L-2-aminobutyric acid gene engineering strain and application thereof

Abstract

The invention provides an L-2-aminobutyric acid gene engineering strain and application thereof. The engineering strain comprises an expression plasmid vector or an expression plasmid vector combination, the vector or combination comprises a first group of polynucleotides and a second group of polynucleotides, and the first group of polynucleotides comprises at least one polynucleotide encoding an L-2-aminobutyric acid synthetic pathway enzyme; the second group of polynucleotides comprising at least one polynucleotide encoding an L-2-aminobutyric acid ex-transit pathway enzyme; and a backbone plasmid capable of autonomously replicating in a host cell. The preparation method comprises the following steps: screening and optimizing threonine lytic enzyme, threonine endophytic protein and L-2-aminobutyric acid efflux protein; the yield of the L-2-aminobutyric acid is increased by lacking the internal transport and absorption protein of the L-2-aminobutyric acid, and the method has important significance for promoting the sustainable development of the industry and enhancing the market competitiveness of the product.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of amino acid synthesis, and particularly relates to an L-2-aminobutyric acid genetically engineered strain and application thereof. BACKGROUND

[0002] As a non-natural amino acid, L-2-aminobutyric acid (2AB, for short 2-aminobutyric acid) has been used as a precursor for the synthesis of many chiral drugs, including anticonvulsant drugs such as brivaracetam and levetiracetam, and ethambutol for treating tuberculosis, and has wide application and commercial value in the pharmaceutical industry.

[0003] At present, most natural L-type natural amino acids can be produced by microbial fermentation. Among them, the annual output of glutamic acid, lysine and threonine exceeds 2 million tons. Compared with chemical method, microbial fermentation method is concerned because of its advantages of cheap and clean resources through the metabolic pathway of microorganisms to synthesize and accumulate L-2-aminobutyric acid in excess.

[0004] There are many domestic enterprises producing L-2-aminobutyric acid by biological method, but there are still problems such as low acid production level, low conversion rate, many by-products and poor product extraction technology. Therefore, improving the fermentation strain and technology of L-2-aminobutyric acid, improving the acid production level and conversion rate, and then reducing the extraction difficulty, have important significance for promoting the sustainable development of the industry and enhancing the market competitiveness of the product. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides an L-2-aminobutyric acid genetically engineered strain and application thereof. The problem of low acid production level in the prior art is solved.

[0006] Threonine dehydratase is a key enzyme in the synthesis pathway of L-2-aminobutyric acid, which is the first step of threonine metabolism and can catalyze threonine to generate the key precursor 2-ketobutyric acid. Its activity is inhibited by feedback of isoleucine. On the other hand, the accumulation of 2-ketobutyric acid will inhibit the growth of bacteria and interfere with the normal metabolism of host bacteria, which is not conducive to the production of L-2-aminobutyric acid. Amino acid dehydrogenase can asymmetrically reduce 2-ketobutyric acid to generate L-2-aminobutyric acid, so it is important to reasonably express amino acid dehydrogenase in the fermentation process for the synthesis and accumulation of L-2-aminobutyric acid. In addition, since 2-aminobutyric acid has certain cytotoxicity, it is necessary to reasonably express the efflux protein to transport the intracellular accumulated 2-aminobutyric acid to the extracellular to promote its continuous synthesis and achieve high concentration accumulation.

[0007] The first aspect of the present application provides an expression plasmid vector or an expression plasmid vector combination, which comprises: a first group of polynucleotides comprising at least one polynucleotide encoding an enzyme of an L-2-aminobutyric acid synthesis pathway; and a second group of polynucleotides comprising at least one polynucleotide encoding an enzyme of an L-2-aminobutyric acid export pathway.

[0008] and a backbone plasmid which can autonomously replicate in a host cell.

[0009] In the detailed description of the present application, the plasmid vector or vector combination is used to promote the host cell to enhance the expression amount of L-2-aminobutyric acid.

[0010] In the detailed description of the present application, the first group of polynucleotides comprises a) a gene encoding threonine deaminase (ilvA) or a mutant thereof, or threonine dehydratase (tdcB) or a mutant thereof; and b) a gene encoding an amino acid dehydrogenase or a mutant thereof.

[0011] In the detailed description of the present application, the threonine deaminase (ilvA) is derived from Escherichia coli, and has a protein accession number of CAA28577, or is an enzyme mutant having a protein accession number of CAA28577, and 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 CAA28577, and maintaining the enzyme activity of the above enzyme. For example, the protein mutation site of the enzyme mutant is L447F, L451A.

