Compositions and methods for improving malonyl-coa biosynthesis using stage 2 dynamic metabolism control

Abstract

Methods and microorganisms for increasing the flux of malonyl-CoA and producing a product with malonyl-CoA as a precursor. Methods include dynamic modulation of nitrogen regulatory proteins in a stationary phase of the method. The method can dynamically modulate more than one gene.

Description

[0001] Priority declaration

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 269,646, filed on March 21, 2022, the entire contents of which are incorporated herein by reference.

[0003] Statement Regarding Federally Funded Research

[0004] This invention was made with government support under Federal Grant HR0011-14-C-0075 awarded by DARPA, Grant #N00014-16-1-2558 awarded by ONR, and Grant #4000181535 awarded by SERDP. The Federal Government has certain rights in this invention. Background Art

[0005] Malonyl-CoA is a central metabolite and intermediate that is a precursor to a variety of products, including biofuels / biopolymers, plant natural products, polyketides, and pharmaceutical intermediates. Efforts to increase malonyl-CoA flux are extensive and include nutrient starvation, dynamic control, and genetic manipulation, including additions and deletions in Escherichia coli and other microbial hosts.

[0006] In Escherichia coli, malonyl-CoA is a tightly regulated metabolite responsible for fatty acid biosynthesis, such that its biosynthesis is coordinated with the rates of fatty acid biosynthesis, phospholipid production, and cell growth. Under normal metabolic conditions, only a small fraction of the acetyl-CoA pool is converted to malonyl-CoA by the action of acetyl-CoA carboxylase (ACCase). Malonyl-CoA flux and pool are regulated by nested feedback loops and connections to whole-cell energy and carbon conditions to a greater extent than most other metabolic branch points and commitment steps, and in native organisms its intracellular concentration is maintained in the low to mid-micromolar range, approximately 10- to 100-fold lower than acetyl-CoA during aerobic growth. The scarcity of metabolites and the complex regulatory connections are widely believed to have hampered bioprocess development of these products. Unraveling central metabolites from host networks is a central part of platform strain development. Summary of the invention

[0007] This Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0008] In some aspects, the invention includes a genetically modified microorganism having an anabolic valve for dynamic selective regulation of a nitrogen regulatory protein (glnB). The anabolic valve can include silencing of genetic expression of the glnB gene, selective enzymatic degradation of the glnB protein, or any combination of the two. The anabolic valve is selectively activated in response to changes in the growth medium in which the genetically modified microorganism is grown, so as to silence the glnB gene and / or degrade the glnB protein.

[0009] In some aspects, the genetically modified microorganism has an additional anabolic valve for silencing a gene, wherein the gene is: enoyl-ACP reductase (fabI), citrate synthase (gltA), glucose-6-phosphate dehydrogenase (zwf), or any combination of these, or an additional anabolic valve for selective enzymatic degradation of a protein, wherein the protein is: enoyl-ACP reductase (fabI), citrate synthase (gltA), glucose-6-phosphate dehydrogenase (zwf), or any combination of these.

[0010] In some aspects, the present invention generally describes a bioprocess for producing a product using a genetically modified microorganism that includes an anabolic valve for dynamic selective regulation of a nitrogen regulatory protein (glnB).

[0011] Other methods, features and / or advantages are, or will become, apparent by examination of the following drawings and detailed description. It is intended that all such additional methods, features and advantages be included within this description and be protected by the following claims.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The novel features of the invention are particularly set forth in the claims. The features and advantages of the invention will be better understood by referring to the following detailed description and the accompanying drawings, which illustrate exemplary aspects of the invention in which the principles of the invention are employed:

[0014] Figures 1A-1D: 1A) depicts a schematic diagram of dynamic control of GltA, Zwf, FabI and GlnB levels to improve THN production. The red double triangle "valve" indicates a central metabolic protein that is targeted for dynamic reduction. Reduced GltA activity reduces the αKG pool and alleviates the inhibition of αKG-mediated PTS-dependent glucose uptake (particularly, PtsI), thereby increasing glucose uptake rate, glycolytic flux and pyruvate production. Reduced Zwf activity reduces NADPH levels, thereby activating the SoxRS regulator, and increases the activity of pyruvate-ferredoxin oxidoreductase (Pfo, ydbK), thereby increasing acetyl-CoA flux. Reduced FabI activity dynamically reduces the acyl-ACP pool to alleviate the inhibition of ACCase. Reduced GlnB levels reduce GlnB-mediated ACCse inhibition (in the absence of α-ketoglutarate). 1B) Overview of two-stage dynamic metabolic control. Biomass grows to a stationary phase, where depletion of the limiting nutrient (i.e., phosphate) "turns off" competing pathways while "turning on" the desired production route. (1C-1D) Genetic circuitry behind the two-stage dynamic control of protein levels upon phosphate depletion during stationary phase. 1C) (i) The engineered strain expresses an array of silencing gRNAs from the low-phosphate-inducible ugpB gene promoter. (ii) The engineered strain also expresses a natural nuclease-deficient E. coli-grade ligase capable of silencing genes, which results in (iii) immediate silencing of the targeted gene upon phosphate depletion. 1D) Inducible proteolysis. (i) The C-terminal DAS+4 degradation determinant tag allows controlled proteolysis once translated. (ii) SspB chaperone expression is controlled by a low-phosphate-inducible promoter, which enables (iii) chaperone-assisted proteolysis by the ClpXP protease under low phosphate conditions.

[0015] Figure 2 is a list of plasmids useful in practicing one aspect of the invention.

[0016] Figure 3 is a table of microbial strains useful in practicing one aspect of the present invention.

