Method for increasing yield of L-isoleucine

Abstract

The invention provides a method for increasing the yield of L-isoleucine. Comprising the steps that an engineering strain containing an expression plasmid vector or an expression plasmid vector combination is cultured, the vector or combination comprises a first group of polynucleotides and a second group of polynucleotides, and the first group of polynucleotides comprise at least one polynucleotide encoding L-isoleucine synthetic pathway enzyme; the second group of polynucleotides comprising at least one polynucleotide encoding an enzyme that modulates the balance of NAD + / NADH and NADP + / NADPH; and a backbone plasmid capable of autonomously replicating in a host cell. According to the invention, the yield of L-isoleucine and the conversion rate of glucose are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a method for improving L-isoleucine yield. BACKGROUND

[0002] L-isoleucine, also known as L-isoleucine, is one of the three branched chain amino acids (BCAA) essential to the human body, has multiple physiological functions, participates in the synthesis of hormones and enzymes in the human body, promotes protein synthesis and inhibits its decomposition, and has important regulating effects on human and animal health, and therefore has wide application and commercial value in the food, medicine and feed industries.

[0003] At present, the main method for producing L-isoleucine in industry is microbial fermentation method, which is a method for biosynthesis and overproduction of L-isoleucine by using the metabolic pathway of microorganisms, including direct fermentation method and precursor-adding fermentation method. The direct fermentation method utilizes the ability of microorganisms to synthesize amino acids required by themselves, and through mutagenesis treatment of specific microorganisms, a nutrition-deficient and amino acid structural analog-resistant mutant strain is selected and bred to eliminate the feedback inhibition and repression in metabolic regulation, so as to achieve the purpose of overproduction of a certain amino acid. At present, most of the strains for producing L-isoleucine by direct fermentation are obtained by mutagenesis breeding of glutamic acid-producing bacteria (Brevibacterium flavum, Corynebacterium glutamicum, Brevibacterium lactofermentum, etc.). The precursor-adding fermentation method, also known as microbial transformation method, uses glucose as the fermentation carbon source and energy source, and adds specific precursors in the fermentation process to avoid the feedback regulation in the amino acid biosynthesis pathway, and the precursors are efficiently transformed into the target amino acid by microorganisms. For L-isoleucine, the precursors mainly include alpha-aminobutyric acid, alpha-hydroxybutyric acid, alpha-ketobutyric acid and threonine, and the microorganisms mainly include Escherichia coli, Bacillus and Pseudomonas.

[0004] At present, there are many domestic enterprises producing L-isoleucine by fermentation method, but there are still problems such as low acid production level, low conversion rate, many by-products and poor fermentation product extraction technology. Therefore, improving the fermentation strain and technology of L-isoleucine, increasing the acid production level and conversion rate, and then reducing the extraction difficulty are of great significance for promoting the sustainable development of L-isoleucine industry and enhancing the market competitiveness of the product. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a method for improving L-isoleucine yield. The method solves the problems of low acid production level and low conversion rate in the prior art.

[0006] A first aspect of the present invention provides an expression plasmid vector or a combination of expression plasmid vectors, the vector or combination comprising: a first group of polynucleotides and a second group of polynucleotides, wherein the first group of polynucleotides comprises at least one polynucleotide encoding an enzyme in the L-isoleucine synthesis pathway; and the second group of polynucleotides comprises at least one polynucleotide encoding a regulatory NAD+ pathway enzyme. + / NADH and NADP + / NADPH-balanced enzymes with polynucleotides;

[0007] And the backbone plasmid, which can replicate autonomously in the host cell.

[0008] In a specific embodiment of the present invention, the plasmid vector or vector combination is used to promote enhanced L-isoleucine expression in host cells.

[0009] In one embodiment of the present invention, the L-isoleucine synthesis pathway includes the pathway from threonine to L-isoleucine as described in KEGG entry M00570, or the pathway from 2-ketobutyric acid to L-isoleucine as described in KEGG entry M00019.

[0010] In one embodiment of the present invention, the first group of polynucleotides includes a gene encoding at least one of the following enzymes: threonine dehydrogenase (ilvA) or a mutant thereof, acetylhydroxybutyrate synthase (ilvIH, ilvBN, ilvGM, alsS) or a mutant thereof, dihydroxy acid dehydrase (ilvD) or a mutant thereof, acetylhydroxy acid reductase (ilvC) or a mutant thereof, and branched-chain amino acid transaminase (ilvE) or a mutant thereof.

