Yarrowia lipolytica ILV6 protein mutant and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly discloses yarrowia lipolytica ILV6 protein mutants and application thereof. The ILV6 protein mutant is obtained by mutating the 81st glycine (Gly) of the amino acid sequence of the ILV6 protein of wild type yarrowia lipolytica. The method comprises the following steps: transforming a yarrowia lipolytica ILV6 protein mutant into a yarrowia lipolytica yeast to construct a yarrowia lipolytica recombinant strain; compared with the Yarrowia lipolytica recombinant engineering bacteria which do not express the Yarrowia lipolytica ILV6 protein mutant or express the wild type ILV6 protein or express the saccharomyces cerevisiae wild type ILV6 protein, the Yarrowia lipolytica recombinant engineering bacteria which express the Yarrowia lipolytica ILV6 protein mutant can tolerate DL-norvaline with relatively high concentration; in addition, the content of intracellular valine in the fermentation process can be obviously improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically designing a mutant of Yersinia lipophila ILV6 protein and its application. Background Technology

[0002] Yarrowia lipolytica ( Yarrowia lipolytica Its broad substrate spectrum and strong resistance make it suitable as a microbial factory for industrial applications. The yeast branched-chain amino acid synthesis pathway is widely used in synthetic biology for the biosynthesis of branched-chain compounds.

[0003] The biosynthesis of branched-chain amino acids is strictly regulated by the activity of acetolactate synthase (e.g. Figure 1 As shown in the diagram, yeast acetyllactate synthase is composed of two protein subunits, ILV2 and ILV6, and exerts its regulatory role in response to the concentration of intracellular branched-chain amino acids. On the one hand, branched-chain amino acids are recognized and bound by ILV6 to specifically inactivate ILV2; on the other hand, branched-chain amino acids use transcription factors to inhibit the transcriptional expression of protein-coding genes such as ILV3, ILV5, and ILV2.

[0004] Current research on the production of branched-chain long-chain alcohols from branched-chain amino acid metabolism in *Yersinia lipolytica* focuses primarily on promoter optimization and exogenous protease screening; no studies related to ILV6 have been reported. Therefore, using the ILV6 mutant to desensitize and / or reduce the sensitivity of the negative feedback system in the endogenous valine synthesis metabolism of *Yersinia lipolytica* is an effective means to expand the application of *Yersinia lipolytica* in the production of branched-chain compounds. Summary of the Invention

[0005] In view of the problems and shortcomings of the existing technology, the purpose of this invention is to provide a mutant of Yersinia lipophila ILV6 protein and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a mutant of the ILV6 protein in *Yersinia lipolytica*, wherein the ILV6 protein mutant is obtained by mutating the 81st glycine (Gly) in the amino acid sequence of the wild-type *Yersinia lipolytica* ILV6 protein; the ILV6 protein mutant is as follows: A1), A2), or A3). A1) The YlILV6 Gly81Asp protein mutant was obtained by mutating glycine (Gly) at position 81 of the amino acid sequence of the wild-type ILV6 protein to aspartic acid (Asp), and its amino acid sequence is shown in Sequence 1. Sequence 1 is as follows: MLGKRFVGPVLTPKGARHSSISALAYKTLHRNRSQPKLPVIETPAWNANTAVSSILYETPMPSKAPIKAHVFNCLVQNEPDVLSRVAGTLASRGFNIDSLVVCNTEVADLSRMTIVLRGQDAVIEQARRQIEDLVPVWAVLDYS NASIIKRELLLARVSILGPEYFQDLLTHHGHEFEDAVLQNDFHPNNIAASEALRHKHQYLDAVTKLAHQFGGKILDISERNVIVELSAKPERVSSFLHLLKPFGILEVARSGMMALPRTPLETPDEEDIKKAEEVVDQTSLPPG.