[0012] In the detailed description of the present application, the threonine dehydratase (tdcB) is derived from Escherichia coli, and has a protein accession number of AAC76152, or is an enzyme mutant having a protein accession number of AAC76152, and 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 AAC76152, and maintaining the enzyme activity of the above enzyme.

[0013] In the detailed description of the present application, the amino acid dehydrogenase is glutamate dehydrogenase (gdhA) or a mutant thereof.

[0014] In the detailed description of the present application, the glutamate dehydrogenase (gdhA) is derived from Escherichia coli, and has a protein accession number of AAC74831, or is an enzyme mutant having a protein accession number of AAC74831, and 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 AAC74831, and maintaining the enzyme activity of the above enzyme. For example, the protein mutation site of the enzyme mutant is K92V, T195S.

[0015] Preferably, the first set of polynucleotides comprises a) a gene encoding threonine deaminase (ilvA) or a mutant thereof; b) a gene encoding glutamate dehydrogenase (gdhA) or a mutant thereof.

[0016] In the detailed description of the application, the first set of polynucleotides further comprises a gene encoding at least one enzyme in the threonine synthesis pathway, and / or the aspartate synthesis pathway.

[0017] In the detailed description of the application, the threonine synthesis pathway comprises the pathway from aspartate to threonine in KEGG entry M00018. In the detailed description of the application, the aspartate synthesis pathway comprises the pathway from phosphoenolpyruvate to aspartate in KEGG entry M00170 or M00171.

[0018] In the detailed description of the application, the enzymes in the threonine synthesis pathway comprise aspartate kinase / homoserine dehydrogenase (thrA) or a mutant thereof, homoserine kinase (thrB) or a mutant thereof, threonine synthase (thrC) or a mutant thereof.

[0019] In the detailed description of the application, the first set of polynucleotides further comprises a gene encoding a threonine / serine importer, preferably the threonine / serine importer comprises at least one of tdcC protein or a mutant thereof, sstT protein or a mutant thereof.

[0020] In the detailed description of the application, the tdcC protein is derived from Escherichia coli, and has the protein accession number AAC76151, or is an enzyme mutant that maintains the above-mentioned enzyme activity and has an amino acid sequence that 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 AAC76151.

[0021] In the detailed description of the application, the first set of polynucleotides further comprises a gene encoding imine / enamine deaminase (ridA) or a mutant thereof.

[0022] In the detailed description of the application, the second set of polynucleotides comprises a gene encoding an L-2-aminobutyrate exporter.

[0023] In the detailed description of the application, the L-2-aminobutyrate exporter comprises alanine exporter (alaE) or a mutant thereof, and / or leucine exporter (leuE) or a mutant thereof. Preferably, the alanine exporter (alaE) or a mutant thereof.

[0024] In the detailed description of the present application, the alanine exporter (alaE) is derived from Escherichia coli, and has a protein accession number AAC75717, or is an enzyme mutant 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 AAC75717, while maintaining the above-mentioned enzyme activity.

[0025] In the detailed description of the present application, the leucine exporter (leuE) is derived from Escherichia coli, and has a protein accession number AAC74868, or is an enzyme mutant 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 AAC74868, while maintaining the above-mentioned enzyme activity.

[0026] In the detailed description of the present application, the polynucleotides in the first and second groups of polynucleotides are homologous or heterologous to the host cell.

[0027] In the detailed description of the present application, the polynucleotides in the first and second groups of polynucleotides comprise codon optimization for the host cell.

[0028] 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. In addition, the nucleotide sequences or polynucleotides can include double-stranded DNA or single-stranded DNA (i.e. sense and antisense strands constituting double-stranded DNA) or RNA. The polynucleotides containing a specific polynucleotide sequence can include fragments and / or mutants of the specific 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 specific 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 specific polynucleotide sequence. It should be understood that such fragments and / or mutants of the specific polynucleotide sequence encode a polypeptide having substantially the same function as the polypeptide encoded by the original specific polynucleotide sequence.

[0029] In the detailed description of the present application, the polynucleotides in the first to second groups of polynucleotides can be operably linked to a promoter, which is homologous or heterologous to the host cell.

[0030] In the detailed description of the 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 backbone plasmids 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.

[0031] The second aspect of the present application provides transformants comprising one or more of the above-mentioned expression plasmid vectors or combinations thereof in a host cell.

[0032] In the present application, a 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(s) by transformation in a host cell that exhibits competence for the expression plasmid vector(s).

[0033] In the detailed description of the 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.

[0034] In the detailed description of the application, the mutant host cell comprises a first set of polynucleotides and a second set of polynucleotides integrated into the chromosome of the host cell.