[0011] In a specific embodiment of the present invention, the threonine deaminase (ilvA) is derived from *Escherichia coli*, with protein accession number CAA28577, or is an enzyme mutant that retains the above-mentioned enzyme activity and has at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence represented by protein accession number CAA28577. For example, the protein mutation sites of the enzyme mutant are L447F and L451A.

[0012] In the detailed description of the application, the acetohydroxy acid synthase (ilvIH) is derived from E. coli, wherein the catalytic subunit of acetohydroxy acid synthase (ilvI) has the protein accession number CAA25755, or an enzyme mutant having the amino acid sequence of at least 96% or 97% or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number CAA25755 and retaining the enzyme activity. The regulatory subunit of acetohydroxy acid synthase (ilvH) has the protein accession number CAA25756, or an enzyme mutant having the amino acid sequence of at least 96% or 97% or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number CAA25756 and retaining the enzyme activity. For example, the protein mutation sites of the enzyme mutant are G14D, S17F. The acetohydroxy acid synthase can also be ilvBN and its mutants derived from E. coli (ilvB protein accession number: CAA26387; ilvN protein accession number: AAC76693), ilvGM and its mutants (ilvG protein accession number: AAA67571; ilvM protein accession number: CAA28574), or alsS derived from B. subtilis (protein accession number: CAB07802).

[0013] In the detailed description of the application, the acetohydroxy acid synthase (ilvIH) is derived from E. coli, wherein the catalytic subunit of acetohydroxy acid synthase (ilvI) has the protein accession number CAA25755, or an enzyme mutant having the amino acid sequence of at least 96% or 97% or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number CAA25755 and retaining the enzyme activity. The regulatory subunit of acetohydroxy acid synthase (ilvH) has the protein accession number CAA25756, or an enzyme mutant having the amino acid sequence of at least 96% or 97% or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number CAA25756 and retaining the enzyme activity. For example, the protein mutation sites of the enzyme mutant are G14D, S17F. The acetohydroxy acid synthase can also be ilvBN and its mutants derived from E. coli (ilvB protein accession number: CAA26387; ilvN protein accession number: AAC76693), ilvGM and its mutants (ilvG protein accession number: AAA67571; ilvM protein accession number: CAA28574), or alsS derived from B. subtilis (protein accession number: CAB07802).

[0014] In the detailed description of the application, the first group of polynucleotides further comprises a gene encoding branched-chain amino acid efflux protein (ygaZH) or its mutants.

[0015] In the detailed description of the application, the branched-chain amino acid efflux protein (ygaZH) is derived from E. coli, wherein the Z subunit of branched-chain amino acid efflux protein (ygaZ) has the protein accession number AAC75729, or an enzyme mutant having the amino acid sequence of at least 96% or 97% or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number AAC75729 and retaining the enzyme activity. The H subunit of branched-chain amino acid efflux protein (ygaH) has the protein accession number AAC75730, or an enzyme mutant having the amino acid sequence of at least 96% or 97% or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number AAC75730 and retaining the enzyme activity.

[0016] In the detailed description of the application, the second set of polynucleotides comprises at least one gene encoding an enzyme selected from the group consisting of NAD kinase (nadK) or a mutant thereof, pyridine nucleotide transhydrogenase (pntAB) or a mutant thereof.

[0017] In the detailed description of the application, the NAD kinase (nadK) is derived from Escherichia coli, and has a protein accession number AAC75664, or is a mutant of the enzyme having at least 96% or 97% or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number AAC75664 and retaining the enzyme activity.

[0018] In the detailed description of the application, the pyridine nucleotide transhydrogenase (pntAB) is derived from Escherichia coli, and the alpha subunit of the pyridine nucleotide transhydrogenase (pntA) has a protein accession number CAA46884, or is a mutant of the enzyme having at least 96% or 97% or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number CAA46884 and retaining the enzyme activity. The beta subunit of the pyridine nucleotide transhydrogenase (pntB) has a protein accession number CAA46885, or is a mutant of the enzyme having at least 96% or 97% or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number CAA46885 and retaining the enzyme activity.

[0019] In the detailed description of the application, the polynucleotides in the first and second sets of polynucleotides are homologous or heterologous to the host cell.

[0020] In the detailed description of the application, the polynucleotides in the first and second sets of polynucleotides are codon optimized for the host cell.

