expression level.

[0166] To dynamically reduce the levels of GltA and Zwf ( Figure 3H-3I Chromosomal modifications were performed on the strains to attach C-terminal DAS+4degron tags to these genes. Furthermore, several strains were designed with C-terminal hyperfolded GFP tags after each gene, some with and some without C-terminal degron tags. Plasmids expressing gRNA were designed to repress... gltAp2 and zwf Promoter expression. Using these strains and plasmids, dynamic control of enzyme levels was monitored by tracking GFP via ELISA during two-stage basal medium microfermentation, as reported by Moreb et al. ELISA was used because protein levels in the engineered strains were too low to use GFP fluorescence as a direct reporter gene. GltA proteolysis and silencing resulted in a 70% and 85% reduction in GltA levels, respectively, with a combined reduction of 90%. In the case of Zwf, proteolysis, silencing, and combinations thereof all resulted in protein levels below the quantitation limit of our assay.

[0167] The effect of the “G” and “Z” valve combination on metabolic flux was measured in basal medium microfermentation without any heterogeneous production pathways. This was because the strain used had a deficiency in the major pathway leading to acetic acid production. poxB and pta-ackA Therefore, pyruvate synthesis was initially evaluated as a measure of metabolic flux via glycolysis. Figures 4A-4D The “G” valve had the greatest impact on pyruvate production, with no detectable product observed in the control strain without SMV. The increased pyruvate production could be attributed to a stoichiometric effect, where a portion of the flux normally entering the TCA cycle is redistributed to spillover metabolites, or a more comprehensive increase in glucose uptake, resulting in greater spillover metabolism and pyruvate synthesis. To evaluate both options, we measured the effect of the “G” valve on glucose uptake. Results are as follows: Figure 4C As shown, the increase in pyruvate production is primarily attributable to an increase in uptake, rather than a redistribution of baseline flux.

[0168] Therefore, the increased glucose uptake via the "G" valve is likely due to the direct regulatory role of α-ketoglutarate (aKG), a metabolite produced by the TCA cycle. αKG, a precursor to glutamate, plays several key regulatory roles, including regulating glucose transport by directly inhibiting PTS-dependent glucose transporter enzyme I. Figure 3A-3I This feedback regulation is one way to coordinate glucose uptake with nitrogen assimilation (glutamate synthesis). We conducted supplementary experiments by adding 20 mM dimethyl-aKG (DM-aKG) to the microfermentations at the start of production. DM-aKG was used instead of aKG because DM-aKG has been shown to cross membranes better and is added to the intracellular aKG pool after hydrolysis. Figures 4A-4D As shown, DM-αKG inhibited sugar uptake in both control cells and strains with a valve that reduced GltA levels. These results collectively support that the dynamic reduction in GltA levels and subsequent decrease in the αKG pool are the primary drivers of increased sugar uptake and pyruvate biosynthesis. We then turned to evaluating pyruvate production in an instrumented bioreactor. Batch fermentation with a basal medium was performed, as previously reported by Menacho-Melgar et al. In this method, phosphate concentration limited biomass levels, and once depleted, induced the silencing of gRNA and SspB molecular chaperone expression. The results of comparing the control host strain with strains exhibiting dynamic control over GltA levels were presented in… Figure 4D As shown in the data. The control strain showed the least transient accumulation of pyruvate, while dynamic control was used to obtain a maximum titer exceeding 30 g / L.

[0169] To evaluate the effect of dynamic control on acetyl-CoA flux, we used leveraged cimic acid synthase, which produces one mole of cimic acid from one mole of pyruvate and one mole of acetyl-CoA. Cimic acid is a precursor to the industrial chemicals itaconic acid and methyl methacrylate, and an intermediate in the biosynthesis of branched-chain amino acids. For cimic acid production, we used a low-phosphate-inducible plasmid expressing a previously reported feedback-resistant mutant cimic acid synthase (cimA3.7). This plasmid was introduced into a group of “G” and “Z” valve strains, and their cimic acid yield in a two-stage microfermentation was evaluated. Figures 5A-5D The best production strains have both "G" and "Z" valves.