[0017] Figures 4A-4D : 4A-4B) Engineered strains expressing silencing gRNA arrays from low phosphate inducible ugpB gene promoter. 4C-4D) Genes were labeled with C-terminal sfGFP (+ / -DAS+4 tags) and protein levels were quantified by ELISA. All data were collected 24 hours after induction by phosphate depletion in microfermentation.

[0018] Figure 5 Graph showing the effect of dynamic control of GltA, Zwf, FabI, GlnB levels on THN / Flaviolin production in minimal medium microfermentation. Figure 1BBoth the proteolytic and transcriptional valves described. All strains were grown in triplicate and normalized to OD = 1 prior to production in phosphate-free medium. Two sample t tests were performed comparing engineered strains to controls. * indicates p-value DETAILED DESCRIPTION

[0019] Malonyl-CoA is a platform chemical that serves as a precursor to a variety of high-value chemicals. In Escherichia coli, malonyl-CoA levels and flux are tightly regulated. We demonstrate the use of two-stage dynamic metabolic control to improve malonyl-CoA biosynthesis in engineered E. coli. We have previously demonstrated the use of two-stage dynamic metabolic control to improve stationary-phase glucose uptake, central metabolism, and product biosynthesis. In this work, we demonstrate that coordinated dynamic reductions in the levels of fabI (enoyl-ACP reductase), gltA (citrate synthase), zwf (glucose-6-phosphate dehydrogenase), and glnB (nitrogen-regulated protein PII-1) during stationary phase lead to synergistic improvements in malonyl-CoA flux and product biosynthesis. Importantly, maximal improvements in flux require coordinated deregulation of all four metabolic valves as well as 1) glucose uptake, 2) acetyl-CoA production, and 3) acetyl-CoA carboxylase activity, which is regulated directly by nitrogen-regulated proteins and allosterically by acyl-ACP produced by enoyl-ACP reductase. This method is widely applicable to the production of malonyl-CoA-dependent products.

[0020] Referring now to Figure 1, two-stage dynamic metabolic control provides a potential route to successfully dysregulate metabolism and untangle central metabolites from host regulatory networks. During the non-growing stationary phase, the levels of central metabolism and regulatory proteins can be pushed beyond the boundary conditions imposed by growing cells, leading to a more optimal dysregulated production state. We have previously demonstrated the use of two-stage dynamic metabolic control in Escherichia coli to dysregulate central metabolism and improve product biosynthesis, as well as process robustness and scalability.

[0021] Specifically, we report the use of a combination of controlled proteolysis and CRISPR-based gene silencing to reduce levels of key metabolic enzymes, including citrate synthase (gltA, “G” valve), glucose-6-phosphate dehydrogenase (zwf, “Z” valve), and enoyl-ACP reductase (fabI, “F” valve), leading to improved stationary-phase biosynthesis of pyruvate, alanine, citrate, and xylitol.

[0022] As shown in Figure 1, the dynamic reduction of citrate synthase ("G") levels leads to a reduction in the level of alpha-ketoglutarate, an inhibitor of PTS-dependent glucose uptake. Therefore, its reduction dysregulates PTS-dependent sugar uptake, thereby enhancing stationary phase metabolism by increasing pyruvate production and glycolytic flux. The dynamic reduction of glucose-6-phosphate dehydrogenase ("Z") has been shown to reduce NADPH levels, thereby activating the SoxRS regulator. When the SoxRS regulator is activated, it increases the oxidation of pyruvate to acetyl-CoA. The combination of the "G" valve and the "Z" valve results in a substantial increase in the biosynthesis of citramalate produced by pyruvate and acetyl-CoA. The dynamic reduction of enoyl-ACP reductase ("F") levels has been shown to dynamically reduce the acyl-ACP pool with multiple regulatory effects. First, the reduced acyl-ACP / CoA pool alleviates the inhibition of their membrane-bound transhydrogenase (PntAB), thereby increasing NADPH flux and stationary phase xylitol biosynthesis. Perhaps more importantly, the reduced acyl-ACP pool of enoyl-ACP relieves inhibition of acetyl-CoA carboxylase. A 2-step dynamic reduction of enoyl-ACP activity using a temperature-sensitive fabI allele has been used to improve 3-hydroxypropionate biosynthesis in Escherichia coli.

[0023] To improve malonyl-CoA biosynthesis, we utilized the ‘G’, ‘Z’, and ‘F’ valves and a dynamic reduction in GlnB levels or the ‘B’ valve. GlnB encodes a nitrogen regulatory protein (P-II) that has been shown to inhibit ACCase in an α-ketoglutarate-dependent manner (Fig. 1). This level of regulation may have evolved to coordinate fatty acid biosynthesis with nitrogen availability and amino acid (glutamate) biosynthesis. Unfortunately, α-ketoglutarate levels have competing regulatory effects, and a reduction in α-ketoglutarate levels (caused by the ‘G’ valve) relieves the inhibition of glucose uptake, thereby improving stationary-phase biosynthesis, but also leads to an increase in GlnB-based inhibition of ACCase, thereby blocking malonyl-CoA synthesis. We hypothesized that a dynamic reduction in GlnB levels would improve malonyl-CoA production in strains with the ‘G’ valve. This work demonstrates a synergistic effect of a combination of the ‘G’, ‘Z’, ‘F’, and ‘B’ valves on malonyl-CoA flux.

[0024] I. Definitions

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

[0026] Unless otherwise specified, "a", "an", "the", "one or more" and "at least one" are used interchangeably. The singular forms "a", "an" and "the" include the plural forms thereof.

[0027] The recitation of numerical ranges by endpoints includes all numbers subsumed within that range (eg, 0.5 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0028] When referring to a value or amount of mass, weight, time, volume, concentration, or percentage, the term "about" means including variations of ±10% from the specified amount. The terms "comprising" and "including" are intended to be equivalent and open ended. The phrase "consisting essentially of means that the composition or method may include additional ingredients and / or steps, but only if these additional ingredients and / or steps do not materially change the basic and novel characteristics of the claimed composition or method. The phrase "selected from the group consisting of..." is meant to include mixtures of the listed groups.