[0021] The nucleotide sequences, polynucleotides and DNA molecules used in the present application are not limited to functional regions, and can include at least one of expression suppression regions, coding regions, leader sequences, exons, introns and expression cassettes. Furthermore, the nucleotide sequences or polynucleotides can include double-stranded DNA or single-stranded DNA (i.e. the sense strand and the anti-sense strand constituting the double-stranded DNA) or RNA. The polynucleotides containing a particular polynucleotide sequence can include fragments and / or mutants of the particular polynucleotide sequence. The fragments of the polynucleotides refer to a portion of the polynucleotides, which encode a polypeptide providing substantially the same function as the polypeptide encoded by the complete polynucleotide. Examples of the mutants of the particular polynucleotide sequence include naturally occurring allelic mutants, artificial mutants and polynucleotide sequences obtained by deletion, substitution, addition and / or insertion of one or more nucleotides in the particular polynucleotide sequence. It should be understood that such fragments and / or mutants of the particular polynucleotide sequence encode a polypeptide having substantially the same function as the polypeptide encoded by the original particular polynucleotide sequence.

[0022] In the detailed description of the present application, the polynucleotides in the first to second groups of polynucleotides are operably linked to a promoter, which is homologous or heterologous to the host cell.

[0023] In the detailed description of the present application, the backbone plasmid can be any plasmid that can replicate in a host cell. In one embodiment, the expression plasmid vector comprises a backbone plasmid that can replicate in E. coli. Examples of the backbone plasmid include, but are not limited to, backbone plasmids that can replicate in E. coli strains, such as pUC (e.g., pUC18 and pUC19 plasmids), pBR322, pSC101, p15a, pACYC, pET, and pSC101 plasmids, and plasmids derived therefrom.

[0024] The second aspect of the present application provides transformants comprising one or more of the above-described expression plasmid vectors or combinations thereof in a host cell.

[0025] In the present application, the transformant is a host cell that has been altered by introducing one or more expression plasmid vectors in the host cell, wherein the one or more expression plasmid vectors are the same or different. In certain embodiments, the transformant is obtained by introducing the expression plasmid vector by transformation in a host cell that exhibits competence for the expression plasmid vector.

[0026] In the detailed description of the present application, the transformant is a mutant host cell, and the expression plasmid vector or combination of expression plasmid vectors is integrated into the chromosome of the host cell.

[0027] In the detailed description of the present application, the mutant host cell comprises the first group of polynucleotides and the second group of polynucleotides integrated into the chromosome of the host cell.

[0028] In the present application, the first and second groups of polynucleotides can be integrated into the chromosome of the host cell according to methods such as plasmid transformation, phage-mediated transformation, and / or genome editing.

[0029] In one embodiment of the present application, the expression of the gene encoding soluble pyridine nucleotide transhydrogenase (sthA) is inhibited, attenuated, or eliminated in the transformant.

[0030] In the detailed description of the present application, the soluble pyridine nucleotide transhydrogenase (sthA) is derived from E. coli, which has a protein accession number AAC76944, or an enzyme mutant having an amino acid sequence that maintains the above-mentioned enzyme activity and has at least 96% or 97% or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number AAC76944.

[0031] In a further preferred embodiment of the present application, the metabolic byproduct succinic acid, lactic acid, ethanol, acetic acid and formic acid production pathways, 2-ketobutyric acid decomposition pathway, threonine decomposition pathway in the transformant are inhibited or blocked.

[0032] In a further preferred embodiment of the present application, the expression of one, two or more of the following genes in the transformant is inhibited, reduced or eliminated: phosphotransacetylase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, methylglyoxal synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavoprotein subunit frdA, 2-ketobutyrate-formate lyase tdcE, threonine dehydrogenase tdh.

[0033] The present application uses gene knockout to inhibit, reduce or eliminate the expression of the corresponding enzyme, and those skilled in the art know that other ways to inhibit enzyme expression or reduce / eliminate enzyme function activity can also be applicable to the construction of genetically engineered strains, such as promoter knockout or replacement, introduction of enzyme inactivation / deactivation mutations, deletion (or partial deletion) of gene transcription / translation essential functional elements or regions, introduction of mutations or nucleic acid sequences that accelerate the degradation of mRNA that guides enzyme synthesis, introduction of mutations or protein tags that accelerate enzyme degradation / decomposition / inactivation, inhibition of signal activation, RNA interference and gene silencing, CRISPRi, etc.; in the specific embodiments of the present application, the expression amount of the enzyme is reduced by more than 30% or the activity of the enzyme is reduced by more than 30%.

[0034] In the detailed description of the present application, the host strain of the transformant is selected from bacteria or fungi; alternatively, the host strain is selected from wild or genetically engineered Escherichia coli, Bacillus, Corynebacterium, Saccharomyces or Streptomyces; alternatively, the host strain is selected from wild or genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; alternatively, the host strain is selected from wild or genetically engineered Escherichia coli. The E. coli cell can be an E. coli strain derived from E. coli K12 (e.g., MG1655, W3110, DH10b, DH1, BW2952 and strains derived therefrom) or any E. coli strain of E. coli B or strains derived therefrom.