[0007] A2) The ILV6 protein mutant is obtained by mutating glycine (Gly) at position 81 of the amino acid sequence of the wild-type ILV6 protein to proline (Pro), and is denoted as YlILV6 Gly81Pro protein mutant. Its amino acid sequence is shown in Sequence 2. Sequence 2 is as follows: MLGKRFVGPVLTPKGARHSSISALAYKTLHRNRSQPKLPVIETPAWNANTAVSSILYETPMPSKAPIKAHVFNCLVQNEPPVLSRVAGTLASRGFNIDSLVVCNTEVADLSRMTIVLRGQDAVIEQARRQIEDLVPVWAVLDYS NASIIKRELLLARVSILGPEYFQDLLTHHGHEFEDAVLQNDFHPNNIAASEALRHKHQYLDAVTKLAHQFGGKILDISERNVIVELSAKPERVSSFLHLLKPFGILEVARSGMMALPRTPLETPDEEDIKKAEEVVDQTSLPPG.

[0008] A3) The ILV6 protein mutant is obtained by mutating glycine at position 81 of the wild-type ILV6 protein amino acid sequence to tyrosine (Tyr), and is denoted as YlILV6 Gly81Tyr protein mutant, whose amino acid sequence is shown in Sequence 3. Sequence 3 is as follows: MLGKRFVGPVLTPKGARHSSISALAYKTLHRNRSQPKLPVIETPAWNANTAVSSILYETPMPSKAPIKAHVFNCLVQNEPYVLSRVAGTLASRGFNIDSLVVCNTEVADLSRMTIVLRGQDAVIEQARRQIEDLVPVWAVLDYS NASIIKRELLLARVSILGPEYFQDLLTHHGHEFEDAVLQNDFHPNNIAASEALRHKHQYLDAVTKLAHQFGGKILDISERNVIVELSAKPERVSSFLHLLKPFGILEVARSGMMALPRTPLETPDEEDIKKAEEVVDQTSLPPG.

[0009] A second aspect of the present invention provides biomaterials related to the ILV6 protein mutant described in the first aspect above, wherein the biomaterials are any of the following: B1) A nucleic acid molecule encoding the ILV6 protein mutant of claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2).

[0010] Based on the above biological materials, preferably, the sequence of the nucleic acid molecule encoding the YlILV6 Gly81Asp protein mutant is as shown in Sequence 4, and Sequence 4 is specifically as follows: ATGCTTGGAAAACGATTTGTGGGTCCCGTGCTGACCCCCAAGGGCGCTCGGCACTCGTCCATCAGTGCCCTGGCGTACAAGACGCTCCACCGAAACCGATCGCAGCCCAAGCTGCCCGTTATCGAGACTCCAGCATGGAACGCCAACACGGCGGTTTCTTCCATTCTGTACGAGACCCCGATGCCGTCCAAGGCGCCCATCAAGGCACACGTGTTCAACTGTCTGGTTCAGAACGAACCCGATGTTCTGTCGCGAGTTGCCGGCACTCTGGCGTCTCGAGGCTTCAACATTGATTCGCTGGTAGTGTGCAACACGGAGGTTGCCGATCTGTCGCGAATGACCATTGTTCTGCGAGGCCAGGATGCCGTGATCGAGCAGGCGCGACGACAGATTGAGGATCTGGTGCCCGTGTGGGCAGTGCTGGACTACTCCAACGCCTCGATCATCAAGCGAGAGCTGCTGCTGGCCCGAGTGTCGATTCTGGGCCCCGAGTACTTCCAGGATCTGCTGACCCACCACGGCCACGAGTTTGAGGACGCCGTGCTGCAGAACGACTTCCACCCCAACAACATTGCCGCCTCCGAGGCTCTGCGACACAAGCACCAGTACCTGGACGCCGTGACCAAGCTGGCCCACCAGTTTGGCGGCAAGATTCTCGACATTTCTGAGCGAAACGTCATTGTCGAGCTCTCGGCCAAGCCCGAGCGAGTCTCCTCCTTCCTGCACCTTCTCAAGCCCTTTGGTATTCTTGAGGTGGCCCGATCCGGAATGATGGCCCTGCCCCGAACTCCTCTTGAGACCCCCGATGAGGAGGACATCAAGAAGGCCGAGGAGGTGGTTGACCAGACCTCTCTGCCTCCTGGTTAG。

