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WO2024222943A1 - Construction et utilisation d'une souche d'ingénierie génétique capable de réaliser la co-production d'acides aminés à chaîne ramifiée - Google Patents

Construction et utilisation d'une souche d'ingénierie génétique capable de réaliser la co-production d'acides aminés à chaîne ramifiée Download PDF

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WO2024222943A1
WO2024222943A1 PCT/CN2024/090312 CN2024090312W WO2024222943A1 WO 2024222943 A1 WO2024222943 A1 WO 2024222943A1 CN 2024090312 W CN2024090312 W CN 2024090312W WO 2024222943 A1 WO2024222943 A1 WO 2024222943A1
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gene
dna molecule
encoding
synthase
relieved
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Chinese (zh)
Inventor
张科春
黄浩
倪梦寒
王靖宇
孙村民
朱湘成
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Mint Biotechnologies Co Ltd
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Mint Biotechnologies Co Ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • 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.
  • Branched-chain amino acids refer to neutral amino acids with branched fatty hydrocarbon chains on the ⁇ -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.
  • 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.
  • 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.
  • branched-chain amino acids There are many methods for producing branched-chain amino acids, including extraction, chemical synthesis, and microbial fermentation.
  • the extraction and chemical synthesis methods are difficult to achieve industrial production due to limited raw material sources and high production costs.
  • microbial fermentation is the main method for industrial production of branched-chain amino acids.
  • Microbial fermentation has low raw material costs and high reaction conditions. Mild, easy to achieve large-scale production.
  • the current technology of 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 a lot of miscellaneous acids, which has become the primary factor affecting the industrial application of branched-chain amino acids in my country.
  • the yield and conversion rate of its production strains are still low, and its current output can no longer meet the growing market demand.
  • 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.
  • 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.
  • 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:
  • 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:
  • At least one DNA molecule encoding a polypeptide the DNA molecule being a gene of acetolactate synthase/acetylhydroxybutyrate synthase, preferably the gene being an acetolactate synthase/acetylhydroxybutyrate synthase substantially freed from inhibition by L-isoleucine and/or L-valine;
  • Acetohydroxybutyrate synthase gene 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;
  • the expression of at least one of the DNA molecules is enhanced.
  • 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.
  • the DNA molecule of 2) is the isopropylmalate dehydratase encoding gene leuCD.
  • the DNA molecule of 3) is the isopropylmalate dehydrogenase encoding gene leuB.
  • the DNA molecule of 4) is the aromatic amino acid transaminase gene tyrB.
  • 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.
  • tdcB threonine dehydratase gene
  • a threonine transporter gene a branched-chain amino acid transporter gene
  • an acetohydroxyacid isomerase gene acetohydroxyacid isomerase gene
  • ilvD dihydroxyacid dehydratase gene
  • ilvE branched-chain amino acid aminotransferase gene
  • a branched-chain amino acid dehydrogenase gene branched-chain amino acid dehydr
  • the second aspect of the present invention provides: an engineered strain for co-producing valine and leucine, wherein the strain expresses:
  • 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;
  • the DNA molecule of 2) is the isopropylmalate dehydratase encoding gene leuCD;
  • the DNA molecule of 3) is the isopropylmalate dehydrogenase encoding gene leuB;
  • the DNA molecule of 4) is the aromatic amino acid transaminase gene tyrB;
  • 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, and further preferably, the gene of acetolactate synthase/acetylhydroxybutyrate synthase substantially relieved of inhibition by L-isoleucine and/or L-valine is a relieved ilvIH gene, a relieved ilvBN gene, a relieved One or more of the ilvGM gene of the removal type and the alsS gene of the release type;
  • the expression of at least one of the DNA molecules is enhanced.
  • 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:
  • 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;
  • the DNA molecule of 2) is the isopropylmalate dehydratase encoding gene leuCD;
  • the DNA molecule of 3) is the isopropylmalate dehydrogenase encoding gene leuB;
  • the DNA molecule of 4) is the aromatic amino acid transaminase gene tyrB;
  • 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);
  • 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;
  • the expression of at least one of the DNA molecules is enhanced.
  • 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.
  • tdcB threonine dehydratase gene
  • a threonine transporter gene a branched-chain amino acid transporter gene
  • an acetohydroxyacid isomerase gene acetohydroxyacid isomerase gene
  • ilvD dihydroxyacid dehydratase gene
  • ilvE branched-chain amino acid aminotransferase gene
  • 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.
  • E. coli Escherichia coli
  • Bacillus Bacillus
  • Brevibacterium Brevibacterium
  • Yeast Yeast
  • Corynebacterium Corynebacterium
  • Streptomyces Streptomyces
  • 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,
  • the method for constructing the engineered strain includes plasmid expression, genome integration such as homologous recombination mediated by CRISPR, lambda-red, phage, etc.
  • 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.
  • 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:
  • 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.
  • 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.
  • FIG. 1 Biosynthetic pathway of branched-chain amino acids (L-isoleucine, L-valine, and L-leucine) (italics represent catalytic The enzyme of this biochemical reaction; the underline represents the fermentation substrate/raw material that can be added)
  • 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.
  • 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.
  • 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.
  • 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.
  • endogenous refers to any substance that originates from or is produced within an organism, cell, tissue, or system.
  • exogenous refers to any substance that is introduced from or produced outside of an organism, cell, tissue or system.
  • expression is defined as the transcription and/or translation of a specific nucleotide sequence driven by its promoter.
  • 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.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.