[0170] In the case of pyruvate, the "Z" valve has no significant impact on production. Figure 4BCitrate and pyruvate are similar products because they are both oxidized and do not require redox cofactors (such as NADPH) for biosynthesis. A key difference between the two products is that citrate requires an additional precursor, acetyl-CoA. The “Z-valve”-dependent improvement in citrate production may depend on improved acetyl-CoA production in strains with reduced Zwf activity. This suggests that Zwf levels, or downstream metabolite levels, have a negative regulatory effect on stationary-phase acetyl-CoA synthesis. It is important to note that strains used to produce pyruvate and citrate... poxB and pflB There is a deficiency in (which can induce acetyl-CoA synthesis), and it was initially assumed that all acetyl-CoA flux is through a well-characterized pyruvate dehydrogenase (PDH) multienzyme complex. Unexpectedly, the proteolytic degradation of Lpd (a subunit of PDH) has no effect on citric acid production. Based on this, we considered the potential of an alternative primary pathway for acetyl-CoA production in stationary cultures, namely, by... ydbK Genetically encoded pyruvate-flavin oxidoreductase / ferroreductin oxidoreductase (Pfo).

[0171] like Figure 3A-3I As shown, Pfo( ydbK It may be partially responsible for acetyl-CoA synthesis during the stationary phase, and this activity is regulated by intermediates in PPP due to its role in the oxidative stress response, and is also known to be involved in the response to oxidative stress. To test this hypothesis, we constructed [a specific PCR method] in cimicifuga strains containing both "G" and "Z" valves. ydbK The deficiency was identified, and the yield of limonolic acid was measured. (Example) Figure 5B As shown, ydbK The absence of Pfo significantly reduced citric acid synthesis, confirming its role in acetyl-CoA flux. Since Pfo has been shown to be induced under oxidative stress via the SoxRS regulator (also regulated by the NADPH pool), its expression is likely due to altered NADPH levels caused by decreased Zwf activity.

[0172] Finally, we evaluated the citrate-producing strains in the instrumented bioreactor. The control strain produced a reasonable citrate titer (~40 g / L), while the introduction of SMV increased the yield. The combined "GZ" valve strain exhibited the highest citrate yield, reaching a titer of ~100 g / L. This process was then intensified by increasing the biomass level from ~10 gCDW / L to ~25 gCDW / L, resulting in a titer of 126 ± 7 g / L. This process... Figure 5CAs shown in the figure, the overall yield of the process is 0.74-0.77 g citrate / g glucose, with the yield approaching 0.80-0.82 g citrate / g glucose during the production phase. The theoretical yield of citrate from glucose is 1 mol / mol or 0.817 g / g.

[0173] Previous studies utilizing dynamic control have primarily relied on stoichiometric frameworks, where pathways are switched "on" and "off" to reduce fluxes of desired products in stoichiometric competition—in other words, pathway redirection. For example, Venayak et al. highlighted the importance of GltA / CS as a central valve candidate for dynamic metabolic control, partly based on stoichiometric models. However, these studies and models have overlooked the importance of downstream metabolite (e.g., αKG) regulation. This work demonstrates that increasing fluxes through the dysregulation of feedback control has a significant impact on production, independent of minimizing stoichiometry or competing pathways. In particular, the unexpected increase in acetyl-CoA flux due to reduced Zwf activity is noteworthy.

[0174] This is the first report of the interaction between stationary minimum Zwf levels, SoxRS activation, and Pfo activity. Furthermore, the magnitude of the metabolic flux through Pfo is unexpected. Although Pfo is an iron sulfur cluster-containing enzyme that has been successfully expressed under both aerobic and anaerobic conditions, it is rapidly inactivated by molecular oxygen in vitro; therefore, it was conventionally considered unlikely to support these types of flux. These data suggest that the Pfo pathway can function as a central metabolic pathway under certain conditions and maintain high levels of activity even under aerobic conditions in vivo. A better understanding may lead to alternative strategies (independent of reducing Zwf levels) to optimize flux through this pathway, such as pathway overexpression and / or enzyme engineering.