[0011] Based on the above biological materials, preferably, the sequence of the nucleic acid molecule encoding the YlILV6 Gly81Pro protein mutant is as shown in Sequence 5, and Sequence 5 is specifically as follows: ATGCTTGGAAAACGATTTGTGGGTCCCGTGCTGACCCCCAAGGGCGCTCGGCACTCGTCCATCAGTGCCCTGGCGTACAAGACGCTCCACCGAAACCGATCGCAGCCCAAGCTGCCCGTTATCGAGACTCCAGCATGGAACGCCAACACGGCGGTTTCTTCCATTCTGTACGAGACCCCGATGCCGTCCAAGGCGCCCATCAAGGCACACGTGTTCAACTGTCTGGTTCAGAACGAACCCCCCGTTCTGTCGCGAGTTGCCGGCACTCTGGCGTCTCGAGGCTTCAACATTGATTCGCTGGTAGTGTGCAACACGGAGGTTGCCGATCTGTCGCGAATGACCATTGTTCTGCGAGGCCAGGATGCCGTGATCGAGCAGGCGCGACGACAGATTGAGGATCTGGTGCCCGTGTGGGCAGTGCTGGACTACTCCAACGCCTCGATCATCAAGCGAGAGCTGCTGCTGGCCCGAGTGTCGATTCTGGGCCCCGAGTACTTCCAGGATCTGCTGACCCACCACGGCCACGAGTTTGAGGACGCCGTGCTGCAGAACGACTTCCACCCCAACAACATTGCCGCCTCCGAGGCTCTGCGACACAAGCACCAGTACCTGGACGCCGTGACCAAGCTGGCCCACCAGTTTGGCGGCAAGATTCTCGACATTTCTGAGCGAAACGTCATTGTCGAGCTCTCGGCCAAGCCCGAGCGAGTCTCCTCCTTCCTGCACCTTCTCAAGCCCTTTGGTATTCTTGAGGTGGCCCGATCCGGAATGATGGCCCTGCCCCGAACTCCTCTTGAGACCCCCGATGAGGAGGACATCAAGAAGGCCGAGGAGGTGGTTGACCAGACCTCTCTGCCTCCTGGTTAG。

[0012] Based on the above biological materials, preferably, the sequence of the nucleic acid molecule encoding the YlILV6 Gly81Tyr protein mutant is as shown in Sequence 6, and Sequence 6 is specifically as follows: ATGCTTGGAAAACGATTTGTGGGTCCCGTGCTGACCCCCAAGGGCGCTCGGCACTCGTCCATCAGTGCCCTGGCGTACAAGACGCTCCACCGAAACCGATCGCAGCCCAAGCTGCCCGTTATCGAGACTCCAGCATGGAACGCCAACACGGCGGTTTCTTCCATTCTGTACGAGACCCCGATGCCGTCCAAGGCGCCCATCAAGGCACACGTGTTCAACTGTCTGGTTCAGAACGAACCCTACGTTCTGTCGCGAGTTGCCGGCACTCTGGCGTCTCGAGGCTTCAACATTGATTCGCTGGTAGTGTGCAACACGGAGGTTGCCGATCTGTCGCGAATGACCATTGTTCTGCGAGGCCAGGATGCCGTGATCGAGCAGGCGCGACGACAGATTGAGGATCTGGTGCCCGTGTGGGCAGTGCTGGACTACTCCAACGCCTCGATCATCAAGCGAGAGCTGCTGCTGGCCCGAGTGTCGATTCTGGGCCCCGAGTACTTCCAGGATCTGCTGACCCACCACGGCCACGAGTTTGAGGACGCCGTGCTGCAGAACGACTTCCACCCCAACAACATTGCCGCCTCCGAGGCTCTGCGACACAAGCACCAGTACCTGGACGCCGTGACCAAGCTGGCCCACCAGTTTGGCGGCAAGATTCTCGACATTTCTGAGCGAAACGTCATTGTCGAGCTCTCGGCCAAGCCCGAGCGAGTCTCCTCCTTCCTGCACCTTCTCAAGCCCTTTGGTATTCTTGAGGTGGCCCGATCCGGAATGATGGCCCTGCCCCGAACTCCTCTTGAGACCCCCGATGAGGAGGACATCAAGAAGGCCGAGGAGGTGGTTGACCAGACCTCTCTGCCTCCTGGTTAG。