[0035] In the present application, the first and second sets of polynucleotides can be integrated into the chromosome of the host cell according to methods such as plasmid transformation, phage-mediated transformation, and genome editing.

[0036] In the detailed description of the application, the expression of one or more of the following genes in the transformant is inhibited, attenuated, or eliminated: a gene encoding an L-2-aminobutyric acid importer, a gene encoding a threonine / homoserine exporter.

[0037] In the detailed description of the application, the L-2-aminobutyric acid importer comprises branched-chain amino acid permease (brnQ) or a mutant thereof, alanine transporter cycA or a mutant thereof, branched-chain amino acid binding protein livJ or a mutant thereof.

[0038] In the detailed description of the application, the threonine / homoserine exporter comprises rhtA protein or a mutant thereof, rhtB protein or a mutant thereof, rhtC protein or a mutant thereof.

[0039] In the detailed description of the present application, the branched-chain amino acid permease (brnQ) is derived from Escherichia coli, and its protein accession number is AAC73504, or an enzyme mutant having an amino acid sequence that maintains the enzyme activity of the above enzyme and has at least 96% or 97% or 98%, or at least 99% sequence identity with the amino acid sequence represented by the protein accession number AAC73504.

[0040] In a further preferred embodiment of the present application, the metabolic byproduct production pathways of succinic acid, lactic acid, ethanol, acetic acid and formic acid, the 2-ketobutyric acid decomposition pathway, and the threonine decomposition pathway excluding the threonine deamination pathway are inhibited or blocked in the transformant.

[0041] In a further preferred embodiment of the present application, the expression of one, two or more of the following genes is inhibited, reduced or eliminated in the transformant: 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).

[0042] 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 detailed description 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%.

[0043] 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.

[0044] In a third aspect of the present application, a composition comprising the above-mentioned transformant or culture thereof is provided.

[0045] In a fourth aspect of the present application, a method for preparing L-2-aminobutyric acid is provided, comprising culturing the above-mentioned transformant to obtain L-2-aminobutyric acid.

[0046] In an embodiment of the present application, the above-mentioned transformant is fermented in a medium with or without threonine to obtain L-2-aminobutyric acid.

[0047] In a fifth aspect of the present application, the use of the above-mentioned expression plasmid vector or expression plasmid vector combination, transformant or culture thereof, composition in the preparation of L-2-aminobutyric acid is provided.

[0048] In a sixth aspect of the present application, a chassis strain is provided, which is an E. coli strain or a Corynebacterium glutamicum strain, wherein the expression of one or two of the following genes is inhibited, attenuated or eliminated: alanine transporter (cycA) or a mutant thereof, branched-chain amino acid binding protein (livJ) or a mutant thereof, branched-chain amino acid permease (brnQ) or a mutant thereof, threonine / sarcosine efflux protein (rhtA, rhtB or rhtC) or a mutant thereof.

[0049] In a further preferred embodiment of the application, the metabolic byproduct formation pathways of succinic acid, lactic acid, ethanol, acetic acid and formic acid, the 2-ketobutyrate decomposition pathway, the threonine decomposition pathway excluding the threonine deaminating pathway are inhibited or blocked in the chassis strain.

[0050] In a further preferred embodiment of the application, the expression of one, two or more of the following genes is inhibited, attenuated or eliminated in the chassis strain: 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).

[0051] Compared with the prior art, the application has the following beneficial effects:

[0052] The application adopts a genetic engineering method to obtain a genetically engineered strain; promotes threonine metabolism by screening different threonine dehydratases / deaminases; strengthens the absorption of the precursor threonine by the strain by introducing a threonine inner membrane transporter to increase the yield of L-2-aminobutyric acid; verifies that the enhancement of the L-2-aminobutyric acid efflux function can improve the yield of L-2-aminobutyric acid by screening different types of amino acid efflux proteins; and finally proves that reducing the absorption of L-2-aminobutyric acid by the cell is also beneficial to improving the yield of L-2-aminobutyric acid by screening different types of amino acid inner membrane transporters. The genetically engineered strain is subjected to fermentation culture, and precursor threonine is added or not added during the culture process, and L-2-aminobutyric acid (2AB) is separated from the culture after the fermentation culture is completed.