[0029] In addition, the present disclosure also contemplates that, in some aspects, any feature or combination of features described herein may be excluded or omitted. For illustration, if the specification states that a complex includes components A, B, and C, it specifically means that any one or combination of A, B, or C may be omitted and abandoned individually or in any combination.

[0030] 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 is true: (a) the nucleic acid sequence is foreign to a given host microorganism (i.e., not naturally occurring); (b) the sequence may be naturally present in a given host microorganism, but in an unnatural amount (e.g., greater than expected); or (c) the nucleic acid sequence comprises two or more subsequences that are differently related to each other in nature. For example, with respect to example (c), a recombinantly produced heterologous nucleic acid sequence will have two or more sequences from unrelated genes that are arranged to produce a new functional nucleic acid, such as a non-native promoter that drives gene expression. The term "heterologous" is intended to include the term "exogenous" as the latter is commonly used in the art. Prior to the introduction of the heterologous nucleic acid sequence, the nucleic acid sequence encoding the enzyme is heterologous with reference to the genome of the host microorganism (regardless of whether the heterologous nucleic acid sequence is introduced into the genome). As used herein, chromosome and native and endogenous refer to the genetic material of the host microorganism.

[0031] As used herein, the term "gene disruption" or its grammatical equivalents (including "destroying enzyme function", "enzyme function disruption", etc.) is intended to refer to genetic modification of a microorganism so that the encoded gene product has a reduced polypeptide activity compared to the polypeptide activity in or from an unmodified microbial cell. The genetic modification can be, for example, the deletion of the entire gene, the deletion or other modification of regulatory sequences required for transcription or translation, the deletion of a gene portion that results in a truncation of a gene product (such as an enzyme), or any of a variety of mutation strategies that reduce the activity of the encoded gene product (including to an undetectable level of activity). The disruption can broadly include the complete or partial deletion 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 a stop codon, a frameshift mutation, the introduction or removal of a portion of a gene, and the introduction of a degradation signal, which affect the level and / or stability of mRNA transcription and change the promoter or repressor upstream of the gene encoding the enzyme.

[0032] Bioproduction, microfermentation or fermentation as used herein may be aerobic, microaerobic or anaerobic.

[0033] When reference is made herein (including the claims) to genetic modification of a gene product (ie, an enzyme), it is to be understood that the genetic modification is of a nucleic acid sequence (eg, or including a gene) that typically encodes the gene product (ie, an enzyme).

[0034] Species and other phylogenetic identifications are made according to classifications known to those skilled in the art of microbiology.

[0035] Enzymes are listed herein by reference to UniProt identification numbers, which are well known to those skilled in the art. The UniProt database can be accessed via http: / / www.UniProt.org / . When reference is made herein (including the claims) to a genetic modification of a gene product (i.e., an enzyme), it is to be understood that the 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).

[0036] Where the methods and steps described herein indicate certain events that occur in a particular order, it will be appreciated by those of ordinary skill in the art that the order of certain steps may be modified and that these modifications are consistent with variations of the present invention. Additionally, certain steps may be performed simultaneously in a parallel process or may be performed sequentially, where possible.

[0037] The meanings of the abbreviations are as follows: “C” refers to Celsius or degrees Celsius, as is clear from the usage, DCW refers to dry cell weight, “s” refers to second, “min” refers to minute, “h”, “hr” or “hrs” refers to hour, “psi” refers to pounds per square inch, “nm” refers to nanometer, “d” refers to day, “μL” or “uL” or “ul” refers to microliter, “mL” refers to milliliter, “L” refers to liter, “mm” refers to millimeter, “nm” refers to nanometer, “mM” refers to millimolar, “μM” or “uM” refers to micromolar, “M” refers to molar, “mmol” refers to millimolar, “μmol” or “uMol” refers to micromole, “g” refers to gram, and “μg” refers to gram. ” or “ug” means microgram, “ng” means nanogram, “PCR” means polymerase chain reaction, “OD” means optical density, “OD600” means optical density measured at a photon wavelength of 600 nm, “kDa” means kilodaltons, “g” means gravity 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-thiogalactoside, “aTc” means anhydrotetracycline, “RBS” means ribosome binding site, “rpm” means revolutions per minute, “HPLC” means high performance liquid chromatography, and “GC” means gas chromatography.

[0038] Overview of Aspects of the Invention

[0039] In some aspects, the invention includes a genetically modified microorganism having an anabolic valve for dynamic selective regulation of a nitrogen regulatory protein (glnB). The anabolic valve can include silencing of genetic expression of the glnB gene, selective enzymatic degradation of the glnB protein, or any combination of the two. The anabolic valve is selectively activated in response to changes in the growth medium in which the genetically modified microorganism is grown, so as to silence the glnB gene and / or degrade the glnB protein.

[0040] In some aspects, the genetically modified microorganism may further include one or more additional anabolic valves for silencing gene expression of one or more genes other than the glnB gene; or selective enzymatic degradation anabolic valves that induce selective enzymatic degradation of one or more proteins other than the glnB protein, or any combination of the two.

[0041] In some aspects, the genetically modified microorganism has an additional anabolic valve for silencing a gene, wherein the gene is: enoyl-ACP reductase (fabI), citrate synthase (gltA), glucose-6-phosphate dehydrogenase (zwf), or any combination of these, or an additional anabolic valve for selective enzymatic degradation of a protein, wherein the protein is: enoyl-ACP reductase (fabI), citrate synthase (gltA), glucose-6-phosphate dehydrogenase (zwf), or any combination of these.