[0175] Example 4: Stabilized glucose uptake and pyruvate synthesis are insensitive to α-ketoglutarate levels in Escherichia coli PTS(-) strains

[0176] Now refer to Figure 7AOverview of glucose uptake in *Escherichia coli* PTS(-) strains. Strain DLF_00286 (genotypes 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 through the overexpression of galP galactose permease (which can also transport glucose) and glucokinase (glk), which activates glucose. Figure 7B Strain DLF_00286 and strain DLF_00286 with dynamically controlled citrate synthase (GltA) levels produced pyruvate in two-stage micro-fermentations. Stationary pyruvate synthesis in strain DLF_00286 was improved compared to the PTS(+) control (DLF_0025). Dynamic control of citrate synthase (gltA) levels did not improve pyruvate synthesis in the host background of DLF_00286. Figure 7C In PTS(-) strains, glucose uptake was insensitive to dimethyl-αKG supplementation. Stationary pyruvate synthesis was improved in strain DLF_00286 compared to the PTS(+) control (DLF_0025). Dynamic control of citrate synthase (gltA levels) did not improve pyruvate synthesis in the host background of DLF_00286. Figure 7D The pyruvate and biomass yields of strain DLF_00286 and its “G” valve derivative were measured. Biomass (grey) and pyruvate yield (blue) of the control strain, and biomass (black) and pyruvate yield (green) of the “G” valve strain were plotted as a function of time.

[0177] Example 5: Acetyl-CoA flux depends on Pfo (YdbK) activity.

[0178] Now refer to Figure 8A The proteolytic degradation of Lpd (lpd-DAS+4, a subunit of the pyruvate dehydrogenase multienzyme complex) and the deletion in ydbK were evaluated in the "GZ" valve background. Figure 8BThe relative activity of the ydbK enzyme during the stationary phase as a function of the "G" and "Z" valves is shown. ydbK activity was measured in the crude lysate using pyruvate and CoA as substrates and methylviologen as the electron acceptor. Figure 8C In the image, the NADPH library (gray bar) and ydbK expression level (green bar) are shown in the engineered strain. The expression of the overfolded GFP (sfGFP) reporter gene is driven by the ydbK promoter.

[0179] Example 6: Acetyl-CoA flux depends on soxS activation and can be improved independently of the "Z" valve.

[0180] Now refer to Figure 9A The strain was engineered for low-phosphate induction of SoxS (independent of NADPH library and SoxR activation). This was achieved by designing an additional SoxS copy on the chromosome, which was generated by a low-phosphate inducible strain. yibD Gene promoter induction. In Figure 9B In this study, citric acid production was observed in the microfermentation of a PTS(+) strain engineered with a combination of a “G” valve and a low-phosphate-inducible soxS. Importantly, strains with a ydbK deletion still exhibited reduced citric acid flux under soxS induction.

[0181] More generally, this invention highlights the potential to manipulate known and unknown feedback regulatory mechanisms to improve enzyme activity and metabolic flux in vivo. This approach can unlock numerous novel engineering strategies and significantly improve productivity, titers, and yields. Furthermore, these results confirm the metabolic potential of stationary-phase cultures. Dynamic metabolic control in two-stage cultures is particularly well-suited for implementing these strategies. Simply overexpressing key enzymes does not bypass natural regulation, and complete removal of central metabolic enzymes and / or metabolites often leads to growth defects and strains, requiring evolutionary compensatory metabolic changes to meet growth demands. In contrast, changes in the levels of central regulatory metabolites during the stationary phase can reconnect regulatory networks and metabolic fluxes without such limitations.

[0182] As described above, while this application has been illustrated by way of description of embodiments, and while the embodiments have been described in considerable detail, it is not intended to limit or in any way restrict the scope of the appended claims to such detail. Those skilled in the art will readily see additional advantages and modifications with the aid of this application. Therefore, this application is not limited in its broader aspects to the specific details and illustrative embodiments shown. Deviations from these details and examples may be made without departing from the spirit or scope of the general inventive concept.

Claims

1. A genetically modified Escherichia coli (E. coli) E. coli Microorganisms, including: Production pathways for citrate acid that include citrate synthase. Conditional induction of SoxS; Conditionally triggered anabolic valves that silence the gene expression of citrate synthase (gltA) and glucose-6-phosphate dehydrogenase (zwf), or conditionally triggered anabolic valves that can selectively proteolytically hydrolyze the citrate synthase (gltA) and glucose-6-phosphate dehydrogenase (zwf). The anabolic valve of the genetically modified *E. coli* microorganism is conditionally triggered during the stationary phase or non-dividing cell state. Among them, under the condition that the limiting nutrients are depleted in the growth medium in which the genetically modified Escherichia coli microorganisms are grown, a stationary phase or non-dividing cell state is induced. During the stationary or non-dividing cell state, under aerobic or partially aerobic conditions, pyruvate-flavin oxidoreductase / ferroreductase activity is increased in the genetically modified *E. coli* microorganisms to produce an acetyl-CoA library; and In this study, sugar uptake was enhanced in the genetically modified *Escherichia coli* microorganisms compared to unmodified microorganisms.

