An optimized method for constructing an L-isoleucine production strain and its application

Abstract

The present invention provides a construction method and application of an optimized L-isoleucine production strain. By further expressing threonine endonucleic acid transporter proteins of different sources and types in the isoleucine production strain, the ability of the cell to absorb threonine is enhanced, thereby increasing the yield and content of L-isoleucine in the fermentation product and reducing the relative yield and content of the byproduct miscellaneous acid (valine). Further, the cell efflux threonine transporter is deleted to obtain a genetically engineered high-yield strain. By fermenting and culturing the engineered strain, threonine is added during the culturing process, and L-isoleucine is isolated from the culture.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to an optimized L-isoleucine strain construction method and application thereof. Background Art

[0002] L-isoleucine, also known as "L-isoleucine", is one of the three essential branched-chain amino acids (BCAA) for the human body. It has multiple physiological functions, participates in the synthesis of hormones and enzymes in the human body, promotes protein production and inhibits its decomposition, and plays an important regulatory role in human and animal health. Therefore, it has a wide range of applications and commercial value in the food, medicine, feed and other industries.

[0003] At present, the main method for producing L-isoleucine in industry is microbial fermentation. Microbial fermentation is a method of biosynthesizing and overaccumulating L-isoleucine by using the metabolic pathway of microorganisms, including direct fermentation and precursor addition fermentation. Among them, the direct fermentation method relies on the ability of microorganisms to synthesize the amino acids they need. Through the mutagenesis of specific microorganisms, nutritional deficiency and amino acid structural analog resistance mutants are selected to relieve the feedback inhibition and repression in metabolic regulation, so as to achieve the purpose of overaccumulating a certain amino acid. At present, most strains for direct fermentation to produce L-isoleucine are bred by mutagenesis of glutamate-producing bacteria (Breibacterium flavum, Corynebacterium glutamicum, Brevibacterium lactofermentum, etc.). The precursor addition fermentation method is also called microbial conversion method. Microorganisms use glucose as the fermentation carbon source and energy source, and add specific precursor substances during the fermentation process to avoid the feedback regulation in the amino acid biosynthesis pathway, and efficiently convert it into the target amino acid through the action of microorganisms. For L-isoleucine, its precursor substances mainly include α-aminobutyric acid, α-hydroxybutyric acid, α-ketobutyric acid and threonine, and the microorganisms used mainly include Escherichia coli, Bacillus and Pseudomonas.

[0004] At present, many domestic companies have produced L-isoleucine by fermentation, but there are still problems such as low acid production level, low conversion rate, many by-products and poor extraction technology of fermentation products. Therefore, improving the fermentation strains and technical processes of L-isoleucine, increasing the acid production level and conversion rate, and then reducing the difficulty of extraction are of great significance to promoting the sustainable development of the L-isoleucine industry and enhancing the market competitiveness of products. Summary of the invention

[0005] In order to improve the above technical problems, the present invention provides a method for increasing the yield of L-isoleucine, which solves the problems of low acid production level, slow acid production and high impurity acid in the prior art.

[0006] The present invention provides an engineered strain for producing isoleucine, wherein the threonine / serine transporter protein gene in the engineered strain is <h2 style=";text-align:left;direction:ltr">tdcC , <h2 style=";text-align:left;direction:ltr"> sstT , <h2 style=";text-align:left;direction:ltr"> sdaC , <h2 style=";text-align:left;direction:ltr"> steT , <h2 style=";text-align:left;direction:ltr"> thrY or <h2 style=";text-align:left;direction:ltr"> ydgC expression was enhanced.

[0007] In one embodiment, the Thr / Ser endonuclease gene <h2 style=";text-align:left;direction:ltr"> tdcC , <h2 style=";text-align:left;direction:ltr"> sstT , <h2 style=";text-align:left;direction:ltr"> sdaC , <h2 style=";text-align:left;direction:ltr"> steT , <h2 style=";text-align:left;direction:ltr"> thrY or <h2 style=";text-align:left;direction:ltr"> ydgC Ways to enhance the expression of include overexpression of endogenous or exogenous corresponding genes, or replacement of strong promoters or knockout of inhibitory elements.

[0008] In one embodiment, the branched-chain amino acid efflux gene <h2 style=";text-align:left;direction:ltr"> ygaZH expression was enhanced.