[0042] In some aspects, in addition to the glnB anabolic valve, the genetically modified microorganism also includes a second anabolic valve for dynamically selectively regulating enoyl-ACP reductase (fabI), which anabolic valve includes: gene expression silencing of the fabI gene, selective enzymatic degradation of the fabI protein, or a combination thereof; and a third anabolic valve for dynamically selectively regulating glucose-6-phosphate dehydrogenase (zwf), which anabolic valve includes: gene expression silencing of the zwf gene, selective enzymatic degradation of the zwf protein, or a combination thereof; and a fourth anabolic valve for dynamically selectively regulating citrate synthase (gltA), which anabolic valve includes: gene expression silencing of the gltA gene, selective enzymatic degradation of the gltA protein, or a combination thereof.

[0043] In some aspects, the change in the growth medium is a depletion of phosphate in the growth medium. In some aspects, the genetically modified microorganism is an E. coli microorganism. In some aspects, the anabolic valve comprises:

[0044] A gene encoding at least one small guide RNA that is specific for targeting more than one gene of an enzyme essential for growth of the genetically modified microorganism.

[0045] In some aspects, the present invention fully describes a bioprocess for producing a product using a genetically modified microorganism, the bioprocess comprising the steps of providing a genetically modified microorganism. The microorganism comprises: a production pathway for the product, the production pathway using malonyl-CoA as a biosynthetic precursor for the product; an anabolic valve for dynamic selective regulation of a nitrogen regulatory protein (glnB), the anabolic valve comprising: gene expression silencing of the glnB gene, selective enzymatic degradation of the glnB protein, or a combination thereof; and one or more additional anabolic valves for dynamic selective regulation of one or more genes other than the glnB gene, or selective enzymatic degradation of one or more proteins other than the glnB protein. The method further comprises the steps of growing the genetically modified microorganism in a culture medium; transitioning from microorganism growth to a stable production phase, the transition comprising: reducing or stopping the growth of the genetically modified microorganism at least in part by controlled depletion of limiting nutrients from the culture medium, activating the anabolic valve, and increasing the available malonyl-CoA pool. The final step of the method is producing the product in the stable production phase.

[0046] The disclosed aspects are non-limiting

[0047] Although various aspects of the present invention have been shown and described herein, it should be emphasized that these aspects are provided as examples only. Without departing from various aspects of the present invention, many changes, modifications and substitutions may be made. Specifically, for whatever reason, for any group of compounds, nucleic acid sequences, polypeptides (including specific proteins, including functional enzymes, metabolic pathway enzymes or intermediates, elements or other compositions), or concentrations described or otherwise presented in lists, tables or other groupings herein, unless otherwise expressly stated, each such grouping provides a basis for identifying various subset aspects, and the subset aspect includes each subset of such grouping in its broadest range, excluding one or more members (or subsets) of the corresponding grouping. In addition, when any range is described herein, unless otherwise expressly stated, the range includes all values ​​therein and all subranges therein.

[0048] Likewise, more generally, based on the disclosure, discussion, embodiments and aspects herein, conventional molecular biology, cell biology, microbiology and recombinant DNA techniques can be employed by those skilled in the art. These techniques are fully explained in the literature. For example, see Sambrook and Russell, "Molecular Cloning: A Laboratory Manual", 2001 Third Edition (Volumes 1-3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Animal Cell Culture, RI Freshney, ed., 1986. These published resources are incorporated herein by reference.

[0049] The following published resources are incorporated herein by reference for descriptions used in conjunction with the inventions described herein, e.g., methods for industrial bioproduction of chemical products from sugar sources, and industrial systems that can be used to achieve such conversions (e.g., Biochemical Engineering Fundamentals, 2nd ed., JE Bailey and DF Ollis, McGraw-Hill, New York, 1986, Chapter 9, pp. 533-657, for bioreactor design; e.g., Unit Operations of Chemical Engineering, 5th ed., WL McCabe et al., McGraw-Hill, New York, 1993, for process and separation technology analysis; e.g., Equilibrium Staged Separations, PC Wankat, Prentice-Hall, Englewood Cliffs, NJ, 1988, for teaching separation technology).

[0050] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety.

[0051] General

[0052] I. Carbon Source

[0053] The biological production substratum used together with the recombinant microorganism in the present invention must comprise suitable carbon source or substrate for growth and production stage.Suitable substrate can include but is not limited to the combination of glucose, sucrose, xylose, mannose, arabinose, oil, carbonic acid gas, carbon monoxide, methane, methanol, formaldehyde or glycerine.It is expected that all above-mentioned carbon substrates and mixtures thereof are all suitable as carbon source of the present invention.

[0054] II. Microorganisms

[0055] The features described herein and claimed can be provided in a microorganism selected from listed herein or another suitable microorganism, which also includes one or more natural, introduced or enhanced product biological production pathways. Therefore, in some aspects, the microorganism includes an endogenous product production pathway (which can be enhanced in some such aspects), and in other aspects, the microorganism does not include an endogenous product production pathway.

[0056] More specifically, based on the various criteria described herein, suitable microbial hosts for bioproduction of chemical products can generally include, but are not limited to, the organisms described in the Methods section.

[0057] The host microorganism or source microorganism of any gene or protein described herein can be selected from the following microorganism list: Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacillus, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacterium, Escherichia, Salmonella, Bacillus, Streptomyces and Pseudomonas. In some aspects, the host microorganism is an Escherichia coli microorganism.

[0058] III. Culture medium and culture conditions

[0059] In addition to a suitable carbon source, e.g., selected from one of the types disclosed herein, the bioproduction medium must also contain suitable minerals, salts, cofactors, buffers and other components known to those skilled in the art to be suitable for growing the culture and promoting bioproduction of the chemical product of the invention.