[0035] In the third aspect of the present application, a composition comprising the transformant or culture thereof is provided.

[0036] In the fourth aspect of the present application, a method for preparing L-isoleucine is provided, which comprises fermenting L-isoleucine using the transformant or composition.

[0037] In one embodiment of the present application, the method comprises fermenting L-isoleucine using the transformant or composition with threonine as a substrate.

[0038] In one embodiment of the present application, the method comprises fermenting L-isoleucine using the transformant or composition with threonine as a substrate.

[0039] In one embodiment of the present application, the fermentation medium containing threonine comprises glucose, yeast powder, ammonium sulfate, magnesium sulfate, calcium chloride, potassium dihydrogen phosphate, manganese sulfate pentahydrate, threonine, betaine, nicotine acid, VB1, IPTG, calcium carbonate.

[0040] In the fifth aspect of the present application, the use of the expression plasmid vector or expression plasmid vector combination, the transformant or culture thereof, or the composition in the preparation of L-isoleucine is provided.

[0041] In a sixth aspect of the present application, a chassis strain is provided, which is an Escherichia coli strain or a Corynebacterium glutamicum strain, and expression of a gene encoding soluble pyridine nucleotide transhydrogenase (sthA) in the chassis strain is inhibited, weakened or eliminated.

[0042] In the detailed description of the present application, the soluble pyridine nucleotide transhydrogenase (sthA) is derived from Escherichia coli, and the protein accession number is AAC76944, or an enzyme mutant having an amino acid sequence having at least 96% or 97% or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number AAC76944, which maintains the above-mentioned enzyme activity.

[0043] In the chassis strain, the metabolic byproduct production pathways of succinic acid, lactic acid, ethanol, acetic acid and formic acid, 2-ketobutyric acid decomposition pathway, threonine decomposition pathway are inhibited or blocked.

[0044] In a further preferred embodiment of the present application, expression of one, two or more of the following genes in the chassis strain is inhibited, weakened or eliminated: phosphotransacetylase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, acetolactate synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavoprotein subunit frdA, 2-ketobutyrate-formate lyase tdcE, threonine dehydrogenase tdh.

[0045] Compared with the prior art, the present application has the following beneficial effects:

[0046] The present application improves the yield of L-isoleucine by expressing Escherichia coli NAD kinase and pyridine nucleotide transhydrogenase. By screening different reducing force module gene combinations for expression in isoleucine-producing bacteria, a genetically engineered high-yield strain is obtained. By fermenting the genetically engineered strain and adding threonine during the culture process, L-isoleucine is isolated from the culture. DETAILED DESCRIPTION

[0047] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustrative and explanatory of the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology implemented based on the above description of the present application is covered within the scope of protection intended by the present application.

[0048] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0049] In the present disclosure, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the terms and experimental procedures steps related to nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, used herein are terms and procedures commonly employed by those skilled in the respective art. Also, to better appreciate the present disclosure, the following definitions and explanations of the relevant terms are provided. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0050] The articles "a" and "the" are used herein to refer to one or more than one (i.e., to "at least one") of the grammatical object of the article. By way of alternative example, use of the alternative (e.g., "or") should be construed to mean either one, but not both, of the alternatives. The term "and / or" should be construed to mean either one or both of the alternatives. As used herein, and unless otherwise indicated, the term "about" means that a measurable value such as an amount, a time period, and the like, specifically recited is varied by less than or equal to 10%, more preferably by less than or equal to 5%, even more preferably by less than or equal to 1%, and still more preferably by less than or equal to 0.1%, from the stated value, as long as this variation does not render said measurement of value ineffective to meaningfully measure the quantity or time period to which it relates.

[0051] As used herein, the term "genetic synthesis" refers to the production using recombinant DNA technology or the use of synthetic DNA or amino acid sequence technology available and well known in the art. "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules having a defined sequence of nucleotides or of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription and translation of mRNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0052] As used herein, the term "endogenous" refers to any substance that is from or produced within an organism, cell, tissue, or system.

[0053] As used herein, the term "exogenous" refers to any substance that is introduced into or produced outside of an organism, cell, tissue, or system.

[0054] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0055] Unless otherwise specified, a "polynucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences which are degenerate versions of each other and which encode the same amino acid sequence. The phrase nucleotide sequence which encodes a protein or an RNA can also include an intron to the extent that the nucleotide sequence encoding the protein can in some version contain an intron(s).

[0056] As used herein, a polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant means, i.e., cloning a nucleic acid sequence from a recombinant library or the genome of a cell, using cloning techniques, etc., or using synthetic means.