[0009] In one embodiment, the branched-chain amino acid efflux gene <h2 style=";text-align:left;direction:ltr"> ygaZH Ways to enhance the expression of include overexpression of endogenous or exogenous corresponding genes, or replacement of strong promoters or knockout of inhibitory elements.

[0010] In one embodiment, it further comprises a gene for threonine deaminase and / or a gene for acetylhydroxybutyrate synthase.

[0011] In one embodiment, the threonine deaminase gene and / or the acetylhydroxybutyrate synthase gene are corresponding genes that are substantially relieved of L-isoleucine inhibition.

[0012] In one embodiment, the gene for the threonine deaminase that is substantially relieved of the inhibition of L-isoleucine is a relieved <h2 style=";text-align:left;direction:ltr"> ilvA The gene of the acetylhydroxybutyrate synthase that substantially relieves the inhibition of L-isoleucine is selected from the group consisting of <h2 style=";text-align:left;direction:ltr"> ilvIH Gene, release type <h2 style=";text-align:left;direction:ltr"> ilvBN Gene, release type <h2 style=";text-align:left;direction:ltr"> ilvGM Gene, release type <h2 style=";text-align:left;direction:ltr"> alsS One or more genes.

[0013] In one embodiment, the engineered strain further comprises a threonine dehydratase gene, a threonine transporter gene, an acetohydroxyacid isomerase gene ( <h2 style=";text-align:left;direction:ltr"> ilvC )、dihydroxyacid dehydratase gene( <h2 style=";text-align:left;direction:ltr"> ilvD ), branched-chain amino acid aminotransferase gene ( <h2 style=";text-align:left;direction:ltr"> ilvE ), one or more of the branched-chain amino acid dehydrogenase genes.

[0014] In one embodiment, the activity of the threonine exporter is attenuated or eliminated.

[0015] In one embodiment, the method of reducing or eliminating the activity of the threonine exporter is to knock out or mutate <h2 style=";text-align:left;direction:ltr"> rhtAC Gene or inhibition <h2 style=";text-align:left;direction:ltr"> rhtAC Gene expression.

[0016] In one embodiment, the engineered strain is modified to maximize the concentration of carbon metabolic flow to pyruvate and direct it to isoleucine, and the production pathway of the metabolic byproducts of miscellaneous acids and alcohols represented by ethanol, acetate, and lactate in the engineered strain is weakened or blocked. <h2 style=";text-align:left;direction:ltr"> ldhA , <h2 style=";text-align:left;direction:ltr"> pta , <h2 style=";text-align:left;direction:ltr"> poxB , <h2 style=";text-align:left;direction:ltr"> adhE , <h2 style=";text-align:left;direction:ltr"> pflB , <h2 style=";text-align:left;direction:ltr"> mgsA , <h2 style=";text-align:left;direction:ltr"> frdA , <h2 style=";text-align:left;direction:ltr"> tdh , <h2 style=";text-align:left;direction:ltr"> tdcE The activity of genes is weakened or eliminated separately or simultaneously.

[0017] In one embodiment, the engineered strain can be a microorganism or other organism including but not limited to wild or genetically engineered Escherichia coli (E. coli), Bacillus, Yeast, Corynebacterium, Brevibacterium or Streptomyces.

[0018] In one embodiment, the polynucleotide sequence encoding the threonine / serine transporter protein is derived from a wild or genetically engineered microorganism or other organism. <h2 style=";text-align:left;direction:ltr"> tdcC , <h2 style=";text-align:left;direction:ltr"> sstT , <h2 style=";text-align:left;direction:ltr"> sdaC , <h2 style=";text-align:left;direction:ltr"> steT , <h2 style=";text-align:left;direction:ltr"> thrY or <h2 style=";text-align:left;direction:ltr"> ydgC Gene.

[0019] In one embodiment, the polynucleotide sequence encoding the threonine / serine transporter protein is derived from a wild or genetically engineered microorganism or other organism. <h2 style=";text-align:left;direction:ltr"> ygaZH Gene.

[0020] In one embodiment, the polynucleotide sequence encoding the threonine export protein comprises a wild or genetically engineered protein derived from a microorganism or other organism. <h2 style=";text-align:left;direction:ltr"> rhtAC The gene(s) are deleted or have mutations that reduce activity / expression.