[0060] Another aspect of the present invention relates to culture media and culture conditions comprising the genetically modified microorganism of the present invention and optional supplements.

[0061] Typically, cells are grown in an appropriate culture medium at a temperature of about 25°C to about 40°C, and for thermophilic microorganisms, up to 70°C. Suitable growth media are well characterized and known in the art. A suitable pH range for bioproduction is between pH 2.0 and pH 10.0, with pH 6.0 to pH 8.0 being a typical pH range for initial conditions. However, actual culture conditions for particular aspects are not meant to be limited to these pH ranges. Bioproduction can be carried out under aerobic, microaerobic or anaerobic conditions, with or without agitation.

[0062] IV. Bioproduction Reactors and Systems

[0063] Utilizing the fermentation system according to the method and / or composition of the present invention is also within the scope of the present invention.Any recombinant microorganism described herein and / or mentioned can be introduced into industrial biological production system, in which microorganism converts carbon source into product in commercially feasible operation.Biological production system comprises introducing this recombinant microorganism into bioreactor container, wherein contains carbon source substrate and the biological production culture medium suitable for growing recombinant microorganism, and biological production system is maintained in suitable temperature range (if reaction is aerobic or micro-aerobic, then keeps dissolved oxygen concentration range) one period of suitable time, to obtain the required conversion of part substrate molecules to selected chemical products.Biological production can be carried out under aerobic, micro-aerobic or anaerobic conditions, with or without stirring.Industrial biological production system and operation thereof are well-known to the technicians in chemical engineering and bioprocess engineering fields.

[0064] The amount of product produced in a bioproduction medium can generally 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).

[0065] V. Gene Modification, Nucleotide Sequence and Amino Acid Sequence

[0066] Aspects of the present invention can be produced by introducing an expression vector into a host microorganism, wherein the expression vector comprises a nucleic acid sequence encoding an enzyme that is ordinarily present or absent in the host microorganism.

[0067] The ability to genetically modify host cells is essential for producing any genetically modified (recombinant) microorganism. The mode of gene transfer technology can be through electroporation, conjugation, transduction or natural transformation. There is a wide range of host binding plasmids and resistance markers to choose from. Based on the properties of antibiotic resistance markers that can play a role in the host, cloning vectors are tailor-made for the host organism. Similarly, as disclosed herein, genetically modified (recombinant) microorganisms can include modifications other than plasmid introduction, including modifications to their genomic DNA.

[0068] More generally, a nucleic acid construct can be prepared that includes an isolated polynucleotide encoding a polypeptide having enzymatic activity operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a microorganism such as Escherichia coli under conditions compatible with the control sequences. The isolated polynucleotide can be manipulated to provide expression of the polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the sequence of the polynucleotide prior to inserting it into the vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods have been established in the art.

[0069] The control sequence can be an appropriate promoter sequence, i.e. a nucleotide sequence recognized by the host cell, for expressing a polynucleotide encoding the polypeptide of the present invention. The promoter sequence can include a transcriptional control sequence that mediates the expression of the polypeptide. The promoter can be any nucleotide sequence that shows transcriptional activity in the selected host cell, including promoters that are mutated, truncated, and hybridized, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell. Technology for modifying and utilizing recombinant DNA promoter sequences has been established in the art.

[0070] For various aspects of the present invention, genetic manipulation can include regulation that is intended to change enzyme, thereby change the operation of the enzymatic activity of the enzyme of the final activity of enzyme or identification in any corresponding approach.This genetic modification may relate to transcription, translation and post-translational modification, which cause enzymatic activity and / or selective variation under selected culture conditions.Genetic manipulation of nucleotide sequence can increase copy number and / or include mutants that use enzymes relevant to product production.The concrete method and mode realizing this genetic modification are well known to those skilled in the art.

[0071] In various fields, in order to play a role more efficiently, microorganism can include one or more gene deletions.For example, in Escherichia coli, the gene encoding the following items can be destroyed (including deletion): 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 dependency Lon protease (lon), outer membrane protease (ompT), arcA transcription dual regulator (arcA) and iclR transcription regulator (iclR). This gene disruption, including deletion, is not meant to be restrictive, and can be realized in various fields with various combinations.Gene deletion can be realized by multiple strategies well known in the art, and the method for mixing exogenous DNA into host chromosome is also the same.

[0072] In various aspects, in order to play a role more efficiently, microorganisms can include one or more anabolic valves, composed of enzymes of a combination of targeted controlled proteolysis, expression silencing or controlled proteolysis and expression silencing. For example, an enzyme encoded by a gene in Escherichia coli or a combination of many enzymes encoded by many genes can be designed as anabolic valves to change metabolism and improve product formation. Representative genes in Escherichia coli may include but are not limited to the following genes: 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 familiar with how to identify these genes and / or homologs of other genes in additional microbial species.

[0073] For all nucleic acid and amino acid sequences provided herein, it should be understood that conservatively modified variants of these sequences are also included, and each aspect thereof is within the scope of the present invention. Functionally equivalent nucleic acid and amino acid sequences (functional variants) can include conservatively modified variants and more widely varied sequences, which are fully within the technical scope of those of ordinary skill in the art, and these functionally equivalent nucleic acid and amino acid sequences and microorganisms comprising these sequences are also within the scope of each aspect of the present invention, as are the methods and systems comprising such sequences and / or microorganisms.

[0074] Thus, as described in the above sections, some compositions, methods, and systems of the invention include providing genetically modified microorganisms that include both production pathways for making desired products from central intermediates and anabolic valves for redistributing flux.

[0075] Aspects of the invention also relate to providing multiple genetic modifications to improve the overall efficiency of microorganisms in converting selected carbon sources into selected products. Specific combinations are shown, such as in the examples, which can significantly increase specific productivity, volumetric productivity, titer and yield compared to more basic genetic modification combinations.