[0057] In various illustrative embodiments, the polynucleotides herein include, but are not limited to, polynucleotides comprising expression vectors, viral vectors, transfer plasmids, expression cassettes, and polynucleotides encoding a polypeptide of a cytokine antibody or antibody fragment or antigen binding fragment.

[0058] As disclosed herein or as known in the art, a polynucleotide can be combined with other DNA sequences, regardless of the length of the coding sequence itself, such as promoters and / or enhancers, untranslated regions (UTRs), polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites, stop codons, transcription termination signals, post-transcriptional response elements, and polynucleotides encoding self-cleaving polypeptides, epitope tags, such that the overall length can vary significantly. Thus, a polynucleotide fragment of almost any length can be employed, with the overall length preferably limited by ease of preparation and use in the intended recombinant DNA protocol.

[0059] As used herein, the term "vector" is a composition of matter that includes an isolated nucleic acid and that can be used to transfer the isolated nucleic acid into a cell. The transferred nucleic acid is typically linked, e.g., inserted, into the vector nucleic acid molecule. The vector can contain sequences that direct autonomous replication in the cell or can contain sequences sufficient to permit integration into the host cell DNA. Many vectors are known in the art, including but not limited to plasmids, phagemids, artificial chromosomes, bacteriophages, and animal viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses.

[0060] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are those non-translated regions of the vector - origin of replication, selectable marker, promoter, enhancer, translation initiation signal intron, post-transcriptional regulatory element, polyadenylation sequence, 5' and 3' untranslated regions - that interact with host cellular proteins to carry out transcription and translation. The length and specific identity of such elements can vary. Any number of suitable transcription and translation elements can be used depending on the vector system and host utilized, including ubiquitous and inducible promoters. In particular embodiments, the polynucleotide is a vector including, but not limited to, expression vectors and viral vectors and includes exogenous, endogenous, or heterologous control sequences, such as promoters and / or enhancers. An "endogenous" control sequence is one that is naturally linked to a given gene in the genome. An "exogenous" control sequence is one that is placed in juxtaposition to a gene by genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. A "heterologous" control sequence is an exogenous sequence from a different species than the cell being genetically manipulated. A "synthetic" control sequence can include elements of one or more endogenous and / or exogenous sequences and / or sequences determined in vitro or in silico that provide optimal promoter and / or enhancer activity for a particular gene therapy. As used herein, the term "promoter" refers to a recognition site of a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes the polynucleotide operably linked to the promoter.

[0061] The term "promoter" refers to a fragment of DNA that contains a sequence capable of furnishing promoter function.

[0062] The term "conditionally expressed" can refer to any type of conditional expression, including but not limited to: inducible expression; repressible expression; expression in cells or tissues having a particular physiological, biological, or disease state, etc. This definition is not intended to exclude cell type or tissue specific expression. Certain embodiments provide for conditional expression of a polynucleotide of interest, e.g., expression is controlled by subjecting a cell, tissue, organism, etc. to a treatment or condition that causes the polynucleotide to be expressed or causes the expression of a polynucleotide encoded by the polynucleotide of interest to increase or decrease.

[0063] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid inducible promoters, such as the promoters of genes encoding glucocorticoid or estrogen receptors, metallothionein promoters; MX-1 promoters; "gene switch" mifepristone regulatable system; tetracycline-dependent regulation system, etc.

[0064] In some embodiments, the genetically modified cell comprises a polynucleotide further comprising a positive selectable marker that enables selection of cells that exhibit a phenotype that is negative in vitro. The positive selectable marker can be a gene that, when introduced into a host cell, expresses a dominant phenotype that allows for the positive selection of cells that carry the gene. This type of gene is known in the art.

[0065] In one embodiment, the positive selectable marker and the negative selectable marker are linked such that loss of the negative selectable element also concomitantly results in loss of the positive selectable marker. In particular embodiments, the positive and negative selectable markers are fused such that loss of one necessarily results in loss of the other.

[0066] As used herein, the term "transfected" or "transformed" or "transduced" refers to a process by which exogenous nucleic acid is transferred or introduced into a recipient strain. A "transfected" or "transformed" or "transduced" strain is one that has been transfected, transformed or transduced with exogenous nucleic acid. The strain includes the primary and its progeny. The recipient strain can be Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus megaterium, Vibrio natriegens, Bacillus amyloliquefaciens, or Saccharomyces cerevisiae, etc.

[0067] In the present specification, the ilvA gene encoding threonine dehydratase substantially relieved of inhibition by L-isoleucine is referred to as "relieved ilvA gene".

