Construction and application of genetically engineered strains capable of achieving co-production of branched-chain amino acids

Abstract

The present invention relates to the construction and application of a genetically engineered strain capable of realizing the co-production of branched-chain amino acids, and can realize the co-production of branched-chain amino acids (BCAA) such as valine, leucine and isoleucine by fermentation. A genetically engineered strain is obtained by a genetic engineering method, wherein the genetically engineered strain is a synthetic pathway of leucine introduced into an Escherichia coli host cell capable of producing isoleucine and / or valine, and isopropylmalate synthase encoding gene leuAm, isopropylmalate dehydrogenase encoding gene leuB, isopropylmalate dehydratase encoding gene leuCD, aromatic amino acid transaminase gene tyrB and leucine efflux protein gene leuE are expressed to release the feedback inhibition of L-leucine. The co-production of two or three branched-chain amino acids is finally realized, and the co-production process has at least 30% improvement in the utilization rate of overall glucose. The genetically engineered strain described in the present invention has a short fermentation cycle, high yield, high conversion rate, and has a high production application value.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering and relates to genetic engineering bacteria for co-producing branched-chain amino acids such as valine, leucine and isoleucine and applications thereof. Background Art

[0002] Branched-chain amino acids (BCAA) refer to neutral amino acids with branched fatty hydrocarbon chains on the α-carbon, including L-leucine, L-isoleucine and L-valine, which are essential amino acids for humans and animals. The biosynthetic pathways of the three branched-chain amino acids partially share the same precursor substances and enzymes. Branched-chain amino acids cannot be synthesized by higher animals themselves and can only be supplemented through food. Branched-chain amino acids account for 40% of essential amino acids, and their sufficient addition and reasonable proportion are very important for the normal development of animals. The main functions of branched-chain amino acids include: increasing cell energy absorption, improving cell vitality, delaying cell aging, scavenging free radicals, improving tissue and organ function, anti-fatigue, skin beauty, improving cardiopulmonary and myocardial capacity, improving cardiovascular and cerebrovascular protection, reducing fat, anti-heart failure, skeletal muscle synthesis and improving immunity. Branched-chain amino acids have important applications in food, medicine, health products, feed, cosmetics and other fields.

[0003] L-valine can supplement nutrition, promote body growth and provide energy. L-valine can also be used in infusions and injections to promote surgical wound healing. L-valine is also one of the five limiting amino acids in crude protein feed for young piglets and broilers, and its long-term deficiency will have an adverse effect on animal growth performance. In recent years, as an important feed dietary amino acid variety, the demand for L-valine has continued to increase. The export volume of feed-grade L-valine has reached more than 30,000 tons, with an average annual growth rate of 20%, and the market prospects are very good. L-leucine plays an important role in stimulating muscle protein synthesis and maintaining glucose homeostasis. L-leucine is also used as a flavoring and lubricant for tablet production. L-isoleucine has the function of promoting the synthesis of proteins, enzymes and peptide hormones in the body, plays an important role in the life metabolism process with various physiological functions, and is widely used in feed, medicine, food and other industries.

[0004] The production methods of branched-chain amino acids include extraction, chemical synthesis, microbial fermentation, etc. The extraction method and chemical synthesis method are difficult to achieve industrial production due to the limited raw material sources and high production costs. Similar to the production of most other L-amino acids, microbial fermentation is the main method for the current industrial production of branched-chain amino acids. The microbial fermentation method has low raw material costs, mild reaction conditions, and is easy to achieve large-scale production. However, the technology for producing BCAA by microbial fermentation in my country is not yet mature, and the fermentation level is not high, resulting in a long fermentation cycle, low acid production, and many miscellaneous acids, which has become the primary factor affecting the industrial application of branched-chain amino acids in my country. In particular, due to the long synthesis pathway of L-leucine and L-isoleucine and the strict feedback regulation mechanism, the yield and conversion rate of its production strains are still low, and its current output can no longer meet the growing market demand.

[0005] The production of branched-chain amino acids by microbial fermentation relies on the ability of microorganisms to synthesize the amino acids they need. Various nutritional deficiency or amino acid structure analogue resistance mutants are selected through strain mutagenesis, such as Seymourne bacillus, lactofermentation bacillus, Corynebacterium glutamicum, Corynebacterium blunt-toothed, Brevibacterium flavum, Escherichia coli, etc., in order to relieve feedback inhibition and repression in metabolic regulation and achieve the purpose of excessive accumulation of branched-chain amino acids.