[0053] The application improves the yield of L-2-aminobutyric acid by screening and optimizing threonine decomposition enzymes, threonine inner membrane transporters and L-2-aminobutyric acid efflux proteins, and deleting L-2-aminobutyric acid inner membrane absorption proteins, which is of great significance to promoting the sustainable development of the industry and enhancing the market competitiveness of the product. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 L-2-aminobutyric acid (2AB) synthesis path schematic diagram (double-line genes represent gene deletion). DETAILED DESCRIPTION

[0055] The technical solutions of the application will be further described in detail below in combination with specific embodiments. It should be understood that the following embodiments are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the application. Any technology realized based on the above content of the application is covered within the scope of protection intended by the application.

[0056] The starting materials and reagents used in the following examples are commercially available or can be prepared by known methods unless otherwise stated.

[0057] Unless otherwise indicated, the scientific and technical terms used in the following examples have the meanings that are commonly understood by one of ordinary skill in the art. Also, the nomenclature used in connection with, and the laboratory procedures and techniques of, nucleic acid chemistry, molecular biology, cell biology, and immunology described herein are those well-known and commonly used in the art. To the extent that there is a conflict or inconsistency between the definitions provided herein and those provided by the United States Patent Office, the definitions provided herein control. In addition, the practice of the present disclosure employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, genetics, and immunology, all within the purview of the skilled artisan. Also, procedures involving the use of recombinant DNA, hybridization with nucleic acids, and the like, follow methods well known in the art. To the extent that particular definitions of terms are used in the following description, those definitions are intended to apply uniformly through the entire description unless an otherwise expressly set out definition applies.

[0058] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By the use of the term "or" herein, it is intended that wherever a choice of items can be made (for example, a choice between alternatives), either one of the items, or both, can be employed. By the use of the term "and / or" herein, it is intended that wherever a choice of items can be made (for example, a choice between alternatives), either one of the items, or both, can be employed. The term "and / or" shall be understood to mean one or the other or both. As used herein, and unless otherwise indicated, the term "about" means that the recited value (e.g. of an amount, a time period, etc.) is subject to a variation of ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, as long as such variation is appropriate for the method being performed.

[0059] 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.

[0060] As used herein, the term "encodes" 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 in biological processes having either a defined sequence of nucleotides or of amino acids, and the biological properties that result 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 the non-coding strand, used as the template for transcription of a gene or cDNA, can be said to encode a protein or other product.

[0061] As used herein, the term "endogenous" refers to any substance that is from or produced within an organism, cell, tissue, or system.

[0062] As used herein, the term "exogenous" refers to any substance that is introduced into or produced outside of an organism, cell, tissue, or system.

[0063] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0064] 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. A polynucleotide sequence encoding a protein (or RNA) can also include introns, to the extent that the polynucleotide sequence encoding the protein can contain intron(s) in certain versions.

[0065] As used herein, polynucleotides include, but are 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.

[0066] 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.

[0067] As disclosed elsewhere herein or as known in the art, regardless of the length of the coding sequence itself, a polynucleotide can be combined with other DNA sequences, 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, polynucleotide fragments 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.

[0068] 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 to, 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.

[0069] "expression control sequences," "control elements," or "regulatory sequences" present in the expression vector are non-translated regions of the vector - origin of replication, selection cassette, promoter, enhancer, translational start signal intron, post-transcriptional regulatory element, polyadenylation sequence, 5' and 3' untranslated regions - that interact with host cell proteins to carry out transcription and translation. The length and specificities of such elements can vary. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters, can be used. 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 a sequence that is naturally linked to a given gene in the genome. An "exogenous" control sequence is a sequence 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. The RNA polymerase initiates and transcribes the polynucleotide operably linked to the promoter.

[0070] As used herein, the term "gene knockout" is a technique that inactivates or deletes a specific gene of an organism by certain approaches. Mainly, the principle of DNA homologous recombination is applied, and a designed homologous fragment is used to replace the target gene fragment, so as to achieve the purpose of gene knockout.

[0071] 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 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 be increased or decreased.

[0072] In some embodiments, inducible promoters 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 systems; tetracycline-dependent regulation systems, etc.

[0073] In some embodiments, the genetically modified cell further comprises a polynucleotide as a negative selectable marker and a positive selectable marker. 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. Genes of this type are known in the art.

[0074] In one embodiment, the positive selectable marker and the negative selectable marker are linked such that the loss of the negative selectable element also accompanies the loss of the positive selectable marker. In particular embodiments, the positive and negative selectable markers are fused such that the loss of one necessarily results in the loss of the other.

[0075] 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.

[0076] In the present specification, the ilvA gene encoding threonine dehydratase substantially relieved of inhibition by L-isoleucine is referred to as "relieved ilvA gene".