[0021] In one embodiment, the method for constructing the engineered strain includes plasmid expression, genome integration such as homologous recombination mediated by CRISPR, lambda-red, phage, etc.

[0022] In another aspect, the present invention provides use of the aforementioned engineered strain in preparing L-isoleucine or preparing food, health products or feed containing L-isoleucine.

[0023] In another aspect, the present invention provides a method for producing L-isoleucine, characterized in that it comprises the steps of:

[0024] (i) cultivating the aforementioned engineered strain;

[0025] (ii) isolating L-isoleucine from the culture of (i).

[0026] In one embodiment, the method comprises adding 2-ketobutyrate or a raw material that can be converted into 2-ketobutyrate, such as threonine, fumarate, aspartic acid, homoserine, propionic acid, 2-aminobutyric acid, etc., under culture conditions with insufficient oxygen, and separating L-isoleucine from the culture of (i).

[0027] In one embodiment, the method comprises adding 2-ketobutyrate or a raw material that can be converted into 2-ketobutyrate, such as threonine, fumarate, aspartic acid, homoserine, propionic acid, 2-aminobutyric acid, etc., under culture conditions with sufficient oxygen, and separating L-isoleucine from the culture of (i).

[0028] In one embodiment, the method comprises adding pyruvate or a raw material that can be converted into pyruvate, such as glucose or alanine, under culture conditions with sufficient oxygen, and isolating the L-isoleucine from the culture of (i).

[0029] In one embodiment, the method comprises adding a raw material such as glucose to separate L-isoleucine from the culture of (i) under oxygen-sufficient or oxygen-deficient culture conditions.

[0030] Beneficial Effects

[0031] The present invention provides a construction method and application of an optimized L-isoleucine production strain. By further expressing threonine endonuclease carriers of different sources and types in the isoleucine production strain, the ability of the cell to absorb threonine is enhanced, thereby increasing the yield and content of L-isoleucine in the fermentation product and reducing the relative yield and content of the byproduct miscellaneous acid (valine). Further, the cell efflux threonine endonuclease carrier is deleted to obtain a genetically engineered high-yield strain. The genetically engineered strain is subjected to fermentation culture, threonine is added during the culture process, and L-isoleucine is separated from the culture. DETAILED DESCRIPTION

[0032] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments 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 implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0033] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0034] The articles "a" and "the" are used herein to refer to one or more than one of the grammatical objects of the article. The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination thereof in the alternatives. The term "and / or" should be understood to mean one or two of the alternatives. As used herein and unless otherwise specified, the term "about" refers to a measurable value such as an amount, a time period, etc., indicating a variation of ±10% from a given value, more preferably ±5%, even better ±1%, and still more preferably ±0.1%, as long as such variation is suitable for implementing the disclosed method.

[0035] As used herein, the term "gene synthesis" refers to the production of recombinant DNA technology or the acquisition of synthetic DNA or amino acid sequence technology available and known in the art. "Encoding" refers to the inherent properties of other polymers and macromolecules synthesized in biological processes as templates by specific sequences of nucleotides in polynucleotides such as genes, cDNAs or mRNAs, which have any of the defined sequences of nucleotides (i.e., rRNA, tRNA and mRNA) or defined sequences of amino acids and the biological properties produced therefrom. Therefore, if the transcription and translation of the mRNA corresponding to that gene produces protein in a cell or other biological system, the gene encodes the protein. The coding strands that are equivalent to the mRNA sequence and are usually provided in the sequence table, and the non-coding strands used as templates for transcribed genes or cDNAs, can be referred to as proteins or other products encoding that gene or cDNA.

[0036] As used herein, the term "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0037] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside an organism, cell, tissue or system.

[0038] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0039] As used herein, the term "threo / serine endorporter" includes a threo / serine-hydrogen ion symporter, a serine-hydrogen ion symporter, a threo / serine transporter, a threonine transporter, and a threo / serine permease.

[0040] 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.

[0041] 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 nucleic acid sequences from a recombinant library or a cell genome, using cloning techniques, etc., or using synthetic means.

[0042] 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 polypeptides of cytokine antibodies or antibody fragments or antigen binding fragments.