[0006] So far, improving the L-branched-chain amino acid production of branched-chain amino acid production strains depends on whether the selected mutant strains have the potential to accumulate a large amount of L-branched-chain amino acids, and on the other hand, it depends on the fermentation production environment conditions and technical processes. Therefore, transforming excellent L-branched-chain amino acid fermentation strains and optimizing technical processes, improving the acid production capacity of strains, and simplifying the extraction process are of great economic value to improving the competitiveness of domestic L-branched-chain amino acid product production. Summary of the invention

[0007] The present invention introduces a leucine synthesis pathway into an Escherichia coli host cell that can produce isoleucine and / or valine, and achieves the co-production of three branched-chain amino acids by expressing an isopropylmalate synthase encoding gene leuAm, an isopropylmalate dehydrogenase encoding gene leuB, an isopropylmalate dehydratase encoding gene leuCD, an aromatic amino acid transaminase gene tyrB, and a leucine efflux protein gene leuE that relieve feedback inhibition of L-leucine. Specific aspects of the present invention include:

[0008] The first aspect of the present invention provides: a genetically engineered strain capable of achieving co-production of branched-chain amino acids, wherein the strain expresses:

[0009] 1) at least one DNA molecule encoding a polypeptide that catalyzes the synthesis of α-isopropylmalic acid;

[0010] 2) at least one DNA molecule encoding a polypeptide that catalyzes the isomerization of α-isopropylmalic acid to β-isopropylmalic acid;

[0011] 3) at least one DNA molecule encoding a polypeptide that catalyzes the dehydrogenation of β-isopropylmalate to produce α-ketoisocaproate;

[0012] 4) at least one DNA molecule encoding a polypeptide that catalyzes the transamination of an aromatic amino acid to α-ketoisocaproic acid to produce leucine;

[0013] as well as:

[0014] 5) at least one DNA molecule encoding a polypeptide that catalyzes the production of ketobutyrate from threonine; preferably, the DNA molecule is a gene for threonine deaminase that is substantially free of inhibition by L-isoleucine; further preferably, the gene for threonine deaminase that is substantially free of inhibition by L-isoleucine is a released ilvA gene;

[0015] and / or

[0016] 6) at least one DNA molecule encoding a polypeptide, the DNA molecule being a gene of acetolactate synthase / acetylhydroxybutyrate synthase, preferably the gene being a gene of acetolactate synthase / acetylhydroxybutyrate synthase substantially relieved of inhibition by L-isoleucine and / or L-valine, further preferably, the gene of acetolactate synthase / acetylhydroxybutyrate synthase substantially relieved of inhibition by L-isoleucine and / or L-valine is one or more of a relieved ilvIH gene, a relieved ilvBN gene, a relieved ilvGM gene, and a relieved alsS gene;

[0017] The expression of at least one of the DNA molecules is enhanced.

[0018] In a specific embodiment of the present invention, the DNA molecule of 1) is the isopropylmalate synthase encoding gene leuA, preferably, it is the isopropylmalate synthase encoding gene that relieves the feedback inhibition of L-leucine, and further preferably, it is the isopropylmalate synthase encoding gene leuAm with the G479C mutation.

[0019] In a specific embodiment of the present invention, the DNA molecule of 2) is the isopropylmalate dehydratase encoding gene leuCD.

[0020] In a specific embodiment of the present invention, the DNA molecule of 3) is the isopropylmalate dehydrogenase encoding gene leuB.

[0021] In a specific embodiment of the present invention, the DNA molecule of 4) is the aromatic amino acid transaminase gene tyrB.

[0022] In a specific embodiment of the present invention, the genetically engineered strain further includes one or more of a threonine dehydratase gene (tdcB), a threonine transporter gene, a branched-chain amino acid transporter gene, an acetohydroxyacid isomerase gene (ilvC), a dihydroxyacid dehydratase gene (ilvD), a branched-chain amino acid aminotransferase gene (ilvE), and a branched-chain amino acid dehydrogenase gene.