2. The genetically modified *Escherichia coli* microorganism according to claim 1, wherein the genetically modified *Escherichia coli* microorganism comprises endogenous... poxB Genes and pflB The absence of genes.

3. The genetically modified *Escherichia coli* microorganism according to claim 1, wherein the increased pyruvate-flavin oxidoreductase activity is due to overexpression of the gene encoding pyruvate oxidoreductase during the stationary phase or non-dividing cell state.

4. The genetically modified *Escherichia coli* microorganism according to claim 1, wherein the pyruvate-flavin oxidoreductase is composed of... ydbK Gene encoding.

5. The genetically modified *Escherichia coli* microorganism according to claim 1, wherein the increased pyruvate ferroredoxin oxidoreductase activity is due to the induction of the oxidative soxRS regulator during the stationary phase or non-dividing cell state.

6. The genetically modified *Escherichia coli* microorganism according to claim 1, wherein the increased pyruvate ferroredoxin oxidoreductase activity is due to a decrease in NADPH levels within the genetically modified *Escherichia coli* microorganism during the stationary phase or non-dividing cell state.

7. The genetically modified Escherichia coli microorganism according to claim 1, wherein during the stationary phase or non-dividing cell state, the activity of at least one sugar transporter is increased to enhance sugar uptake.

8. The genetically modified Escherichia coli microorganism according to claim 7, wherein constitutive expression of the sugar transporter gene increases the sugar transporter activity within the genetically modified Escherichia coli microorganism.

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

10. The genetically modified *Escherichia coli* microorganism according to claim 8, wherein the sugar transporter is composed of... pts Gene encoding.

11. The genetically modified *Escherichia coli* microorganism of claim 1, wherein the anabolic valve achieves gene silencing via CRISPR interference, the anabolic valve further comprising a CASCADE guide array comprising two or more genes encoding small guide RNAs, each small guide RNA specifically targeting a different gene to simultaneously silence multiple genes, the guide array comprising more than one promoter for each gene.

12. A biological method for producing protein products from genetically modified *Escherichia coli* microorganisms as described in claim 1, the biological method comprising: In the first stage, the genetically modified *E. coli* microorganisms were grown in the culture medium, and In the second stage, after depletion of limiting nutrients from the growth medium, a stationary or non-dividing cell state is induced. The genetically modified *E. coli* microorganisms in the stable phase or non-dividing cell state produce the product at a rate of 30 g / L or higher.

13. The biological method of claim 12, wherein the increase in pyruvate-flavin oxidoreductase / ferreductase activity is caused by: overexpression of a gene encoding active pyruvate ferreductase, induction of an oxidative soxRS regulator, reduction of NADPH levels, reduction of glucose-6-phosphate dehydrogenase levels using an anabolic valve, or a combination thereof, said anabolic valve being designed to silence genes. zwf A gene or selective protein hydrolyzing glucose-6-phosphate dehydrogenase, the valve of which is activated during the stationary phase or non-dividing cell state.

14. The biological method according to claim 12, wherein the activity of at least one sugar transporter is increased.

15. The biological method of claim 12, wherein the product is citric acid, the enzyme in the production pathway includes citric acid synthase, and the biological method produces citric acid at a concentration of 100 g / L or greater.

16. The biological method of claim 12, wherein the cimolic acid synthase is encoded by the cimA3.7 gene.

17. The biological method of claim 12, wherein the genetically modified *Escherichia coli* microorganism comprises a plasmid containing a citrate synthase gene operatively linked to a low-phosphate-inducible promoter.

18. The biological method of claim 12, wherein the genetically modified *Escherichia coli* microorganism comprises endogenous... poxB Genes and pflB The absence of genes.