[0043] As disclosed 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, polynucleotide fragments of almost any length can be used, with the total length preferably being limited by ease of preparation and use in the intended recombinant DNA protocol.

[0044] 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.

[0045] "Expression control sequences", "control elements" or "regulatory sequences" present in expression vectors are those non-translated regions of the vector - origin of replication, selection cassette, promoter, enhancer, translation start signal intron, post-transcriptional regulatory element, 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 can be used, including ubiquitous promoters and inducible promoters. 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. "Endogenous" control sequences are sequences naturally connected to a given gene in a genome. "Exogenous" control sequences are sequences that are placed in juxtaposition with a gene by genetic manipulation (i.e., molecular biotechnology) so that the transcription of that gene is guided by the connected enhancer / promoter. "Heterologous" control sequences are exogenous sequences from species different from the cells of genetic manipulation. "Synthetic" control sequences can include one or more elements of endogenous and / or exogenous sequences and / or sequences determined in vitro or in silico to provide optimal promoter and / or enhancer activity for a particular gene therapy. 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 a promoter.

[0046] The term "promoter" refers to a fragment of DNA containing sequences capable of providing promoter functions.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] As used herein, the term "transfection" or "conversion" or "transduction" refers to a process by which exogenous nucleic acids are transferred or introduced into a recipient strain. A "transfected" or "converted" or "transduced" strain is a strain that has been transfected, transformed or transduced with an exogenous nucleic acid. The strain includes a primary generation and its progeny. The recipient strain can be Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus megaterium, Vibrionatriegens, Bacillus amyloliquefaciens, or Saccharomyces cerevisiae, etc.

[0052] In the present specification, the ilvA gene encoding the threonine dehydratase that is substantially relieved of inhibition by L-isoleucine is referred to as a "relieving type ilvA gene".

[0053] Materials and Methods

[0054] LB medium: 1% peptone, 0.5% yeast extract, 1% NaCl, adjust pH to 7.2 with 30% NaOH, sterilize at 1×105 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.

[0055] Shake flask fermentation medium: glucose 40g / L, yeast powder 5g / L, ammonium sulfate 10g / L, magnesium sulfate 2mM, calcium chloride 0.1mM, potassium dihydrogen phosphate 0.8g / L, manganese sulfate pentahydrate 0.02g / L, threonine 25g / L, betaine 1g / L, nicotinic acid 0.01g / L, VB1 0.005g / L, IPTG 0.3mM, calcium carbonate 10g / L, the medium was adjusted to pH 7.3~7.4 with concentrated ammonia water and sterilized at 11℃ for 15min.

[0056] Shake flask fermentation method: Pick a single clone from the activated strain on the LB plate (containing an appropriate concentration of antibiotics) at 37°C and inoculate it into LB liquid culture medium (containing an appropriate concentration of antibiotics), and culture it at 37°C for 12-16h at 220rpm. Inoculate 1% of the LB overnight culture into a 250ml shake flask (containing 10g / L CaCO3 as a pH stabilizer) containing 25ml fermentation medium with appropriate concentrations of ampicillin and kanamycin; seal the flask with a breathable membrane and culture it at 30°C, 220 rpm. When the OD600 reaches 0.6, add IPTG (final concentration of 0.2 mM) to induce plasmid expression. Stop fermentation after 40 hours of culture and take samples.

[0057] Determination of amino acid concentration: Amino acid standards were purchased from Sigma-Aldrich (www.sigmaaldrich.cn). Take 1 mL of fermentation broth, centrifuge at 10,000 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 to an appropriate multiple, and 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, and the chromatographic column is Agilent ZORBAX Eclipse Plus C18, 4.6×250mm 5μm. 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 all set according to the above method.