[0023] The second aspect of the present invention provides: an engineered strain for co-producing valine and leucine, wherein the strain expresses:

[0024] 1) at least one DNA molecule encoding a polypeptide that catalyzes the synthesis of α-isopropylmalic acid;

[0025] 2) at least one DNA molecule encoding a polypeptide that catalyzes the isomerization of α-isopropylmalic acid to β-isopropylmalic acid;

[0026] 3) at least one DNA molecule encoding a polypeptide that catalyzes the dehydrogenation of β-isopropylmalate to produce α-ketoisocaproate;

[0027] 4) at least one DNA molecule encoding a polypeptide that catalyzes the transamination of an aromatic amino acid to α-ketoisocaproic acid to produce leucine;

[0028] Preferably, the DNA molecule of 1) is the isopropylmalate synthase encoding gene leuA, preferably, isopropylmalate synthase encoding gene that relieves feedback inhibition of L-leucine, and further preferably isopropylmalate synthase encoding gene leuAm with G479C mutation;

[0029] Preferably, the DNA molecule of 2) is the isopropylmalate dehydratase encoding gene leuCD;

[0030] Preferably, the DNA molecule of 3) is the isopropylmalate dehydrogenase encoding gene leuB;

[0031] Preferably, the DNA molecule of 4) is the aromatic amino acid transaminase gene tyrB;

[0032] as well as

[0033] 6) at least one DNA molecule encoding a polypeptide, the DNA molecule being a gene of acetolactate synthase / acetylhydroxybutyrate synthase, preferably the gene being a gene of acetolactate synthase / acetylhydroxybutyrate synthase substantially relieved of inhibition by L-isoleucine and / or L-valine, further preferably, the gene of acetolactate synthase / acetylhydroxybutyrate synthase substantially relieved of inhibition by L-isoleucine and / or L-valine is one or more of a relieved ilvIH gene, a relieved ilvBN gene, a relieved ilvGM gene, and a relieved alsS gene;

[0034] The expression of at least one of the DNA molecules is enhanced.

[0035] The third aspect of the present invention provides: a genetically engineered strain for co-producing leucine and isoleucine or co-producing three branched-chain amino acids, wherein the strain expresses:

[0036] 1) at least one DNA molecule encoding a polypeptide that catalyzes the synthesis of α-isopropylmalic acid;

[0037] 2) at least one DNA molecule encoding a polypeptide that catalyzes the isomerization of α-isopropylmalic acid to β-isopropylmalic acid;

[0038] 3) at least one DNA molecule encoding a polypeptide that catalyzes the dehydrogenation of β-isopropylmalate to produce α-ketoisocaproate;

[0039] 4) at least one DNA molecule encoding a polypeptide that catalyzes the transamination of an aromatic amino acid to α-ketoisocaproic acid to produce leucine;

[0040] Preferably, the DNA molecule of 1) is the isopropylmalate synthase encoding gene leuA, preferably, isopropylmalate synthase encoding gene that relieves feedback inhibition of L-leucine, and further preferably isopropylmalate synthase encoding gene leuAm with G479C mutation;

[0041] Preferably, the DNA molecule of 2) is the isopropylmalate dehydratase encoding gene leuCD;

[0042] Preferably, the DNA molecule of 3) is the isopropylmalate dehydrogenase encoding gene leuB;

[0043] Preferably, the DNA molecule of 4) is the aromatic amino acid transaminase gene tyrB;

[0044] as well as

[0045] 5) at least one DNA molecule encoding a polypeptide that catalyzes the production of ketobutyric acid from threonine; preferably, the DNA molecule is a gene for threonine deaminase that is substantially free of inhibition by L-isoleucine, and more preferably, the gene for threonine deaminase that is substantially free of inhibition by L-isoleucine is a released ilvA gene; and / or, the DNA molecule is a gene for threonine dehydratase (tdcB);

[0046] and

[0047] 6) at least one DNA molecule encoding a polypeptide, the DNA molecule being a gene of acetolactate synthase / acetylhydroxybutyrate synthase, preferably the gene being a gene of acetolactate synthase / acetylhydroxybutyrate synthase substantially relieved of inhibition by L-isoleucine and / or L-valine, further preferably, the gene of acetolactate synthase / acetylhydroxybutyrate synthase substantially relieved of inhibition by L-isoleucine is one or more of a relieved ilvIH gene, a relieved ilvBN gene, a relieved ilvGM gene, and a relieved alsS gene;

[0048] The expression of at least one of the DNA molecules is enhanced.