[0013] The third aspect of this invention provides the use of the ILV6 protein mutant described in the first aspect or the biological material described in the second aspect in any of the following: C1) Application in the preparation of DL-valine-tolerant recombinant bacteria and / or the enhancement of DL-valine-tolerant recombinant bacteria; C2) Applications in the production of amino acids or / and in increasing amino acid yield; C3) Application in the preparation of recombinant bacteria for producing branched-chain long-chain alcohols and / or recombinant bacteria for increasing the yield of branched-chain long-chain alcohols; C4) Applications in the production of branched long-chain alcohols and / or in increasing the yield of branched long-chain alcohols; C5) Application in the preparation of recombinant bacteria for producing short-chain fatty acids or / and recombinant bacteria for increasing the yield of short-chain fatty acids; C6) Applications in the production of short-chain fatty acids or / and in increasing the yield of short-chain fatty acids.

[0014] According to the above application, preferably, the amino acid is at least one of valine, leucine, and isoleucine.

[0015] According to the above application, preferably, the branched long-chain alcohol is at least one of isoamyl alcohol, isobutanol, 2-methyl-1-butanol, and 2,4-dimethyl-3-pentanol.

[0016] According to the above application, preferably, the short-chain fatty acid is at least one of isobutyric acid, isovaleric acid, and 2-methyl-1-butyric acid.

[0017] The fourth aspect of the present invention provides a recombinant *Yersinia lipophila* strain, wherein the recombinant *Yersinia lipophila* strain contains the nucleic acid molecule described in B1) of the second aspect above, or contains the expression cassette described in B2), or contains the recombinant vector described in B3).

[0018] According to the above-mentioned recombinant bacteria, preferably, the recombinant bacteria of *Yersinia lipolytica* is prepared by transforming the nucleic acid molecule described in B1) or the expression cassette described in B2) or the recombinant vector described in B3) of the second aspect into *Yersinia lipolytica* using *Yersinia lipolytica* as the chassis cell.

[0019] According to the above-mentioned recombinant strain of Yersinia lipolytica, preferably, the Yersinia lipolytica is Yersinia lipolytica Po1f strain, Yersinia lipolytica Po1f ΔKu70 strain, or Yersinia lipolytica Po1f ΔKu70 YLT3::Ilvs recombinant strain.

[0020] According to the above-mentioned recombinant *Yarrowia lipolytica* strain, preferably, the *Yarrowia lipolytica* Po1f ΔKu70 YLT3::Ilvs recombinant strain uses *Yarrowia lipolytica* Po1f ΔKu70 strain as the chassis cell, and the genome of *Yarrowia lipolytica* Po1f ΔKu70 strain is... YLT3 Random integration at the site in tandem in the form of monocistronic units YlIlv3 Gene, YlIlv5 Gene, YlIlv2* The gene was constructed to obtain the stated YlIlv2* Genes are... YlIlv2 The gene obtained by deleting the ClaI restriction site in the coding region of the gene. In the recombinant strain *Yarrowia lipophila* Po1f ΔKu70 YLT3::Ilvs... YlIlv3 Gene, YlIlv5 Gene, YlIlv2* Gene expression regulation is all derived from the pYLXP'2 vector. TEF promoters and XPR2t Termination of sub-control.