  • 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.
  • operably connected refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that produces expression of the latter.
  • first nucleic acid sequence 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.
  • promoter affects transcription or expression of a coding sequence
  • the promoter is operably connected to the coding sequence.
  • operably connected DNA sequences are adjacent, wherein two protein coding regions must be connected in the same reading frame.
  • nucleotide as used herein is defined as a chain of nucleotides.
  • 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 PCRTM, etc., and synthetic means.
  • 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.
  • 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.
  • promoters and/or enhancers 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 cod
  • 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.
  • 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.
  • “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.
  • any number of suitable transcription and translation elements may be used, including ubiquitous promoters and inducible promoters.
  • 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.
  • 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).
  • 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.
  • 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.
  • promoter/enhancer refers to a segment of DNA that contains a sequence that can provide the functions of both a promoter and an enhancer.
  • conditional expression may refer to any type of conditional expression, including but not limited to: inducible expression; repressible 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, 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 that increases or decreases the expression of a polynucleotide encoded by the polynucleotide of interest.
  • enhanced expression 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:
  • 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.
  • 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.
  • 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.
  • the positive and negative selectable markers are fused so that loss of one necessarily results in loss of the other.
  • 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.
  • the recipient strain can be Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus megaterium, Vibrio natriegens, Bacillus amyloliquefaciens, or Saccharomyces cerevisiae, etc.
  • LB medium 1% peptone, 0.5% yeast extract, 1% NaCl, adjusted to pH 7.2 with 30% NaOH, sterilized at 1 ⁇ 10 5 Pa for 20 min. Add 1.5% agar when making plates. When screening for resistance, add ampicillin and/or kanamycin at a final concentration of 100 mg/L according to the type of resistance gene.
  • 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 ⁇ m, dilute it to an appropriate multiple, 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.
  • HPLC https://www.agilent.com/library/applications/5990-4547EN.pdf
  • the high-performance liquid chromatograph is Shimadzu Nexera LC-40, and the chromatographic column is Agilent ZORBAX Eclipse Plus C18, 4.6 ⁇ 250mm 5 ⁇ m.
  • 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.
  • 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.
  • Table 1 Genes (enzymes) related to leucine synthesis in various examples
  • Table 2 Genes (enzymes) related to valine or isoleucine synthesis in various embodiments
  • biomaterials constructed by the present invention are shown in the following table:
  • Example 1 Construction of recombinant plasmids pZE-ilvIHDCmEygaZH, pZE-alsS-ilvDCmEygaZH, pZS-ilvAIH and pZA-leuAmBCD-tyrB-leuE
  • 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.
  • the acetolactate synthase gene alsS (Genbank GeneID: 936852) was cloned from the genome of Bacillus subtilis 168 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.
  • 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.
  • 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.
  • mutant primers were designed for circular PCR to mutate the glycine at position 479 of isopropylmalate synthase LeuA to cysteine, thereby relieving the feedback inhibition of LeuA by L-leucine and obtaining the recombinant plasmid pZA-leuAmBCD-tyrB-leuE.
  • Plasmids pZE-ilvCmDE-ygaZH and pZA-ilvAIH have been mentioned and used in Chinese patent (publication number: CN114410701A).
  • pZE-ilvIHDCmEygaZH and pZA-leuAmBCD-tyrB-leuE were co-transformed into the strain DV10 ( ⁇ ldhA ⁇ pta ⁇ poxB ⁇ adhE ⁇ pflB ⁇ mgsA ⁇ frdA ⁇ dadA ⁇ avtA ⁇ ilvE) (China Patent Publication No.: CN114410701A) based on the Escherichia coli BW25113 (DSM 27469/CGSC 7636) 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.
  • 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.
  • 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.
  • Example 2 Fermentation of recombinant strains to produce branched-chain amino acids (leucine and valine)
  • Fermentation was performed using a fermentation medium (containing 4% glucose, 0.5% yeast extract, 0.12% MgSO 4 , 0.01% CaCl 2 , 1% NH 4 Cl, 0.05% NaCl, 0.6% Na 2 HPO4 , 0.3% KH 2 PO 4 , 0.0005% VB1, pH adjusted to 7.0-7.2 with NaOH and hydrochloric acid, and sterilized 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 h at a speed of 240 rpm.
  • the LB culture was inoculated at a 10% (V/V) amount into 150 mL of the above 30 mL fermentation medium containing appropriate concentrations of antibiotics.
  • mL shake flask containing 40g/L CaCO 3 as pH stabilizer
  • a breathable membrane sealed with a breathable membrane; cultured at 37°C, 220rpm until OD 600 is 2, IPTG induction (final concentration is 0.3mM), and continued to culture at 30°C, 220rpm until 48h to stop fermentation.
  • the yield of each branched-chain amino acid is shown in Table 4.
  • Example 3 Fermentation of recombinant strains to produce branched-chain amino acids (leucine, isoleucine and valine)
  • Fermentation was carried out using a fermentation medium (including 4% glucose, 2% threonine, 0.5% yeast extract, 0.12% MgSO 4 , 0.01% CaCl 2 , 1% NH 4 Cl , 0.05% NaCl, 0.6% Na 2 HPO4 , 0.3% KH 2 PO 4 , 0.0005% VB1, pH adjusted to 7.0-7.2 with NaOH and hydrochloric acid, sterilized at 115°C for 15 min).
  • the fermentation process and method are as follows: strain M-LIV was inoculated into LB medium containing appropriate concentrations of antibiotics, cultured at 37°C for 10 h, and the rotation speed was 240 rpm.
  • the LB culture was inoculated at 10% (V/V) into a 250mL shake flask containing the above 30mL fermentation medium containing an appropriate concentration of antibiotics (containing 40g/L CaCO 3 as a pH stabilizer), and the air-permeable membrane was sealed; cultured at 37°C, 220rpm until OD 600 was 2, IPTG was added for induction (final concentration of 0.3mM), and continued to culture at 30°C, 220rpm until 48h to stop fermentation.
  • the yield of each branched-chain amino acid is shown in Table 5.
  • the co-production of the three branched-chain amino acids has 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.