[0076] In addition to the genetic modifications described above, various aspects provide for genetic modifications that include anabolic valves to increase the pool and availability of cofactors NADPH and / or NADH that may be consumed in product production.

[0077] VI. Anabolic Valve

[0078] The use of anabolic valves allows for a simpler model of metabolic fluxes and physiological requirements during the production phase, transforming proliferating cells into stationary phase biocatalysts. These anabolic valves can be used to shut down essential genes and redirect carbon, electron and energy fluxes to product formation in multi-stage fermentation processes. The following one or more provide the synthetic valves: 1) transcriptional gene silencing or inhibition techniques combined with 2) induction and selective enzyme degradation and 3) nutrient limitation to induce a stable or non-dividing cell state. SMVs can be extended to any pathway and microbial host. These anabolic valves allow for novel rapid metabolic engineering strategies that can be used to produce renewable chemicals and fuels and any product that can be produced by whole-cell catalysis.

[0079] In particular, the present invention describes the construction of anabolic valves comprising one or more or a combination of: controlled gene silencing and controlled proteolysis. It should be understood that several methods for gene silencing and controlled proteolysis are known to those skilled in the art.

[0080] VI.A Gene Silencing

[0081] In particular, the present invention describes the use of controlled gene silencing to control metabolic flux in a controlled multi-stage fermentation process. Several methods of controlled gene silencing are known in the art, including but not limited to mRNA silencing or RNA interference, silencing by transcriptional repressors, and CRISPR interference. Agrawal et al. teach methods and mechanisms of RNA interference, "RNA Interference: Biology, Mechanism, and Applications" Microbiology and Molecular Biology Reviews, December 2003; 67(4) pp. 657-685. DOI: 10.1128 / MMBR.67.657-685.2003. Qi et al. teach methods and mechanisms of CRISPR interference, "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression" Cell, February 2013; 152(5) pp. 1173-1183. DOI: 10.1016 / j.cell.2013.02.022. In addition, Luo et al. teach methods and mechanisms for CRISPR interference using the natural E. coli CASCADE system, "Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression" NAR., October 2014; DOI: 10.1093. In addition, many transcriptional repressor systems are well known in the art and can be used to shut down gene expression.

[0082] VI.B Controlled proteolysis

[0083] In particular, the present invention describes the use of controlled protein degradation or proteolysis to control metabolic flux in a controlled multi-stage fermentation process. Several methods for controlled protein degradation are known in the art, including but not limited to targeted protein cleavage by specific proteases and controlled targeting of proteins for degradation by specific peptide tags. McGinness et al. teach a system for controlled protein degradation using the E. coli clpXP protease, "Engineering controllable protein degradation", Mol Cell., 2006 Jun; 22(5) pp. 701-707. The method relies on the addition of a specific C-terminal peptide tag, such as a DAS4 (or DAS+4) tag. Proteins with such tags are not degraded by the clpXP protease until the expression of the chaperone sspB is specifically enhanced. sspB induces the degradation of DAS4-tagged proteins by the clpXP protease. In addition, many site-specific protease systems are well known in the art. Proteins can be engineered to contain specific target sites for a given protease and then cleaved after controlled expression of the protease. In some aspects, cleavage may result in inactivation or degradation of the protein. For example, Schmidt et al. ("ClpS is the recognition component for Escherichiacoli substrates of the N-end ruledegradation pathway", Molecular Microbiology, Mar. 2009. 72(2), 506-517. doi: 10.1111) taught that an N-terminal sequence can be added to a protein of interest to provide clpS-dependent clpAP degradation. Additionally, this sequence can also be masked by an additional N-terminal sequence that can be controllably cleaved by, for example, a ULP hydrolase. This allows for controlled N-rule degradation that is dependent on the expression of a hydrolase. Thus, proteins can be tagged at the N-terminus or terminally for controlled proteolysis. The preference for using an N-terminal tag versus a C-terminal tag depends largely on whether either tag will affect protein function before controlled degradation begins.

[0084] The present invention describes the use of controlled protein degradation or proteolysis in Escherichia coli to control metabolic flux in a controlled multi-stage fermentation process. Several methods are known in the art for controlled protein degradation in other microbial hosts, including various Gram-negative and Gram-positive bacteria, yeast and even archaea. In particular, systems for controlled proteolysis can be transferred from natural microbial hosts to non-natural hosts for use. 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, teach the expression and use of the E. coli clpXP protease in Saccharomyces cerevisiae. This method can be used to transfer the method of synthesizing metabolic valves to any genetically tractable host.

[0085] VI.C Anabolic Valve Control

[0086] The present invention describes the use of anabolic valves to control metabolic flux in a multi-stage fermentation process. Many methods of inducing expression are known in the art and can be used for transitions between stages in a multi-stage fermentation. These include, but are not limited to, artificial chemical inducers, including: tetracycline, anhydrotetracycline, lactose, IPTG (isopropyl-β-D-1-thiogalactoside), arabinose, raffinose, tryptophan and many other inducers. Systems that link the use of these well-known inducers to 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 a multi-stage fermentation process.

[0087] In addition, it may be desirable to control the transition between growth and production in a multi-stage fermentation by depleting one or more limiting nutrients consumed during the growth process. Limiting nutrients include, but are not limited to, phosphate, nitrogen, sulfur, and magnesium. Natural gene expression systems that respond to these nutrient limitations can be used to practicably link control of gene expression silencing and / or controlled proteolysis to the transition between growth and production phases in a multi-stage fermentation process.

[0088] Example

[0089] In order to promote the understanding of the principles of the present invention, reference will now be made to the preferred aspects, and specific language will be used to describe these preferred aspects. However, it should be understood that this is not intended to limit the scope of the present disclosure, and such changes and further modifications to the present disclosure as shown herein are generally expected by those skilled in the art to which the present disclosure relates.