[0049] The genetically engineered strain as described in any one of the first to third aspects of the present invention, further comprises one or more of a threonine dehydratase gene (tdcB), a threonine transporter gene, a branched-chain amino acid transporter gene, an acetohydroxyacid isomerase gene (ilvC), a dihydroxyacid dehydratase gene (ilvD), a branched-chain amino acid aminotransferase gene (ilvE), and a branched-chain amino acid dehydrogenase gene.

[0050] As described in any one of the first to third aspects of the present invention, the genetically engineered strain maximizes the carbon metabolic flow to pyruvate and directs it to the target amino acid through genetic engineering modification, and the activities of at least the following genes ldhA, adhE, pta, and poxB are weakened or eliminated separately or simultaneously.

[0051] The genetically engineered strain as described in any one of the first to third aspects of the present invention, wherein the engineered strain is wild or genetically engineered Escherichia coli (E. coli), Bacillus (Bacillus), Brevibacterium (Brevibacterium), Yeast (Yeast), Corynebacterium (Corynebacterium) or Streptomyces (Streptomyces).

[0052] A genetically engineered strain as described in any one of the first to third aspects of the present invention, wherein the engineered strain has an ability to absorb BCAA from the culture medium that is more than 30% lower than that of the wild-type strain, and or the engineered strain has an ability to secrete BCAA from the intracellular to the extracellular efflux that is more than 30% higher than that of the wild-type strain; preferably, the branched-chain amino acid-cation symporter activity or the branched-chain amino acid-phenylalanine ABC transport system activity in the engineered strain is weakened or eliminated separately or simultaneously, and further preferably, the brnQ gene is knocked out or mutated or the expression of the brnQ gene is inhibited; further preferably, one or more of the livJKHMGF genes are knocked out or mutated or the expression of one or more of the livJKHMGF genes is inhibited; preferably, the expression of the transporter (efflux) gene ygaZH, brnEF, yjeH or LeuE is enhanced, and further preferably, the method of enhancing the expression of ygaZH, brnEF or yjeH includes overexpressing endogenous or exogenous corresponding genes, or replacing or knocking out inhibitory elements with strong promoters.

[0053] For the genetically engineered strain as described in any one of the first to third aspects of the present invention, the method for constructing the engineered strain includes plasmid expression, genome integration such as homologous recombination mediated by CRISPR, lambda-red, phage, etc.

[0054] The fourth aspect of the present invention provides: use of the genetically engineered strain according to any one of the first to third aspects of the present invention in the co-production of branched-chain amino acids, or use in the preparation of foods, health products or feeds containing branched-chain amino acids.

[0055] The fifth aspect of the present invention provides: a method for co-producing branched-chain amino acids, characterized in that it comprises the steps of:

[0056] (i) culturing the genetically engineered strain according to any one of the first to third aspects of the present invention in a culture medium;

[0057] (ii) isolating branched-chain amino acids from the culture of (i).

[0058] In a specific embodiment of the present invention, a raw material that can be converted into 2-ketobutyric acid is added to the culture medium; preferably, the raw material that can be converted into 2-ketobutyric acid includes one or more of threonine, fumaric acid, aspartic acid, homoserine, propionic acid, and diaminobutyric acid.

[0059] Beneficial effects:

[0060] The present invention realizes the co-production of two or three branched-chain amino acids, and the co-production process improves the overall glucose utilization rate by at least 30%; the genetically engineered strain of the present invention has a short fermentation cycle, high yield, high conversion rate, and high production application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 : Biosynthetic pathway of branched-chain amino acids (L-isoleucine, L-valine and L-leucine) (italics represent enzymes that catalyze the biochemical reaction; underlines represent fermentation substrates / raw materials that can be added) DETAILED DESCRIPTION

[0062] The following will be further described in detail with reference to specific examples. It should be understood that the following examples are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies realized based on the foregoing of the present invention are encompassed within the scope that the present invention is intended to protect.

[0063] IDefinition of Terms

[0064] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0065] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0066] The articles "a," "an," and "the" are used herein to refer to one or more than one of the grammatical object of the article.

[0067] The use of alternatives (eg, "or") should be understood to mean one, two, or any combination of the alternatives. The term "and / or" should be understood to mean one or both of the alternatives.

[0068] As used herein and unless otherwise specified, the term "about" when referring to a measurable value such as an amount, a time period, etc., is meant to include variations of ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the given value, as long as such variations are suitable for practicing the disclosed methods.

[0069] As used herein, the term "gene synthesis" refers to the production using recombinant DNA technology or the acquisition using synthetic DNA or amino acid sequence technology available and known in the art.