[0021] According to the above-mentioned recombinant *Yarrowia lipolyticis* strain, preferably, the construction method of the recombinant *Yarrowia lipolyticis* Po1f ΔKu70 YLT3::Ilvs strain is as follows: (1) Yeast extract from lipophilic yeast YLT3 The gene fragment was ligated into the pUC57 vector to obtain the recombinant plasmid pUC57-YLT3; the recombinant plasmid pUC57-YLT3 was amplified to obtain a 3050 bp linearized L-pUC57-YLT3 gene fragment. (2) The pYLXP'2 vector CAREER3 The gene fragment was ligated with the linearized pYLXP' vector to obtain the recombinant plasmid pYLXP'-URA3; the lox71 and lox66 sequences were inserted into the AvrII and NheI sites of the recombinant plasmid pYLXP'-URA3, respectively, to obtain the recombinant plasmid pYLXP'-URA3-loxP; the recombinant plasmid pYLXP'-URA3-loxP was amplified to obtain a 2273 bp linearized L-URA3-LoxP gene fragment; the linearized L-pUC57-YLT3 gene fragment was ligated with the linearized L-URA3-LoxP gene fragment to obtain the recombinant plasmid pUC57-YLT3-URA3-loxP; (3) Yeast extract Po1f YlIlv3 Gene, YlIlv5 Gene, YlIlv2 The gene was tandemly linked in monocistronic form onto the vector pYLXP'2 to obtain the recombinant plasmid pYLXP'2-YlILV3-YlILV5-YlILV2 * ; (4) The recombinant plasmid pUC57-YLT3-URA3-loxP was double-digested to obtain the pUC57-YLT3-URA3 backbone fragment; the recombinant plasmid pYLXP'2-YlILV3-YlILV5-YlILV2 was digested with enzymes.* Double enzyme digestion was performed to obtain YlILV3-YlILV5-YlILV2. * Gene fragments, combining the pUC57-YLT3-URA3 backbone fragment with YlILV3-YlILV5-YlILV2 * The gene fragments were ligated to obtain the recombinant plasmid pUC57-YLT3-URA3-loxP-YlILV3-YlILV5-YlILV2 * ; (5) The recombinant plasmid pUC57-YLT3-URA3-loxP-YlILV3-YlILV5-YlILV2 * Double enzyme digestion yielded YLT3-URA3-loxP-YlILV3-YlILV5-YlILV2. * Gene fragment, YLT3-URA3-loxP-YlILV3-YlILV5-YlILV2 * The gene fragment was transformed into Yersinia lipolyticis Po1f ΔKu70, and the recombinant strain Yersinia lipolyticis Po1f ΔKu70 YLT3::URA3-Ilvs was obtained by screening with auxotrophic plates. (6) The pYLXP'-Cre vector was transformed into the recombinant strain of Yersinia lipolytica Po1f ΔKu70 YLT3::URA3-Ilvs. After screening on leucine auxotrophic medium containing 5-fluoroorotic acid (SD-Leu-FOA medium), the recombinant strain of Yersinia lipolytica Po1f ΔKu70 YLT3::Ilvs / pYLXP'-Cre was obtained. The recombinant strain of Yersinia lipolytica Po1f ΔKu70 YLT3::Ilvs / pYLXP'-Cre lost the pYLXP'-Cre plasmid after passage under nutrient-free selection pressure, and the recombinant strain of Yersinia lipolytica Po1f ΔKu70YLT3::Ilvs was obtained.

[0022] The fifth aspect of this invention provides the use of the recombinant *Yarrowia lipolytica* strain described in the fourth aspect above in any of the following: D1) Applications in the production of amino acids and / or increasing amino acid yield; D2) Applications in the production of branched long-chain alcohols and / or in increasing the yield of branched long-chain alcohols; D3) Applications in the production of short-chain fatty acids or / and in increasing the yield of short-chain fatty acids.

[0023] According to the above application, preferably, the amino acid is at least one of valine, leucine, and isoleucine.

[0024] According to the above application, preferably, the branched long-chain alcohol is at least one of isoamyl alcohol, isobutanol, 2-methyl-1-butanol, and 2,4-dimethyl-3-pentanol.

[0025] According to the above application, preferably, the short-chain fatty acid is at least one of isobutyric acid, isovaleric acid, and 2-methyl-1-butyric acid.