[0077] LB medium: peptone 1%, yeast extract 0.5%, NaCl 1%, pH adjusted to 7.0 with 30% NaOH, 1 x 10 5 Pa was sterilized for 20 min. Agar 1.5% was additionally added when plating. [Plasmid strains were added with kanamycin (pZAlac) and / or spectinomycin (pZSlac) at a final concentration of 50 mg / L according to the type of resistance gene carried by the plasmid].

[0078] Shaking flask fermentation medium (without threonine): glucose 4%, yeast extract 0.2%, MgS04-7H20 0.1%, NH4(S04)2 1.7%, KH2P04 0.2%, VB10.0002%, FeS04-7H20 0.001%, and MnS04-7H20 0.001%. The pH was adjusted to about 7.0 with NaOH and sterilized at 121°C for 15 min.

[0079] Shaking flask fermentation medium (with threonine): glucose 4%, threonine 2%, yeast extract 0.2%, MgSO40.2%, NH4(SO4)20.8%, KH2PO40.2%, VB10.0002%, betaine 0.1%, nicotinic acid 0.01%. Adjust pH to about 7.0 with NaOH, sterilize at 121°C for 15 min.

[0080] Shaking flask fermentation method: inoculate the strain into LB medium with suitable concentration of antibiotic, incubate at 37°C for 10 hours at 220 rpm. Inoculate the LB culture into the fermentation medium at 10% (V / V) inoculation amount, and ferment in a 250 mL flask (30 mL liquid, containing 40 g / L sterile CaCO3 powder); add IPTG for induction (final concentration 0.4 mM) at the time of inoculation, and stop fermentation after 40 h of incubation.

[0081] Fermenter fermentation strain preparation process: strain ultra-low temperature freezer -80°C storage → plate activation → shaking flask culture → 1L or 5L fermenter. Culture strain activation plate: inoculate 0.12 mL of bacterial solution into each plate with a 1 mL pipette, and incubate at 37°C for 24 h; pick single colonies with normal morphology; LB seed shaking flask culture (culture volume 100 mL / 500 mL flask): temperature 37°C, rotation speed 200 r / min, pH about 7.0, culture time 8-10 h, sample every 2 h during the process, and measure pH and OD600 values; inoculate the fermenter when the OD meets the requirements. Fermentation medium components and feeding conditions are described in the specific content description of the corresponding examples.

[0082] Amino acid concentration determination: L-amino acid standards were purchased from Sigma-Aldrich (www.sigmaaldrich.cn). Centrifuge 1 mL of fermentation broth at 10000 r / min for 5 min to remove the bacterial cells, filter the resulting filtrate through a filter membrane with a pore size of 0.22 μm, dilute it to an appropriate multiple, and then determine the branched-chain amino acid concentration in the sample by high performance liquid chromatography (HPLC) (https: / / www.agilent.com / library / applications / 5990-4547EN.pdf). The high performance liquid chromatograph is Shimadzu Nexera LC-40, the chromatographic column is Agilent ZORBAX Eclipse Plus C18, 4.6 x 250 mm 5 μm. The detector is a DAD diode array detector, the detection wavelength is 338 nm, and the reference wavelength is 390 nm. The composition, proportion change, flow rate and column temperature of the mobile phase are set according to the above method.

[0083] Table 1 Enzyme types involved in the present application

[0084]

[0085]

[0086] The biological material constructed in the present application is shown in Table 2 below:

[0087] Table 2 Biological material involved in the present application

[0088]

[0089]

[0090] Construction of chassis strain and plasmid

[0091] Using λ-Red recombination technology 1 The pflB gene encoding pyruvate-formate lyase in the genome of E. 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 plated on an LB plate containing 100 mg / L ampicillin and incubated 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 E. coli BW25113ΔpflB was obtained. In the same way, the starting strain DA7 (BW25113ΔldhAΔptaΔpoxBΔadhEΔpflBΔmgsAΔfrdA) was obtained using the same method and strategy. This strain knocks out most of the mixed acid fermentation genes of E. coli to prevent the production of by-products such as mixed acids during fermentation, which interferes with the subsequent product separation process. Further, strains 2AB-00 to 2AB-04 and 98082D1 were constructed in the same way. The strains and genotypes are shown in Table 1.