[0068] Materials and Methods

[0069] LB medium: peptone 1%, yeast extract 0.5%, NaCl 1%, pH adjusted to 7.2 with 30% NaOH, 1 x 10 5 Pa was sterilized for 20 min. Agar 1.5% was added when plating. Ampicillin at a final concentration of 100 mg / L and / or kanamycin at a final concentration of 50 mg / L were added according to the type of resistance gene during resistance screening.

[0070] Shaking flask fermentation medium: glucose 30 g / L, yeast powder 5 g / L, ammonium sulfate 8 g / L, magnesium sulfate 2 mM, calcium chloride 0.1 mM, potassium dihydrogen phosphate 0.8 g / L, manganese sulfate pentahydrate 0.02 g / L, threonine 20 g / L, betaine 1 g / L, nicotine acid 0.01 g / L, VB1 0.005 g / L, IPTG 0.1 mM, calcium carbonate 10 g / L, the medium is adjusted to pH 7.3-7.4 with concentrated ammonia water, sterilized for 15 min at 11 ℃.

[0071] Shaking flask fermentation method: single colony was picked from LB plate (containing appropriate concentration of antibiotics) activated at 37 ℃ and inoculated into LB liquid medium (containing appropriate concentration of antibiotics) at 37 ℃ for 12-16 h at a speed of 220 rpm. The LB overnight culture was inoculated into 250 ml shaking flask containing 25 ml fermentation medium (containing 10 g / L CaCO3 as pH stabilizer) at a 1% inoculation amount; after being sealed with a gas permeable membrane, it was placed in a culture at 30 ℃, 220 rpm, and when OD600 was 0.6, IPTG (final concentration 0.2 mM) was added to induce plasmid expression, and fermentation was stopped after 24 hours of culture, and samples were taken.

[0072] Determination of amino acid concentration: amino acid standard products were purchased from Sigma-Aldrich Company (www.sigmaaldrich.cn). 1 mL of fermentation broth was centrifuged at 10,000 r / min for 5 min to remove the bacterial cells, and the obtained filtrate was filtered through a filter membrane with a pore size of 0.22 μm, diluted to an appropriate multiple, and then the branched-chain amino acid concentration in the sample was determined by high performance liquid chromatography (https: / / www.agilent.com / library / applications / 5990-4547EN.pdf). The high performance liquid chromatograph was Shimadzu Nexera LC-40, the chromatographic column was Agilent ZORBAX Eclipse Plus C18, 4.6×250mm 5μm. The detector was a DAD diode array detector, the detection wavelength was 338 nm, and the reference wavelength was 390 nm. The composition, proportion change, flow rate and chromatographic column temperature of the mobile phase were set according to the above method.

[0073] Glucose determination conditions: the high performance liquid chromatograph was Shimadzu Nexera LC-40, the chromatographic column was Bio-Rad Aminex HPX-87H 300×7.8 mm. The column oven was set to 40 ℃; the detector was a RID differential refractometer detector (detector set to constant temperature 40 ℃). The mobile phase was 5 mM sulfuric acid solution, and the flow rate was 0.6 ml / min.

[0074] Table 1 Enzyme types involved in the present application

[0075]

[0076] The biological material constructed by the present application is as shown in Table 2 below:

[0077] Table 2 Biological material involved in the present application

[0078]

[0079] Example 1 Construction of isoleucine production strain and plasmid

[0080] Using λ-Red recombination technology 1 The pflB gene encoding pyruvate-formate lyase in the genome of Escherichia coli K background strain BW25113 was knocked out and replaced with a kanamycin resistance gene Kan to obtain a recombinant strain BW25113ΔpflB::Kan. The plasmid pCP20 was transformed into the above Kan-resistant transformant, which was coated on an LB plate containing 100 mg / L ampicillin and cultured at 30°C for 24 h. The correct transformant in which the kanamycin resistance gene Kan gene was removed from the target gene site was identified by PCR, and an antibiotic-free Escherichia coli BW25113ΔpflB was obtained. In the same way, the starting strain DA9 (BW25113ΔldhAΔptaΔpoxBΔadhEΔpflBΔmgsAΔfrdAΔtdhΔtdcE) was obtained using the same method and strategy. Knocking out most of the mixed acid fermentation genes of Escherichia coli can prevent the interference of by-products such as mixed acids produced by the chassis cell during fermentation, and reduce the substrate conversion rate; deletion of tdh can reduce the decomposition of precursor threonine, and deletion of tdcE and pflB can reduce the decomposition of precursor 2-ketobutyric acid, which is conducive to guiding the isoleucine synthesis metabolic flow to the synthesis of isoleucine. The strain and plasmid genotypes are shown in Table 1 and Table 2.