[0026] The sixth aspect of the present invention provides a method for producing valine using the recombinant *Yersinia lipolyticis* strain described in the fourth aspect above. The method comprises: activating the recombinant *Yersinia lipolyticis* on an SD-Ura plate, inoculating it into SD-Ura liquid medium, and culturing it at 28-32°C and 150-250 rpm for 11-16 h with shaking; more preferably, the method comprises: activating the recombinant *Yersinia lipolyticis* on an SD-Ura plate, inoculating it into SD-Ura liquid medium, and culturing it at 30°C and 200 rpm for 14 h with shaking.

[0027] Compared with the prior art, the positive and beneficial effects achieved by the present invention are as follows: This invention obtains a *Y. lipolyticum* ILV6 protein mutant by site-directed mutagenesis of the glycine at position 81 of the amino acid sequence of the wild-type *Y. lipolyticum* ILV6 protein. This mutant is then transformed into *Y. lipolyticum* yeast to construct a recombinant strain. Compared to recombinant *Y. lipolyticum* strains that do not express the *Y. lipolyticum* ILV6 protein mutant, or express either the wild-type ILV6 protein or the wild-type *Saccharomyces cerevisiae* ILV6 protein, the recombinant strain expressing the *Y. lipolyticum* ILV6 protein mutant can tolerate higher concentrations of DL-valine and significantly increases intracellular valine content during fermentation. Among these, the recombinant strain expressing the Gly81Asp protein mutant shows the best valine content increase (86.7%) compared to the wild-type ILV6 protein, and its tolerance to DL-valine is [IC50]. 50 The tolerance of the recombinant strain expressing the Gly81Pro protein mutant with DL-valine was improved by 94.1%; the IC50 showed the best improvement. 50 The tolerance of DL-valine was increased by 94.9%, and the corresponding valine content also increased by 68.2%; the tolerance of recombinant bacteria expressing the Gly81Tyr protein mutation was also significantly improved, with an IC50 value of 94.9%. 50 The content of valine increased by 62.1%, and the valine content increased by 24.2%. Therefore, the Yersinia lipolytica ILV6 protein mutant of this invention provides an effective component for the application of Yersinia lipolytica as a chassis cell for the biosynthesis of valine-derived compounds. Attached Figure Description

[0028] Figure 1This diagram illustrates the negative feedback regulation of branched-chain amino acid synthesis through acetolactate synthase activity; the pathway consists of branched-chain amino acid metabolism, acetolactate synthase, and branched-chain amino acids. Figure 2 This is a schematic diagram of the structure of the recombinant plasmid pYLXP'2-YlILV6. Figure 3 The effects of ILV6 from different sources and mutants on the DL-valine tolerance of engineered bacteria; Figure 4 Results of site-directed saturation mutant library screening for endogenous ILV6 protein at position 81 in Yersinia lipophila; Figure 5 The content of branched-chain amino acids in engineered bacteria with high tolerance to DL-valine; where a, b, and c represent the results of one-way ANOVA. Detailed Implementation

[0029] To enable those skilled in the art to more clearly understand the technical solutions of the present invention, the technical solutions of the present invention will be described in detail below with reference to specific embodiments. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Experimental methods in the following embodiments that do not specify specific conditions all employ conventional techniques in this technical field or follow the conditions recommended by the manufacturer; reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0030] 1. The culture media and storage solutions used in the following examples are as follows: Luria-Bertani (LB) medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, solid LB medium with 1.8% agar powder added, sterilized at 121°C for 15 min.

[0031] Yeast Extract Peptone Dextrose (YPD) medium: 10 g / L yeast extract powder, 20 g / L peptone, 20 g / L glucose, solid YPD medium with 1.8% agar powder added, sterilized at 115°C for 15 min.

[0032] Ampicillin stock solution (100 mg / mL): Dissolve 1 g of ampicillin in 10 mL of ddH2O, filter to remove bacteria, and store at -20°C. Dilute 1000 times to a final concentration of 100 μg / mL before use.