[0092] The ilvA gene fragment (the encoded IlvA protein has L447F and L451A mutations) was amplified from plasmid pZA-ilvAIH (see Chinese Patent Publication No. CN 114410701A) by PCR reaction 2 The gdhA gene fragment (GdhA glutamate dehydrogenase encoded by GDH2 has K92V and T195S mutations) was amplified from plasmid pZElac_GDH2 3 The tdcC gene fragment, the ridA gene fragment, the tdcB gene fragment, the leuE gene fragment, the alaE gene fragment, and the ygaZH gene fragment were amplified from the E. coli genome. The gene fragments shown in Table 2 were combined and ligated to the vector plasmid fragment pZAlac using a recombination cloning kit by seamless cloning method.3 The recombinant plasmids pAB1 to pAB8 were obtained. The schematic diagram of L-2-aminobutyric acid (2AB) synthesis pathway is shown in FIG. 1. Figure 1

[0093] Example 2 Expression of threonine dehydratase tdcB and ilvA to produce 2-aminobutyric acid (2AB)

[0094] The recombinant plasmids pAB1-pAB4 constructed in Example 1 were transformed into the chassis strain 2AB-01 respectively to obtain 2AB fermentation strains 2AB-05 to 2AB-08. The overnight cultures of the above strains were inoculated into fermentation medium containing threonine, and shake flask fermentation experiments were performed, and the results are shown in Table 3. We can see that the 2AB production yield of the fermentation group expressing the permissive ilvAL447F L451A is higher than that of the fermentation group expressing TdcB. At the same time, after overexpressing the threonine inner carrier TdcC, although the 2AB yield of the TdcB overexpression group has a large increase, it is still lower than that of the corresponding IlvA expression group. The above experimental phenomena show that the decomposition efficiency of TdcB to threonine is poor; overexpression of the threonine inner carrier TdcC can increase the catalytic efficiency of threonine degrading enzyme TdcB and IlvA by enhancing the absorption of extracellular threonine and maintaining a high intracellular threonine concentration.

[0095] Table 3 Shake flask fermentation results

[0096] Strain Threonine degradative enzymes Threonine importers 2AB yield (g / L) 2AB-05 IlvA None 3.79±0.04 2AB-06 TdcB None 1.27±0.11 2AB-07 IlvA TdcC 6.16±0.18 2AB-08 TdcB TdcC 2.63±0.20

[0097] Example 3 Screening of 2-aminobutyric acid (2AB) efflux proteins

[0098] Based on the shake flask fermentation results of Example 2, it is speculated that further strengthening the efflux of 2-aminobutyric acid may further increase the concentration of the fermentation broth, so we overexpressed the efflux proteins YgaZH, LeuE and AlaE that may be involved in the efflux of 2-aminobutyric acid, and based on the ilvA system (pAB3) with the highest 2AB yield, plasmids pAB5 to pAB7 were constructed; these three plasmids were transformed into the chassis strain 2AB-01 respectively to obtain 2AB production strains 2AB-09 to 2AB-11. As in Example 2, shake flask fermentation experiments were performed, and the 2-aminobutyric acid yield is shown in Table 4. The results show that the branched-chain amino acid efflux protein YgaZH cannot efflux 2AB, but the leucine efflux protein LeuE and the alanine efflux protein AlaE can efflux 2AB, and the alanine efflux protein AlaE has the most significant effect on the production of 2AB.

[0099] Table 4 Shake flask fermentation results

[0100] Strain Efflux protein overexpression 2AB yield (g / L) 2AB-05 None 6.21±0.12 2AB-09 AlaE 12.50±0.36 2AB-10 YgaZH 6.05±0.19 2AB-11 LeuE 8.93±0.41

[0101] Example 4 Screening of 2-aminobutyric acid (2AB) inner transport proteins​

[0102] Based on the shake flask fermentation results of Example 3, we further speculate that if the reabsorption of 2AB by cells in the fermentation broth is reduced during fermentation, it may have a positive effect on the production of 2AB, so we deleted the branched-chain amino acid transport protein BrnQ, LivJ and alanine inner transport protein CycA in the chassis strain 2AB-01 to obtain strains 2AB-02, 2AB-03 and 2AB-04 to test the effect of the deletion of the related genes on the yield of 2AB. The plasmid pAB5 was transformed into the above strains to obtain 2AB production strains 2AB-12, 2AB-13 and 2AB-14. As in Example 2, shake flask fermentation experiments were carried out, and the 2-aminobutyric acid (2AB) yield is shown in Table 5. The results show that only the deletion of the branched-chain amino acid transport (absorption) protein BrnQ has an effect on the yield of 2AB, and it is speculated that only BrnQ in E. coli may be involved in the absorption of 2AB.