[0070] "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA or mRNA as a template for the synthesis of other polymers and macromolecules in biological processes, which have any of a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, which is equivalent to the mRNA sequence and is usually provided in the sequence listing, and the non-coding strand, which is used as a template for transcribing a gene or cDNA, can be referred to as encoding a protein or other product of that gene or cDNA.

[0071] As used herein, the term "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0072] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside of an organism, cell, tissue or system.

[0073] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0074] As used herein, the term "nucleic acid construct" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The nucleic acid construct includes sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Nucleic acid constructs include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0075] Unless otherwise specified, "a polynucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, to the extent that the nucleotide sequence encoding the protein may contain intron(s) in certain versions.

[0076] As used herein, the term "operably connected" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that produces expression of the latter. For example, when a first nucleic acid sequence is in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably connected to the second nucleic acid sequence. For example, if a promoter affects transcription or expression of a coding sequence, the promoter is operably connected to the coding sequence. Typically, operably connected DNA sequences are adjacent, wherein two protein coding regions must be connected in the same reading frame.

[0077] The term "polynucleotide" as used herein is defined as a chain of nucleotides. In addition, nucleic acids are polymers of nucleotides. Therefore, nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. Polynucleotides as used herein 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 nucleic acid sequences from recombinant libraries or cell genomes using common cloning techniques and PCR™, etc., and synthetic means.

[0078] In various illustrative embodiments, polynucleotides contemplated herein include, but are not limited to, polynucleotides comprising expression vectors, viral vectors, transfer plasmids, expression cassettes, and polynucleotides encoding polypeptides of cytokine antibodies or antibody fragments or antigen binding fragments.

[0079] As disclosed elsewhere herein or as known in the art, regardless of the length of the coding sequence itself, the 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, so that its overall length can vary significantly. Therefore, it is contemplated that polynucleotide fragments of almost any length can be employed, with the total length preferably being limited by ease of preparation and use in the intended recombinant DNA protocol.

[0080] As used herein, the term "vector" is a material composition that includes an isolated nucleic acid, and it can be used to transfer an isolated nucleic acid to the interior of a cell. The nucleic acid transferred is usually connected to, for example, inserted into a carrier nucleic acid molecule. The vector may include a sequence that guides the autonomous replication in the cell or may include a sequence that is sufficient to allow 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. Therefore, the term "vector" includes autonomously replicating plasmids or viruses.

[0081] Illustrative methods for delivering the polynucleotides contemplated in particular embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistic methods, virosomes, liposomes, immunoliposomes, polycationic or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene guns, and heat shock, among others.

[0082] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are those non-translated regions of the vector - origin of replication, selection cassette, promoter, enhancer, translation start signal intron, post-transcriptional regulatory elements, polyadenylation sequence, 5' and 3' untranslated regions - which interact with host cell proteins for transcription and translation. The length and specificity of such elements can vary. Depending on the vector system and host utilized, any number of suitable transcription and translation elements may be used, including ubiquitous promoters and inducible promoters.

[0083] In a specific embodiment, the polynucleotide is a vector including but not limited to expression vectors and viral vectors and comprises exogenous, endogenous or heterologous control sequences, such as promoters and / or enhancers. An "endogenous" control sequence is a sequence naturally connected to a given gene in a genome. An "exogenous" control sequence is a sequence that is placed in juxtaposition with a gene by genetic manipulation (i.e., molecular biotechnology) so that the transcription of that gene is guided by the enhancer / promoter connected. A "heterologous" control sequence is an exogenous sequence from a species different from the cell of the genetic manipulation. A "synthetic" control sequence can include elements of one or more endogenous and / or exogenous sequences and / or provide the best promoter and / or enhancer activity for use in a specific gene therapy in vitro or in silico determined sequence.

[0084] As used herein, the term "gene knockout" is a technique for inactivating or deleting a specific gene in an organism by a certain pathway. It is mainly achieved by using the principle of DNA homologous recombination to replace the target gene fragment with a designed homologous fragment, thereby achieving the purpose of gene knockout. The introduction of homologous fragments into an organism can also be achieved by DNA electroporation or phage transduction (such as Escherichia coli).

[0085] As used herein, the term "promoter" refers to a recognition site for a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter.