[0033] Synthetic Defined (SD) medium: 20 g / L glucose, 5 g / L ammonium sulfate, 1.79 g / L YNB, 1.29 g / L DO-Supplement-Ura / Leu. The specific procedure for preparing 100 mL of SD medium is as follows: Dissolve 2 g of glucose in 80 mL of water and autoclave at 115°C for 15 min. After the medium cools to below 60°C, add 10 mL of 10×YNB stock solution and 10 mL of 10× amino acid mixed stock solution. Add 1.8% agar powder to the solid SD medium. SD-Ura represents SD medium lacking uracil, and SD-Leu represents SD medium lacking leucine.

[0034] SD-Leu-FOA medium: Dissolve 0.1 g of 5-FOA in 1 mL of dimethyl sulfoxide, prepare fresh and use immediately. After filtration through an organic filter membrane, add to 100 mL of SD-Leu medium to obtain SD-Leu-FOA medium.

[0035] 2. The reagent information used in the following examples is as follows: The ClonExpress Ultra One Step Cloning Kit V3, T4 DNA ligase, 2×Phanta Max Master Mix, and 2×Taq PCR Master Mix were purchased from Nanjing Novizan Biotechnology Co., Ltd.; Deoxyribonucleic acid, single-stranded from salmon testes, was purchased from Sigma-Aldrich; DO-Supplement-Leu and DO-Supplement-Ura were purchased from Shanghai Maokang Biotechnology Co., Ltd.; DNA markers (DL5000 DNA Marker and DL15000 DNA Marker) were purchased from Thermo Scientific; and Yeast nitrogen base without amino acids (YNB) was purchased from Shanghai Bioengineering Co., Ltd. for the preparation of synthetic culture media. The DNA gel purification kit, bacterial plasmid extraction kit, yeast genome extraction kit, PCR primer synthesis, and sequencing services were provided by Sangon Biotech (Shanghai) Technology Service Co., Ltd.

[0036] 3. The conventional technical methods used in the following embodiments are as follows: (1) PCR amplification reaction: PCR system: 25 μL of 2× Phanta Max Master Mix or 2× TaqPCR Master Mix, 0.5 μL of forward primer, 0.5 μL of reverse primer, 1 μL of template, and 23 μL of ddH2O.

[0037] PCR reaction program: Pre-denaturation: 95°C for 5 min; Cycle program is executed 32 times, including denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 3 min; final extension at 72°C for 10 min; finally, cool down to 4°C for 3 min to finish.

[0038] (2) Preparation of competent Escherichia coli cells: Will E. coli JM109 was streaked on LB agar plates and incubated at 37°C for approximately 14 h. Then, a single colony was picked and inoculated into 1 mL of LB liquid medium and incubated at 37°C for approximately 14 h at 200 rpm. Finally, 0.5 mL of the bacterial culture was transferred to 50 mL of LB liquid medium and incubated at 37°C for 200 rpm until OD (dose elapsed). 600 Approximately 0.6. Aliquot 25 mL of bacterial culture into pre-chilled 50 mL centrifuge tubes, incubate on ice for 10 min, then centrifuge at 4000 rpm for 10 min at 4°C to collect the bacterial cells and discard the supernatant. Add 25 mL of pre-chilled CaCl2-MgCl2 solution (80 mmol / L MgCl2, 20 mmol / L CaCl2) to each tube of bacterial cells, resuspend the cells on ice, and centrifuge at 4000 rpm for 10 min to collect the cells and discard the supernatant. Resuspend the cells in 5 mL of pre-chilled 0.1 M CaCl2-glycerol solution (containing 0.1 mol / L CaCl2 and 25% glycerol), mix well, and aliquot 100 μL into pre-chilled 1.5 mL centrifuge tubes. Store at -80°C for later use.

[0039] (3) Validation of recombinant plasmids: All recombinant plasmids were transformed into Escherichia coli JM109. Single colonies were picked and cultured in LB-Amp medium. Plasmids were obtained using a plasmid miniprep kit and Sanger sequencing (NCBI Reference Sequences: XP_501650.1 and NP_009918.1) was performed to confirm the successful construction of the recombinant plasmids.