[0103] Table 5 Shake flask fermentation results

[0104] Strain Importer deletion 2AB yield (g / L) 2AB-09 None 12.43±0.16 2AB-12 BrnQ 14.91±0.58 2AB-13 LivJ 12.19±0.24 2AB-14 CycA 10.22±0.21

[0105] Example 5 Fermentor performance test of 2-aminobutyric acid production strain 2AB-12

[0106] Based on the shake flask fermentation results of Example 4, the fermentation performance of the strain was tested by scaling up the culture of strain 2AB-12 in a 1-L automatic control fermentor. The specific fermentor setting method: the seed liquid was inoculated into the fermentation medium containing 50 mg / L kanamycin (glucose 20 g / L, ammonium sulfate 8 g / L, yeast extract 2 g / L, potassium dihydrogen phosphate 2 g / L, threonine 20 g / L, betaine 2 g / L, nicotinic acid 0.2 g / L, VB1 0.02 g / L) in a 1-L automatic control fermentor at a 5% inoculation amount, the temperature of the fermentation broth was controlled at 37°C, air was introduced and the aeration rate was maintained at 1 vvm, and the stirring speed was automatically adjusted to maintain 20% dissolved oxygen (speed 200-1000 rpm). Other control conditions are as follows: automatically add 50% ammonia water to control the pH at 6.8-7.2; add appropriate amount of antifoam agent; control the residual sugar to be within 1% by adding glucose solution with a concentration of 500 g / L; at the same time, add threonine mother liquor with a concentration of 100 g / L to maintain the threonine concentration in the fermentation broth at 2-5 g / L (sample every 0.5 hour, adjust according to the measured amino acid concentration of the sample); add IPTG to a final concentration of 0.3 mM when the OD600 of the bacteria in the fermentor reaches about 15-20; stop fermentation at 40 h. The yield of 2-aminobutyric acid of strain 2AB-12 is 47.6 g / L when the fermentor is discharged.

[0107] Example 6 De novo synthesis of 2-aminobutyric acid (2AB) based on glucose

[0108] Based on the results of previous examples, to demonstrate that the modification of the discovered 2AB exporters and importers in this application also apply to the de novo synthesis of 2AB based on glucose (i.e. without exogenous addition of threonine), we performed shake flask fermentation to produce 2AB in the threonine production strain Escherichia coli ATCC 98082 background 3 The relevant strains and plasmids are listed in Table 2. Shake flask fermentation was performed using shake flask fermentation medium (without threonine). The results are shown in Table 6, which indicate that overexpression of 2AB exporter AlaE and deletion of 2AB importers / transporters BrnQ are also effective to improve 2AB production when 2-aminobutyric acid is de novo synthesized based on glucose.

[0109] Table 6. Shake flask fermentation results

[0110] Strain Threonine degradative enzymes 2AB efflux protein overexpression 2AB importer deletion 2AB yield (g / L) 2AB-15 IlvA None None 3.84±0.10 2AB-16 IlvA AlaE None 6.17±0.13 2AB-17 IlvA AlaE BrnQ 6.93±0.14 2AB-18 TdcB None None 1.27±0.05 2AB-19 TdcB AlaE None 2.89±0.17 2AB-20 TdcB AlaE BrnQ 3.54±0.09

[0111] Finally, it is to be understood that the methods of the present application for screening the optimal improvement of L-2-aminobutyric acid (2AB) production can be modified by those skilled in the art with reference to the content herein. In particular, it is to be noted that all similar substitutions and modifications are obvious to those skilled in the art and are deemed to be included in the present application. The methods and applications of the present application have been described by preferred examples, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present application to realize and apply the present application technology.

[0112] References:

[0113] 1. Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; Datsenko, K. A.; Tomita, M.; Wanner, B. L.; Mori, H., Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology 2006, 2 (1), 2006.0008.

[0114] 2. 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.

[0115] 3. Zhang, K.; Li, H.; Cho, K. M.; Liao, J. C., Expanding metabolism for total biosynthesis of the nonnatural amino acid L-homoalanine. Proceedings of the National Academy of Sciences 2010, 107 (14), 6234-6239.

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 L-2-aminobutyric acid synthesis pathway enzyme; and a second set of polynucleotides comprising at least one polynucleotide encoding an L-2-aminobutyric acid export pathway enzyme. and a backbone plasmid 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-2-aminobutyric acid.