[0086] The term "enhancer" refers to a segment of DNA that contains a sequence that can provide enhanced transcription and, in some cases, can function independently of its orientation relative to another control sequence. An enhancer can function cooperatively or additively with a promoter and / or another enhancer element. The term "promoter / enhancer" refers to a segment of DNA that contains a sequence that can provide the functions of both a promoter and an enhancer.

[0087] The term "conditional expression" may refer to any type of conditional expression, including but not limited to: inducible expression; repressible expression; expression in cells or tissues having a specific 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, for example, expression is controlled by subjecting a cell, tissue, organism, etc. to a treatment or condition that causes the polynucleotide to be expressed or that increases or decreases the expression of a polynucleotide encoded by the polynucleotide of interest.

[0088] The term "enhanced expression" or "expression is enhanced" refers to increasing the expression of a corresponding gene, or enhancing the expression of a corresponding enzyme or protein, or increasing the activity of a corresponding enzyme or protein by at least one of the following means:

[0089] 1) Enhanced by introducing a plasmid carrying the gene;

[0090] 2) Enhanced by increasing the copy number of the gene on the chromosome;

[0091] 3) Enhanced by changing the promoter sequence of the gene on the chromosome;

[0092] 4) Enhanced by operably linking a strong promoter to the gene;

[0093] 5) Enhancement by introduction of enhancers;

[0094] 6) Enhancement by using genes or alleles encoding high activity of the corresponding enzymes or proteins.

[0095] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid or estrogen receptors, metallothionein promoters; MX-1 promoter, the "gene switch" mifepristone-regulatable system, tetracycline-dependent regulatory systems, etc.

[0096] In certain embodiments, the genetically modified cell comprises a polynucleotide further comprising a positive marker for the selection of a cell that belongs to an external negative selective phenotype. The positive selective marker can be a gene that, when introduced into a host cell, expresses a dominant phenotype that allows positive selection of cells carrying the gene. This type of gene is known in the art.

[0097] In one embodiment, the positive selectable marker and the negative selectable marker are linked so that loss of the negative selectable element is also accompanied by loss of the positive selectable marker. In a specific embodiment, the positive and negative selectable markers are fused so that loss of one necessarily results in loss of the other.

[0098] As used herein, the term "transfection" or "transformation" or "transduction" 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 a strain that has been transfected, transformed or transduced with exogenous nucleic acid. The strain includes the primary and its progeny.

[0099] The recipient strain can be Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus megaterium, Vibrio atriegens, Bacillus amyloliquefaciens, or Saccharomyces cerevisiae, etc.

[0100] II. Reagents and Methods

[0101] Unless otherwise specified, the raw materials and reagents used in the following examples are generally commercially available or can be prepared by known methods.

[0102] LB medium: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.2 adjusted with 30% NaOH, 1×10 5 Pa was sterilized for 20 min. When making the plate, 1.5% agar was added. When screening for resistance, 100 mg / L of ampicillin and / or 50 mg / L of kanamycin were added according to the type of resistance gene.

[0103] Determination of amino acid concentration: L-isoleucine, L-leucine and L-valine standards were purchased from Sigma-Aldrich (www.sigmaaldrich.cn). Take 1 mL of fermentation broth, centrifuge at 10000 r / min for 5 min to remove the bacteria, filter the filtrate through a filter membrane with a pore size of 0.22 μm, dilute it by appropriate times, and use high-performance liquid chromatography HPLC (https: / / www.agilent.com / library / applications / 5990-4547EN.pdf) to determine the concentration of branched-chain amino acids in the sample. The high-performance liquid chromatograph is Shimadzu Nexera LC-40, and the chromatographic column is Agilent ZORBAX Eclipse Plus C18, 4.6×250 mm5μm. The detector is a DAD diode array detector with a detection wavelength of 338 nm and a reference wavelength of 390 nm. The mobile phase composition, ratio change, flow rate and column temperature are set according to the above method.

[0104] Unless otherwise specified, the genes (enzymes) involved in leucine synthesis in each embodiment are shown in Table 1, and the genes (enzymes) involved in valine and isoleucine synthesis are shown in Table 2.