[0058] Table 1 Enzyme types involved in the present invention

[0059]

[0060] The biomaterials constructed by the present invention are shown in Table 2 below:

[0061] Table 2 Biomaterials involved in the present invention

[0062]

[0063] Example 1: Construction of isoleucine production strain and plasmid

[0064] Using λ-Red recombination technology, the gene encoding pyruvate-formate lyase in the genome of Escherichia coli K bacteria background strain BW25113 was transformed into <h2 style=";text-align:left;direction:ltr"> pflB The gene was knocked out and replaced with the kanamycin resistance gene Kan to obtain the recombinant strain BW25113 ΔpflB::Kan. The plasmid pCP20 was transformed into the above Kan-resistant transformant, spread on an LB plate containing 100 mg / L ampicillin, and cultured at 30°C for 24 h. The correct transformant in which the kanamycin resistance gene Kan was removed in the target gene site was identified by PCR to obtain the non-resistant Escherichia coli BW25113 Δ <h2 style=";text-align:left;direction:ltr"> pflB By analogy, the same method and strategy were used to obtain the starting strain DA9 (BW25113 Δ <h2 style=";text-align:left;direction:ltr"> ldhA Δ <h2 style=";text-align:left;direction:ltr"> pta Δ <h2 style=";text-align:left;direction:ltr"> poxB Δ <h2 style=";text-align:left;direction:ltr"> adhE Δ <h2 style=";text-align:left;direction:ltr"> pflB Δ <h2 style=";text-align:left;direction:ltr"> mgsA Δ <h2 style=";text-align:left;direction:ltr"> frdA Δ <h2 style=";text-align:left;direction:ltr"> tdh Δ <h2 style=";text-align:left;direction:ltr"> tdcE This strain knocks out most of the genes of Escherichia coli mixed acid fermentation to prevent the production of byproducts such as mixed acids by the chassis cells during the fermentation process, which interferes with the subsequent product separation process and reduces the substrate conversion rate; the deletion of tdh can reduce the decomposition of the precursor threonine, and the deletion of <h2 style=";text-align:left;direction:ltr"> tdcE and <h2 style=";text-align:left;direction:ltr"> pflB It can reduce the decomposition of the precursor 2-ketobutyrate, which is beneficial to direct the isoleucine anabolism flux from threonine to isoleucine synthesis. Detailed strains and plasmid genotypes are shown in Tables 1 and 2. The genes rhtA and rhtC encoding threonine efflux in DA9 were further deleted in combination to obtain strains DL10-DL12.

[0065] ilvDCEygaZH (ilvC encodes acetohydroxyacid reductoisomerase with L67E, R68F and K75E mutations) and ilvAIH fragment (encoded released IlvA protein with L447F and L451A mutations; ilvH gene carries mutations encoding G14D and S17F) were amplified from plasmids pZE-ilvDCmEygaZH and pZA-ilvAIH (plasmid derived from Chinese patent CN114410701A) by PCR, and 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-ilvAIHDCEygaZH (pZE-ILE).

[0066] The sdaC, tdcC and sstT gene fragments were amplified from the genome of Escherichia coli; <h2 style=";text-align:left;direction:ltr"> steT Gene fragments, the thrY gene fragment was amplified from the genome of Corynebacterium glutamicum ATCC13032; the ydgC gene fragment was amplified from the genome of Lactococcus lactis subsp. lactis ATCC19435; using a recombinant cloning kit, the above gene fragments were connected to the vector plasmid fragment pZAlac4 as shown in Table 2 by a seamless cloning method to obtain recombinant plasmids pLT1 to pLT6.

[0067] The above recombinant plasmid combinations were transformed into BW25113, DA9, DL10, DL11 or DL12 strains. For details of specific plasmid and strain combinations, please refer to the Materials and Methods section.

[0068] Example 2: Shake flask fermentation of L-isoleucine producing bacteria

[0069] Plasmids pZE-ILE and pLT1-pLT6 were transformed into chassis strains BW25113 or DA9 as shown in Table 2, and the obtained recombinant strains ILE-TC and ILE-T0 to ILE-T6 were fermented according to the shake flask fermentation method, with recombinant strains ILE-TC and ILE-T0 as controls. The results are shown in Table 3. We can see that after the introduction of the pLT series plasmids expressing the threonine internal transport vector, the isoleucine (ILE) production of the strain expressing the threonine internal transport vector was increased compared to the control strain, and the production of valine (VAL) as a by-product was relatively reduced. The isoleucine content in the fermentation product was higher (expressed as ILE / VAL), especially in the overexpression strain. <h2 style=";text-align:left;direction:ltr"> ydgC The ILE-T4 strain with the gene had the best effect.