19. A genetically modified microorganism comprising: A production pathway for the production of a product from an acetyl-CoA precursor, comprising at least one enzyme, and Silencing citrate synthase (gltA) and glucose-6-phosphate dehydrogenase (GLP-6-phosphate dehydrogenase) zwf A conditionally triggered anabolic valve for gene expression of the gene, or a conditionally triggered anabolic valve capable of selectively proteasically hydrolyzing the citrate synthase (gltA) and glucose-6-phosphate dehydrogenase (zwf). as well as endogenous poxB and pflB Gene deletion; The anabolic valve of the genetically modified microorganism is conditionally triggered during the stationary phase or non-dividing cell state. Among them, under the condition that the limiting nutrients are depleted from the growth medium in which the genetically modified microorganisms grow, a stationary phase or non-dividing cell state is induced. During the stationary or non-dividing cell state, under aerobic or partially aerobic conditions, pyruvate-flavin oxidoreductase / ferredoxin oxidoreductase activity increases in the genetically modified microorganisms to produce an acetyl-CoA library; and Among them, when compared with unmodified microorganisms, the genetically modified microorganisms exhibited enhanced sugar uptake. The genetically modified microorganisms mentioned therein are Escherichia coli.

20. The genetically modified microorganism of claim 19, wherein the increased pyruvate-flavin oxidoreductase activity is due to overexpression of the gene encoding pyruvate oxidoreductase during the stationary phase or non-dividing cell state.

21. The genetically modified microorganism according to claim 19, wherein the pyruvate-flavin reductin / ferroreductin oxidoreductase is composed of... ydbK Gene encoding.

22. The genetically modified microorganism of claim 19, wherein the increased pyruvate ferroreductin oxidoreductase activity is due to the induction of the oxidative soxRS regulator during the stationary phase or non-dividing cell state.

23. The genetically modified microorganism of claim 19, wherein the increased pyruvate ferroreductase activity is due to a decrease in NADPH levels within the genetically modified microorganism during the stationary phase or non-dividing cell state.

24. The genetically modified microorganism of claim 19, wherein the activity of at least one sugar transporter is increased to enhance sugar uptake.

25. The genetically modified microorganism of claim 19, wherein constitutive expression of the sugar transporter gene increases the sugar transporter activity within the genetically modified microorganism.

26. The genetically modified microorganism of claim 24, wherein the sugar transporter is conditionally overexpressed during the stationary phase or non-dividing cell state.

27. The genetically modified microorganism of claim 24, wherein the sugar transporter is composed of... pts Gene encoding.

28. The genetically modified microorganism according to claim 19, wherein the enzyme in the production pathway is citrate synthase.

29. The genetically modified microorganism according to claim 19, wherein the product is pyruvate.

30. The genetically modified microorganism of claim 19, wherein the anabolic valve achieves gene silencing via CRISPR interference, the anabolic valve further comprising a CASCADE guide array comprising two or more genes encoding small guide RNAs, each small guide RNA specifically targeting a different gene to simultaneously silence multiple genes, the guide array comprising more than one promoter for each gene.

31. A biological method for producing a protein product from a genetically modified microorganism as described in claim 19, the biological method comprising: In the first stage, the genetically modified microorganisms are grown in a culture medium, and In the second stage, after depletion of limiting nutrients from the growth medium, a stationary or non-dividing cell state is induced. The genetically modified microorganisms in the stationary or non-dividing cell state produce products at a rate of 30 g / L or higher.

32. The biological method of claim 31, wherein the increase in pyruvate-flavin oxidoreductase / ferreductase activity is caused by: overexpression of a gene encoding active pyruvate ferreductase, induction of an oxidative soxRS regulator, reduction of NADPH levels, reduction of glucose-6-phosphate dehydrogenase levels using an anabolic valve, or a combination thereof, said anabolic valve being designed to silence genes. zwf A gene or selective protein hydrolyzing glucose-6-phosphate dehydrogenase, the valve of which is activated during the stationary phase or non-dividing cell state.

33. The biological method according to claim 31, wherein the activity of at least one sugar transporter is increased.

34. The biological method of claim 31, wherein the product is citric acid, the enzyme in the production pathway includes citric acid synthase, and the biological method produces citric acid at a concentration of 100 g / L or greater.

35. The biological method of claim 31, wherein the cimolic acid synthase is encoded by the cimA3.7 gene.

36. The biological method of claim 31, wherein the genetically modified microorganism comprises a plasmid containing a citrate synthase gene operably linked to a low-phosphate-inducible promoter.

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