[0105] Table 1: Genes (enzymes) related to leucine synthesis in various examples

[0106]

[0107] Table 2: Genes (enzymes) related to valine or isoleucine synthesis in various embodiments

[0108]

[0109] The biomaterials constructed by the present invention are shown in the following table:

[0110] Table 3: Biomaterials constructed in various examples

[0111]

[0112] Example

[0113] Example 1: Construction of recombinant plasmids pZE-ilvIHDCmEygaZH, pZE-alsS-ilvDCmEygaZH, pZS-ilvAIH and pZA-leuAmBCD-tyrB-leuE

[0114] The ilvDCmEygaZH and ilvIH fragments were amplified from plasmids pZE-ilvDCmEygaZH and pZA-ilvAIH by PCR, respectively. The gene fragments were seamlessly connected to the pZElac vector PCR fragment in one step using a recombinant cloning kit to obtain the recombinant plasmid pZE-ilvIHDCmEygaZH.

[0115] The acetolactate synthase gene alsS (Genbank GeneID: 936852) was cloned from the Bacillus subtilis 168 genome by PCR, and the ilvDCmEygaZH fragment was amplified from the plasmid pZE-ilvDCmEygaZH by PCR. The gene fragment was seamlessly connected to the pZElac vector PCR fragment in one step using a recombinant cloning kit to obtain the recombinant plasmid pZE-alsS-ilvDCmEygaZH.

[0116] The ilvAIH fragment was amplified from the plasmid pZA-ilvAIH by PCR, and the pZSlac vector (pSC101 replicon, spectinomycin resistance) fragment was amplified from the plasmid pZS-Pcsc-cscBKA (Chinese patent application number 2022116667471) by PCR. The ilvAIH gene fragment was seamlessly connected to the pZSlac vector in one step using a recombinant cloning kit to obtain the recombinant plasmid pZS-ilvAIH.

[0117] Using the E. coli BW25113 genome as a template, PCR cloned the leuABCD gene cluster fragment, aromatic amino acid transaminase tyrB gene fragment and leucine efflux protein leuE gene fragment, and used a recombinant cloning kit to seamlessly connect the gene fragments to the pZAlac vector fragment obtained by PCR in one step to obtain the recombinant plasmid pZA-leuABCD-tyrB-leuE. Using pZA-leuABCD-tyrB-leuE as a template, designed mutation primers for circular PCR, mutated the 479th glycine of isopropylmalate synthase LeuA to cysteine, relieved the feedback inhibition of LeuA by L-leucine, and obtained the recombinant plasmid pZA-leuAmBCD-tyrB-leuE.

[0118] Plasmids pZE-ilvCmDE-ygaZH and pZA-ilvAIH have been mentioned and used in Chinese patent (publication number: CN114410701A).

[0119] pZE-ilvIHDCmEygaZH and pZA-leuAmBCD-tyrB-leuE were co-transformed into the strain DV10 (ΔldhAΔptaΔpoxBΔadhEΔpflBΔmgsAΔfrdAΔdadAΔavtAΔilvE) (Chinese Patent Publication No.: CN114410701A) based on the Escherichia coli BW25113 (DSM27469 / CGSC7636) background, and the resulting strain was named M-LV1; pZE-alsS-ilvDCmEygaZH and pZA-leuAmBCD-tyrB-leuE were co-transformed into the Escherichia coli DV10 strain, and the resulting strain was named M-LV2. At the same time, the plasmids pZA-leuAmBCD-tyrB-leuE, pZE-ilvIHDCmEygaZH and pZE-alsS-ilvDCmEygaZH were transformed into the DV10 strain respectively to obtain the control strains M-LC, M-VC1 and M-VC2.

[0120] pZE-ilvCmDE-ygaZH, pZS-ilvAIH and pZA-leuAmBCD-tyrB-leuE were co-transformed into E. coli DV10 strain, and the resulting strain was named M-LIV. pZE-ilvCmDE-ygaZH and pZS-ilvAIH were co-transformed into E. coli DV10 strain to obtain the control strain M-LIVC.

[0121] Example 2: Fermentation of recombinant strains to produce branched-chain amino acids (leucine and valine)