[0070] Table 3 Shake flask fermentation results

[0071]

[0072] Example 3: Shake flask fermentation of L-isoleucine producing bacteria

[0073] Plasmids pZE-ILE and pLT4 were transferred into DL10-DL12 strains as described in Table 2 to obtain recombinant strains ILE-T7, ILE-T8 and ILE-T9, which were fermented in shake flasks. The results are shown in Table 4. It can be seen that the deletion of the threonine exogenous vector can further increase the yield and content of isoleucine in the fermentation product.

[0074] Table 4 Shake flask fermentation results

[0075]

[0076] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A genetically engineered strain for producing isoleucine, wherein the genetically engineered strain is Escherichia coli, and the Thr / Serine endonuclease protein gene from Lactococcus lactis is contained in the genetically engineered strain. ydG and branched-chain amino acid efflux genes from Escherichia coli Yg The expression of ydG The sequence accession number of the gene encoding protein is AAK04454. Yg The sequence accession numbers for the gene-encoded proteins are AAC75729 and AAC75730; The genetically engineered strain also includes a gene for threonine deaminase that is substantially free of L-isoleucine inhibition, a gene for acetylhydroxybutyrate synthase, a gene for acetohydroxyacid isomerase from Escherichia coli ( ilvC ), dihydroxyacid dehydratase gene from Escherichia coli ( ilvD ), branched-chain amino acid aminotransferase gene from Escherichia coli ( ilvE ), ilvC The sequence accession number of the gene encoding protein is AAC76779. ilvD The sequence accession number of the gene encoding protein is CAA28576. ilvE The sequence accession number of the gene-encoded protein is CAA28575; The gene for the threonine deaminase that substantially releases the inhibition of L-isoleucine is a released type gene derived from Escherichia coli. ilv The gene of acetylhydroxybutyrate synthase substantially relieved of L-isoleucine inhibition is a relieved type gene from Escherichia coli iV Gene, ilv The sequence accession number of the gene encoding protein is CAA28577. iV The sequence accession numbers for the proteins encoded by the genes are CAA25755 and CAA25756; The genetically engineered strain is modified to maximize the concentration of carbon metabolic flow to pyruvate and direct it to isoleucine. oeLh , pta , poxB , adhE , pB , oeLh , frdA , td , oeLh The gene is knocked out; The Thr / Ser endonuclease gene ydG and branched-chain amino acid efflux genes Yg Ways to enhance the expression of a gene include overexpressing endogenous or exogenous corresponding genes.

2. The genetically engineered strain according to claim 1, further comprising: oeLh Gene or inhibition oeLh The expression of the gene is reduced and the activity of threonine export protein is weakened or eliminated.

3. The genetically engineered strain according to claim 1 or 2, wherein the method for constructing the genetically engineered strain comprises plasmid expression and genome integration, and the genome integration comprises CRISPR, lambda-red, and phage-mediated homologous recombination.

4. Use of the engineered strain according to any one of claims 1 to 3 in the preparation of L-isoleucine or in the preparation of food, health products or feed containing L-isoleucine.

5. A method for producing L-isoleucine, characterized in that: Includes steps: (i) cultivating the genetically engineered strain according to any one of claims 1 to 4; (ii) isolating L-isoleucine from the culture of (i).

6. The method according to claim 5, comprising adding 2-ketobutyric acid or a raw material that can be converted into 2-ketobutyric acid under culture conditions with insufficient oxygen, wherein the raw material that can be converted into 2-ketobutyric acid includes threonine, fumaric acid, aspartic acid, homoserine, propionic acid and 2-aminobutyric acid, and separating L-isoleucine from the culture of (i).

7. The method according to claim 5, comprising adding 2-ketobutyric acid or a raw material that can be converted into 2-ketobutyric acid under culture conditions with sufficient oxygen, wherein the raw material that can be converted into 2-ketobutyric acid includes threonine, fumaric acid, aspartic acid, homoserine, propionic acid and 2-aminobutyric acid, and separating L-isoleucine from the culture of (i).

8. The method according to claim 5, comprising adding pyruvate or a raw material that can be converted into pyruvate under culture conditions with sufficient oxygen, wherein the raw material that can be converted into pyruvate includes glucose and alanine, and isolating the L-isoleucine from the culture of (i).

9. The method according to claim 5, comprising adding glucose to separate L-isoleucine from the culture of (i) under oxygen-sufficient or oxygen-deficient culture conditions.

Images