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Abstract

L'invention concerne la construction et l'utilisation d'une souche d'ingénierie génétique capable de réaliser la co-production d'acides aminés à chaîne ramifiée. La souche d'ingénierie génétique peut réaliser la co-production d'acides aminés à chaîne ramifiée (BCAA) tels que la valine, la leucine et l'isoleucine au moyen d'un procédé de fermentation. Une souche d'ingénierie génétique est obtenue à l'aide d'un procédé d'ingénierie génétique. En introduisant une voie de synthèse de leucine dans une cellule hôte d'Escherichia coli capable de produire de l'isoleucine et/ou de la valine, et en exprimant un gène de codant pour une isopropylmalate synthétase LeuAm, un gène codant pour une isopropylmalate déshydrogénase leuB, un gène codant pour une isopropylmalate déshydratase leuCD, un gène de transaminase d'acide aminé aromatique tyrB et un gène de protéine d'expulsion de leucine LeUE qui soulagent l'inhibition de rétroaction de la L-leucine, la souche d'ingénierie génétique réalise finalement la coproduction de deux ou trois acides aminés à chaîne ramifiée, et le procédé de co-production augmente le taux d'utilisation du glucose total d'au moins 30 %. La souche d'ingénierie génétique a une courte période de fermentation, un rendement élevé et un taux de conversion élevé, ayant ainsi une valeur d'application de production élevée.
PCT/CN2024/090312 2023-04-28 2024-04-28 Construction et utilisation d'une souche d'ingénierie génétique capable de réaliser la co-production d'acides aminés à chaîne ramifiée Pending WO2024222943A1 (fr)

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