[0090] method

[0091] Unless otherwise stated, all materials and reagents were of the highest possible grade.

[0092] Reagents and media

[0093] LB broth (Lennox) preparations were used for strain culture and plasmid propagation and construction. SM10++ and SM10 phosphate-free media were prepared as reported previously. Appropriate antibiotics were used in all media at the following working concentrations: kanamycin: 35 μg / mL, chloramphenicol: 35 μg / mL, zeocin: 100 μg / mL, blasticidin: 100 μg / mL, gentamicin: 10 μg / mL, and ampicillin: 100 μg / mL.

[0094] Strains & plasmid construction

[0095] Oligonucleotides and synthetic linear DNA for strain construction (Gblocks TM ) sequences were obtained from Integrated DNA Technologies (IDT, Coralville, IA). Figure 2 The plasmids used in this study are provided in the table. Figure 3 The strains used in this study are provided in the table. New strains were prepared using standard recombineering methods by integration of antibiotic resistance cassettes and direct selection. The recombineering plasmid pSIM5 was a gift from Donald Court (https: / / redrecombineering.ncifcrf.gov / court-lab.html). Chromosomal modifications were confirmed by PCR amplification and sequencing (Genewiz / Azenta). The pCASCADE silencing plasmid was constructed as reported previously. The stability of the guide RNA array was confirmed by colony PCR prior to strain evaluation as described previously.

[0096] Micro-fermentation

[0097] As previously described, 96-well plate-based micro-fermentations (in triplicate) were performed by first inoculating 5 ul of frozen glycerol stock with appropriate antibiotics (kanamycin and chloramphenicol) in 145 ul of LB at 37 degrees with shaking at 300 rpm for 16 hours. 5 ul of the culture was inoculated in 145 ul of SM10 ++ 16 hours. Collect the cells by centrifugation and then wash with 150ul of SM10 phosphate-free medium. Resuspend the cells in 100ul of SM10 phosphate-free medium. Dilute 10ul of each culture to 200ul for OD 600Read. Normalize each well to OD=1 by adding the appropriate volume of 100ul culture to a final volume of 150ul (phosphate-free medium) in a fresh plate. Shake the plate at 37 degrees at 300 rpm for 24 hours. After 24 hours, take the OD for each well. 600 , and the volume was transferred to a U-bottom 96-well plate. The cells were pelleted by centrifugation at 3500 g for 10 min, and the supernatant was isolated for measurement.

[0098] Spectrophotometric Measurement of THN / Flaviolin

[0099] Relative THN / flaviolin levels were determined using a Spectramax 384-plus microplate reader as reported previously. The absorbance at 495 nm was measured and divided by the OD of the post-production culture. 600 Measure values ​​and non-quantitatively determine THN / flaviolin production in 96-well flat bottom plates. Multiply both measurements by the appropriate volume path length in the 96-well plate and dilute prior to analysis.

[0100] Quantification of FabI and GlnB levels

[0101] Protein level quantification was performed using strain variants with a C-terminal sfGFP tag (see Figure 2 GFP levels were quantified by ELISA using a quantification kit from AbCam (Cambridge, UK, product # ab171581) according to the manufacturer's instructions. Briefly, samples were obtained from microfermentations 24 hours after phosphate depletion, washed in water and solubilized with the provided extraction buffer.

[0102] Embodiment 1: Valve Construction & Verification

[0103] We have previously shown that metabolic valves that rely on a combination of silencing and proteolysis (Figure 1) result in a >95% reduction in Zwf levels and an 80% reduction in GltA levels. Our previous studies did not include validation of dynamic control of GlnB levels, nor did they include successful use of proteolysis combined with silencing to reduce FabI levels. We previously demonstrated that leaky expression of gRNA silencing of fabI resulted in growth defects and gRNA instability, requiring the reconstruction of host strains that more tightly control the silencing mechanism. Therefore, in our previous work, dynamic control of FabI levels relied solely on proteolysis. In this study, we first used these more stable host strains to evaluate combinations of silencing and proteolysis for FabI and GlnB levels. To do this, we utilized strains that append a C-terminal sfGFP tag with and without a degron tag to either fabI or glnB ( Figure 2). These tags were used to measure GFP using ELISA to achieve absolute quantification of protein levels. Figure 4 presents the results of these studies. In the case of GlnB, we evaluated gRNAs that silenced the two promoters driving GlnB expression, both individually and in combination. The combination of proteolysis and silencing reduced FabI levels and GlnB levels by 99% and 95%, respectively. For GlnB, the combination of proteolysis and silencing of the glnBp2 promoter reduced protein levels the most. Therefore, this silencing gRNA was used in subsequent strain combinations.

[0104] Embodiment 2: In vivo measurement of relative malonyl-CoA flux

[0105] To rapidly quantify the increase in malonyl-CoA flux, we utilized a single enzyme (THNS synthase, encoded by the rppA gene of S. griseolis) that is able to generate 1 mol of 1,3,6,8-tetrahydroxynaphthalene (THN) from 5 mol of malonyl-CoA. The use of 1,3,6,8-tetrahydroxynaphthalene (THN) as a "malonyl-CoA reporter" is well established. Relative THN levels can be quantified by absorbance at 495 nm, and additionally, THN spontaneously oxidizes to the purple product, flavilin. Specifically, we cloned and expressed rppA behind a tightly controlled, robust, low-phosphate-inducible promoter of the yibD gene.