[0081] The ilvDCEygaZH (ilvC encoding acetyl-hydroxy acid reductase with L67ER68F K75E mutations) and ilvAIH fragments (encoding IlvA protein with L447F and L451A mutations; ilvH gene with G14D S17F encoding mutations) were amplified from plasmids pZE-ilvDCmEygaZH and pZA-ilvAIH (plasmid from Chinese patent publication No. CN114410701A) by PCR, respectively. 2 3 The recombinant plasmid pZE-ilvAIHDCEygaZH (pZE-ILE) was obtained by using a recombination cloning kit to perform one-step seamless ligation of the gene fragments to the pZElac 4 vector PCR fragment. ​

[0082] The nadK gene fragment and the pntAB gene fragment were amplified from the genome of E. coli, and the gene fragments shown in Table 2 were combined and ligated to the vector plasmid fragment pZAlac by the seamless cloning method using a recombination cloning kit. 4 The recombinant plasmids pN1 to pN3 were obtained.

[0083] The above recombinant plasmids were transformed into the DA9 or DA10 strain, and the details of the combination of the plasmids and the strains are shown in the Materials and Methods section.

[0084] Example 2 Shake flask fermentation of L-isoleucine production strain

[0085] The pZE-ILE and pN1-pN3 plasmids were transformed into the L-isoleucine production chassis strain DA9 as shown in Table 2, and the resulting recombinant strains were fermented according to the shake flask fermentation method. ILE-N0 was the control, and the results are shown in Table 3. We can see that after the introduction of the pN1-pN3 series of plasmids, the yield of isoleucine was increased to varying degrees compared to the control strain. Among them, the simultaneous overexpression of NadK and PntAB had the best effect. And the introduction of the above plasmids also slightly improved the glucose conversion rate.

[0086] Table 3 Shake flask fermentation results

[0087]

[0088]

[0089] Example 3 Shake flask fermentation of L-isoleucine production strain

[0090] The strain DA10 was further obtained by deleting the gene sthA in DA9, and the plasmids pZE-ILE and pN3 were introduced into DA10 to obtain the recombinant strain ILE-N4. ILE-N4 was subjected to shake flask fermentation, and the results are shown in Table 4. It can be seen that the yield of isoleucine was increased compared to ILE-N3.

[0091] Table 4 Shake flask fermentation results

[0092] Strain name Isoleucine (g / L) Glucose conversion rate ILE-N3 16.3±0.79 34.5％ ILE-N4 17.9±0.34 34.9％

[0093] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present application, and they should all be covered in the scope of the claims of the present application.

[0094] References;

[0095] 1. Datsenko, K. A.; Wanner, B. L., One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences 2000, 97 (12), 6640-6645. Escherichia coli

[0096] 2. Hao, Y.; Ma, Q.; Liu, X.; Fan, X.; Men, J.; Wu, H.; Jiang, S.; Tian, D.; Xiong, B.; Xie, X., High-yield production of L-valine in engineered Escherichia coli by a novel two-stage fermentation. Metabolic Engineering 2020, 62, 198-206.

[0097] 3. Park, J. H.; Oh, J. E.; Lee, K. H.; Kim, J. Y.; Lee, S. Y., Rational Design of Escherichia coli for l-Isoleucine Production. ACS Synthetic Biology 2012, 1 (11), 532-540.

[0098] 4. Zhang, K.; Sawaya, M. R.; Eisenberg, D. S.; Liao, J. C., Expanding metabolism for biosynthesis of nonnatural alcohols. Proceedings of the National Academy of Sciences 2008, 105 (52), 20653-20658.​

Claims

1. An expression plasmid vector or combination of expression plasmid vectors, characterized in that: The vector or combination comprises: a first set of polynucleotides comprising at least one polynucleotide encoding an enzyme of an L-isoleucine synthesis pathway; and a second set of polynucleotides comprising at least one polynucleotide encoding an enzyme that modulates NAD+ / NADH and NADP+ / NADPH balance; and a backbone plasmid that is capable of autonomous replication in the host cell.

2. The expression plasmid vector or combination of expression plasmid vectors according to claim 1, characterized in that The plasmid vector or vector combination is used to facilitate the host cell to enhance the expression amount of L-isoleucine.