3. The expression plasmid vector or combination of expression plasmid vectors according to claim 1, characterized in that The first set of polynucleotides comprises a) a gene encoding threonine deaminase (ilvA) or a mutant thereof, or threonine dehydratase (tdcB) or a mutant thereof; and b) a gene encoding an amino acid dehydrogenase or a mutant thereof. Preferably, the amino acid dehydrogenase is glutamate dehydrogenase (gdhA) or a mutant thereof. Preferably, the first set of polynucleotides comprises a) a gene encoding threonine deaminase (ilvA) or a mutant thereof; and b) a gene encoding glutamate dehydrogenase (gdhA) or a mutant thereof. Preferably, the first set of polynucleotides further comprises a gene encoding at least one enzyme in the threonine synthesis pathway, and / or the aspartate synthesis pathway. Preferably, the threonine synthesis pathway comprises the pathway from aspartate to threonine in KEGG entry M00018. Preferably, the aspartate synthesis pathway comprises the pathway from phosphoenolpyruvate to aspartate in KEGG entry M00170 or M00171. Preferably, the enzymes of the threonine synthesis pathway comprise aspartate kinase / high serine dehydratase (thrA) or a mutant thereof, high serine kinase (thrB) or a mutant thereof, threonine synthase (thrC) or a mutant thereof. Preferably, the first set of polynucleotides further comprises a gene encoding a threonine / serine importer. Preferably, the threonine / serine importer comprises at least one of tdcC protein or a mutant thereof, sstT protein or a mutant thereof. Preferably, the first set of polynucleotides further comprises a gene encoding an imine / enamine deaminase (ridA) 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 an L-2-aminobutyric acid exporter. Preferably, the L-2-aminobutyric acid exporter comprises alanine exporter (alaE) or a mutant thereof, and / or leucine exporter (leuE) 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: The transformant is a host cell altered by introduction of one or more of the expression plasmid vectors or combinations thereof into the host cell, wherein the one or more expression plasmid vectors are the same or different. The transformant is a host cell altered by introduction of one or more of the expression plasmid vectors or combinations thereof into the host cell, wherein the one or more expression plasmid vectors are the same or different. Preferably, the transformant is obtained by transforming the expression plasmid vector or combination thereof into a competent host cell; Preferably, 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; Preferably, the mutant host cell comprises a first set of polynucleotides and a 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 the methods of plasmid transformation, phage-mediated transformation and genome editing; Preferably, the expression of one or more of the following genes in the transformant is inhibited, attenuated or eliminated: a gene encoding an L-2-aminobutyric acid importer, a gene encoding a threonine / high serine exporter; Preferably, the L-2-aminobutyric acid importer comprises branched-chain amino acid permease (brnQ) or a mutant thereof, alanine transporter cycA or a mutant thereof, branched-chain amino acid binding protein livJ or a mutant thereof; Preferably, the threonine / high serine exporter comprises rhtA protein or a mutant thereof, rhtB protein or a mutant thereof, rhtC protein or a mutant thereof; 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 excluding the threonine deamination pathway are inhibited or blocked in the transformant; Preferably, the expression of one, two or more of the following genes in the transformant is inhibited, attenuated 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 characterized in that: Comprising the transformant of claim 6 or a culture thereof.

8. A process for the preparation of L-2-aminobutyric acid, characterized by: Comprising culturing the transformant of claim 6 to obtain L-2-aminobutyric acid; Preferably, the transformant is fermented in a culture medium with or without threonine to obtain L-2-aminobutyric acid.

9. Use of the expression plasmid vector or combination of expression plasmid vectors 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-2-aminobutyric acid.

10. A chassis strain characterized in that: It is an E. coli strain or a C. glutamicum strain, and the expression of one or two of the following genes in the chassis strain is inhibited, attenuated or eliminated: alanine transporter (cycA) or a mutant thereof, branched-chain amino acid binding protein (livJ) or a mutant thereof, branched-chain amino acid permease (brnQ) or a mutant thereof, threonine / high serine exporter (rhtA, rhtB or rhtC) or a mutant thereof; 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 excluding the threonine deamination pathway are inhibited or blocked in the chassis strain; Preferably, the expression of one, two or more of the following genes is inhibited, attenuated or eliminated in the chassis strain: phosphotransacetylase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, methylglyoxal synthase mgsA, D-lactate dehydrogenase IdhA, fumarate reductase flavoprotein subunit frdA, 2-ketobutyrate-formate lyase (tdcE), threonine dehydrogenase (tdh). Preferably, the expression of one, two or more of the following genes is inhibited, attenuated or eliminated in the chassis strain: phosphotransacetylase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, methylglyoxal synthase mgsA, D-lactate dehydrogenase IdhA, fumarate reductase flavoprotein subunit frdA, 2-ketobutyrate-formate lyase (tdcE), threonine dehydrogenase (tdh). Preferably, the expression of one, two or more of the following genes is inhibited, attenuated or eliminated in the chassis strain: phosphotransacetylase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruv

Images