[0106] Using this construct, we evaluated the effects of dynamic control of our "valves" on the two-stage production of THN. Specifically, the strains tested included a set of "G" (citrate synthase) valves, "Z" (glucose 6-phosphate dehydrogenase) valves, "F" (enoyl-ACP reductase) valves, and "B" (nitrogen regulatory protein P-II) valves (Figure 1). A list of plasmids and strains used in this study is shown in Table 1. As hypothesized, the "G" valve resulted in reduced THN production compared to the no-valve control, likely due to reduced α-ketoglutarate levels and GlnB-mediated ACCase inhibition. The "B" valve alone had no effect on THN synthesis. Addition of the "Z" valve ("G", "Z" strains) increased THN production to 149% of the control. Since acetyl-CoA is a known activator and substrate of ACCase, increasing acetyl-CoA levels is expected to increase malonyl-CoA flux. The best producing strains had all valve combinations ("G", "Z", "F" and "B") ( Figure 5). When all valves were present, there was a 916% improvement compared to any one valve (G, B, F) alone, highlighting the synergistic effect of the valves. The combination of “G”, “Z” and “B” did not improve yield compared to the “GZ” strain. This was somewhat unexpected considering the final four-valve combination. We hypothesize that in the “GZ” background, acyl-ACP levels are the primary regulator of malonyl-CoA flux and that GlnB-based inhibition becomes limiting only after the acyl-ACP pool is reduced due to the “F” valve. In previous work, the “Z” valve was not tested alone due to its relatively low improvement in enhancing acetyl-CoA flux.

[0107] The complete disclosures of all patents, patent applications and publications cited herein and electronically available materials are incorporated herein by reference. The above detailed description and examples are intended only for clear understanding. Unnecessary limitations should not be understood thereby. The present invention is not limited to the exact details shown and described, as variations obvious to those skilled in the art will be included in the invention defined by the claims.

Claims

1. A genetically modified microorganism comprising: The first anabolic valve is used to dynamically and selectively regulate the nitrogen regulatory protein (glnB), and the anabolic valve comprises: The gene expression of glnB gene was silenced. Selective enzymatic degradation of the glnB protein, or Their combination, Wherein, the anabolic valve is selectively activated in response to a change in a growth medium in which the genetically modified microorganism is grown, so as to silence the glnB gene and / or degrade the glnB protein.

2. The genetically modified microorganism according to claim 1, further comprising: At least one additional anabolic valve comprising: a gene expression silencing anabolic valve that silences gene expression of one or more genes other than the glnB gene; or a selective enzymatic degradation anabolic valve which induces the selective enzymatic degradation of one or more proteins other than said glnB protein, or A combination of them.

3. The genetically modified microorganism according to claim 2, wherein the additional anabolic valve is used to silence a gene, wherein the gene is: enoyl-ACP reductase (fabI), citrate synthase (gltA), glucose-6-phosphate dehydrogenase (zwf), or a combination thereof; Alternatively, the additional anabolic valve is used to selectively enzymatically degrade a protein, wherein the protein is: enoyl-ACP reductase (fabI), citrate synthase (gltA), glucose-6-phosphate dehydrogenase (zwf) or a combination thereof.

4. The genetically modified microorganism according to claim 1, further comprising: A second anabolic valve is used for dynamically and selectively regulating enoyl-ACP reductase (fabI), wherein the anabolic valve comprises: The gene expression of the fabI gene is silenced. Selective enzymatic degradation of the fabI protein, or combinations thereof; and The third anabolic valve is used for dynamically and selectively regulating glucose-6-phosphate dehydrogenase (zwf), and the anabolic valve comprises: The gene expression of zwf gene is silenced. Selective enzymatic degradation of the ZWF protein, or combinations thereof; and A fourth anabolic valve, for dynamically and selectively regulating citrate synthase (gltA), the anabolic valve comprising: The gene expression of gltA gene is silenced. Selective enzymatic degradation of the gltA protein, or A combination of them.

5. The genetically modified microorganism of claim 1, wherein the change in the growth medium is the depletion of phosphate in the growth medium. The genetically modified microorganism according to claim 1 , wherein the genetically modified microorganism is an Escherichia coli microorganism.

7. The genetically modified microorganism of claim 2, wherein the anabolic valve comprises: A gene encoding at least one small guide RNA that specifically targets more than one gene of an enzyme essential for the growth of the genetically modified microorganism.

8. A bioprocess for producing a product using a genetically modified microorganism, the bioprocess comprising: (a) providing a genetically modified microorganism, the microorganism comprising: (i) a production pathway for a product that uses malonyl-CoA as a biosynthetic precursor for the product; (ii) anabolic valve for dynamically and selectively regulating nitrogen regulatory protein (glnB), the anabolic valve comprising: The gene expression of glnB gene was silenced. Selective enzymatic degradation of the glnB protein, or combinations thereof; and (iii) one or more additional anabolic valves for dynamic selective regulation of one or more genes other than the glnB gene; or selectively enzymatically degrading one or more proteins other than the glnB protein; (b) growing the genetically modified microorganism in a culture medium; (c) transitioning from a microbial growth phase to a stable production phase, said transition comprising: (i) reducing or stopping the growth of the genetically modified microorganism at least in part by controlled depletion of a limiting nutrient from the culture medium; (ii) activation of anabolic valves; (iii) increase the available malonyl-CoA pool; (d) producing a product during said steady state production phase.

9. The bioprocess according to claim 7, wherein the genetically modified microorganism is an Escherichia coli microorganism.

10. The bioprocess according to claim 7, wherein the additional anabolic valve is used to silence genes, wherein the genes are: enoyl-ACP reductase (fabI), citrate synthase (gltA), glucose-6-phosphate dehydrogenase (zwf) or a combination thereof; or the additional anabolic valve is used to selectively enzymatically degrade proteins, wherein the proteins are: enoyl-ACP reductase (fabI), citrate synthase (gltA), glucose-6-phosphate dehydrogenase (zwf) or a combination thereof.

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