3. The expression plasmid vector or combination of expression plasmid vectors according to claim 1, characterized in that The L-isoleucine synthesis pathway comprises the pathway from threonine to L-isoleucine in KEGG entry M00570, or the pathway from 2-ketobutyrate to L-isoleucine in KEGG entry M00019; Preferably, the first set of polynucleotides comprises a gene encoding at least one of the following enzymes: threonine dehydrogenase (ilvA) or a mutant thereof, acetohydroxy acid synthase (ilvIH, ilvBN, ilvGM, alsS) or a mutant thereof, dihydroxy acid dehydratase (ilvD) or a mutant thereof, acetohydroxy acid reductoisomerase (ilvC) or a mutant thereof, branched-chain amino acid transaminase (ilvE) or a mutant thereof; Preferably, the first set of polynucleotides further comprises a gene encoding branched-chain amino acid efflux protein (ygaZH) or a mutant thereof.

4. The expression plasmid vector or combination of expression plasmid vectors according to claim 1, characterized in that: The second set of polynucleotides comprises a gene encoding at least one of the following enzymes: NAD kinase (nadK) or a mutant thereof, pyridine nucleotide transhydrogenase (pntAB) or a mutant thereof.

5. The expression plasmid vector or combination of expression plasmid vectors according to claim 1, characterized in that The polynucleotides in the first and second sets of polynucleotides are homologous or heterologous to the host cell; Preferably, the polynucleotides in the first and second sets of polynucleotides are codon-optimized for the host cell; Preferably, the polynucleotides in the first to second sets of polynucleotides are operably linked to a promoter, which is homologous or heterologous to the host cell.

6. A transformant characterized in that: comprises one or more expression plasmid vectors or combinations thereof according to any one of claims 1-5; Preferably, the transformant is obtained by introducing one or more expression plasmid vectors into the host cell, wherein the one or more expression plasmid vectors are the same or different; Preferably, the transformant is a mutant host cell, and the expression plasmid vector or expression plasmid vector combination is integrated into the chromosome of the host cell; Preferably, the mutant host cell comprises the first set of polynucleotides and the second set of polynucleotides integrated into the chromosome of the host cell; Preferably, the first set and the second set of polynucleotides are integrated into the chromosome of the host cell according to a plasmid transformation, a phage-mediated transformation, and / or a genome editing method; Preferably, the expression of a gene encoding soluble pyridine nucleotide transhydrogenase (sthA) in the transformant is inhibited, attenuated, or eliminated; Preferably, the metabolic byproduct production pathways of succinic acid, lactic acid, ethanol, acetic acid, and formic acid, the 2-ketobutyrate decomposition pathway, and the threonine decomposition pathway are inhibited or blocked in the transformant. Preferably, expression of one, two or more of the following genes in the transformant is inhibited, weakened or eliminated: phosphotransacetylase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, methylglyoxal synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavoprotein subunit frdA, 2-ketobutyrate-formate lyase tdcE, threonine dehydrogenase tdh; Preferably, the host strain of the transformant is selected from bacteria or fungi.

7. A composition comprising the transformant of claim 6 or a culture thereof.

8. A process for the preparation of L-isoleucine, characterized by: The fermentation production of L-isoleucine using the transformant of claim 6 or the composition of claim 7; Preferably, the fermentation production of L-isoleucine using the transformant or the composition as a substrate of threonine; Preferably, the fermentation production of L-isoleucine by inoculating the transformant into a fermentation medium containing threonine; Preferably, the fermentation medium containing threonine comprises glucose, yeast powder, ammonium sulfate, magnesium sulfate, calcium chloride, potassium dihydrogen phosphate, manganese sulfate pentahydrate, threonine, betaine, nicotine acid, VB1, IPTG, calcium carbonate.

9. Use of the expression plasmid vector or expression plasmid vector combination of any one of claims 1-5, the transformant of claim 6 or a culture thereof, the composition of claim 7 in the preparation of L-isoleucine.

10. A chassis strain, which is an Escherichia coli strain or a Corynebacterium glutamicum strain, wherein expression of a gene encoding soluble pyridine nucleotide transhydrogenase (sthA) is inhibited, weakened or eliminated; Preferably, the soluble pyridine nucleotide transhydrogenase (sthA) is derived from Escherichia coli, and has a protein accession number AAC76944, or is an enzyme mutant having an amino acid sequence having at least 96% or 97% or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number AAC76944 and maintaining the enzyme activity of the above enzyme; Preferably, the chassis strain is inhibited or blocked in the metabolic byproduct production pathways of succinic acid, lactic acid, ethanol, acetic acid and formic acid, 2-ketobutyric acid decomposition pathway, threonine decomposition pathway; Preferably, expression of one, two or more of the following genes in the chassis strain is inhibited, weakened or eliminated: phosphotransacetylase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, methylglyoxal synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavoprotein subunit frdA, 2-ketobutyrate-formate lyase tdcE, threonine dehydrogenase tdh.

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