[0122] Fermentation was carried out using a fermentation medium (4% glucose, 0.5% yeast extract, 0.12% MgSO4, 0.01% CaCl2, 1% NH4Cl, 0.05% NaCl, 0.6% Na2HPO4, 0.3% KH2PO4, 0.0005% VB1. Adjust pH to 7.0-7.2 with NaOH and hydrochloric acid, and sterilize at 115°C for 15 min). The fermentation process and method are as follows: strains M-LV1 and M-LV2 were inoculated into LB medium containing appropriate concentrations of ampicillin and kanamycin, respectively, and cultured at 37°C for 10 hours at a speed of 240 rpm. The LB culture was inoculated at a 10% (V / V) amount into a 150 mL shake flask containing the above 30 mL fermentation medium containing an appropriate concentration of antibiotics (containing 40 g / LCaCO3 as a pH stabilizer), and sealed with a breathable membrane; cultured at 37°C and 220 rpm until OD 600The fermentation temperature was 2, IPTG was added for induction (final concentration was 0.3 mM), and the culture was continued at 30 ° C and 220 rpm until 48 h to stop fermentation. The yield of each branched-chain amino acid is shown in Table 4. According to the fermentation results, we can see that although valine and leucine partially overlap in the upstream of the synthesis pathway, the total utilization rate of the carbon source (glucose) has been greatly improved after the co-production of the two. Compared with the single production of valine, the utilization rate of the carbon source (glucose) during co-production increased by 35.2%-41.6%; in addition, in actual industrial production, when valine and leucine are produced in the form of a final mixed product (such as feed additives, etc.), our strains and processes will bring cost advantages in energy consumption, labor, operation and separation.

[0123] Table 4: Branched-chain amino acid fermentation results of each strain in Example 2

[0124]

[0125] Example 3: Fermentation of recombinant strains to produce branched-chain amino acids (leucine, isoleucine and valine)

[0126] Fermentation was carried out using a fermentation medium (4% glucose, 2% threonine, 0.5% yeast extract, 0.12% MgSO4, 0.01% CaCl2, 1% NH4Cl, 0.05% NaCl, 0.6% Na2HPO4, 0.3% KH2PO4, 0.0005% VB1. Adjust the pH to 7.0-7.2 with NaOH and hydrochloric acid, and sterilize at 115°C for 15 minutes). The fermentation process and method are as follows: The strain M-LIV was inoculated into an LB medium containing an appropriate concentration of antibiotics, and cultured at 37°C for 10 hours at a speed of 240 rpm. The LB culture was inoculated at a 10% (V / V) amount into a 250 mL shake flask (containing 40 g / LCaCO3 as a pH stabilizer) containing the above 30 mL fermentation medium containing an appropriate concentration of antibiotics, and sealed with a breathable membrane; cultured at 37°C and 220 rpm until OD 600 2, IPTG was added for induction (final concentration was 0.3 mM), and the culture was continued at 30°C and 220 rpm until 48 hours to stop fermentation. The yield of each branched-chain amino acid is shown in Table 5. Similar to Example 2, the co-production of three branched-chain amino acids achieved a significant improvement in the total utilization rate of the carbon source (glucose); the utilization rate of the carbon source (glucose) was increased by 37.5% during co-production; in addition, in actual industrial production, when the three branched-chain amino acids are produced in the form of a final mixed product (such as a feed additive, etc.), our strains and processes will also bring cost savings in energy consumption, labor, operation and separation.

[0127] Table 5: Branched-chain amino acid fermentation results of each strain in Example 3

[0128]

Claims

1. An engineered strain for co-producing valine and leucine, the strain expressing: 1) G479C mutation in the isopropylmalate synthase encoding gene leuAm; 2) Isopropylmalate dehydratase encoding gene leuCD; 3) Isopropylmalate dehydrogenase encoding gene leuB; 4) Aromatic amino acid transaminase gene tyrB; as well as 5) A DNA molecule encoding a polypeptide, wherein the DNA molecule comprises the following combination: The acetolactate synthase gene alsS, the acetohydroxyacid reductoisomerase gene ilvCm containing the L67E, R68F, and K75E mutations, the dihydroxyacid dehydratase gene ilvD, the branched-chain amino acid aminotransferase gene ilvE, and the branched-chain amino acid efflux gene ygaZH; The expression of the DNA molecule is enhanced; The engineered strain is Escherichia coli, and the following genes are knocked out: ldhA, pta, poxB, adhE, pflB, mgsA, frdA, dadA, avtA and ilvE.

2. The engineered strain according to claim 1, wherein the method for constructing the engineered strain comprises plasmid expression or genome integration.

3. Use of the engineered strain according to claim 1 in the co-production of valine and leucine, or in the preparation of food, health products or feed containing valine and leucine.

4. A method for co-producing valine and leucine, characterized in that: Includes steps: (i) culturing the engineered strain according to claim 1 in a culture medium; (ii) isolating valine and leucine from the culture of (i).

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