[0040] (4) Lithium acetate conversion method using Yersinia lipophila: Single colonies were picked and spread onto YPD solid medium and incubated at 30°C for 12 h. In a 1.5 mL centrifuge tube, 90 μL of PEG4000 (50% w / v), 5 μL of 1.0 M lithium acetate, 5 μL of deoxyribonucleic acid (single stranded from salmon testes), and 50-200 ng of plasmid or linearized fragment were added sequentially and mixed thoroughly. A sufficient amount of bacterial cells was picked up with an inoculation loop and added to the centrifuge tube, and the mixture was mixed thoroughly again. The 1.5 mL centrifuge tube was placed in a 30°C water bath for 30 min, with shaking performed every 10 min to mix. After heat shock at 39°C for 10 min, the cells were transferred to auxotrophic plates and incubated at 30°C for 3 days.

[0041] (5) Construction method of Yersinia lipophila Po1f ΔKu70 YLT3::Ilvs: The lithium acetate conversion method converts linearized fragments (containing YLT3 homologous arms, LoxP sequence-regulated Ura3 auxotrophic tags, and tandem segments in monocistronic form) YlIlv3 Gene, YlIlv5 Gene, YlIlv2 *Gene( YlIlv2* Genes are... YlIlv2 The gene obtained by deleting the ClaI restriction site in the coding region was transformed into *Yersinia lipolytica* Po1f ΔKu70. Positive colonies containing the linearized fragment were screened using SD-Ura medium—the *Yersinia lipolytica* Po1f ΔKu70 YLT3::URA3-Ilvs recombinant strain. The Ura3 tag was recovered using the Cre-LoxP system, and the pYLXP'-Cre plasmid was transformed into *Yersinia lipolytica* Po1f ΔKu70 YLT3::URA3-Ilvs recombinant strain using the lithium acetate method. Positive colonies were then screened using SD-Leu-FOA medium—*Yersinia lipolytica* Po1f ΔKu70. The recombinant YLT3::Ilvs / pYLXP'-Cre strain was prepared by passaged twice in YPD liquid medium and then plated onto YPD solid medium to remove the pYLXP'-Cre plasmid. Single colonies were picked and simultaneously imaged onto YPD and SD-Leu media. Positive bacteria that could only grow on YPD solid medium were selected, namely the engineered YLT3::Ilvs strain Po1f ΔKu70. Among them, the YlILV2 protein (accession number XP_501277.1 in the NCBI database) was encoded. YlIlv2 The gene's name in GeneBank is YALI1_C00293gThe protein encoding YlILV3 (serial number XP_502180.2 in the NCBI database) is... YlIlv3 The gene's name in GeneBank is YALI1_C32276g The protein encoding YlILV5 (serial number XP_502354.2 in the NCBI database) is... YlIlv5 The gene's name in GeneBank is YALI1_D03952g .

[0042] (6) Yeast Extract Culture Method: Single colonies were picked from agar plates and inoculated into 2 mL of fresh YPD or SD medium, and cultured in a shaker at 30°C and 220 rpm for approximately 24 h. Then, they were transferred to 250 mL Erlenmeyer flasks containing 25 mL of YPD or SD medium, allowing the initial OD in the flasks to adjust. 600 The concentration was 0.05, and the mixture was incubated in a constant temperature shaker at 30°C and 220 rpm for 36-72 h.

[0043] (7) Screening method for DL-valine tolerance in Yersinia lipolytica: Using SD-Ura medium, at 30 o C. The bacterial cells were activated at 200 rpm for 2 days; they were then inoculated at a 2% (v / v) inoculation rate into 25 mL of SD-Ura medium containing different concentrations (0-2000 mg / L) of DL-valine for further culture and screening; the OD600 value of the bacterial cells in the shake flasks was detected by an enzyme-linked immunosorbent assay (ELISA) reader to reflect the change in bacterial cell mass after 36 h of culture; DL-valine tolerance was measured by the bacterial cell OD600 value. 600 The results and the corresponding DL-valine content in the culture medium were obtained by calculating the IC50 using dose-response curves fitted with GraphPad Prism software. 50 .

[0044] (8) Sample preparation method for amino acid detection in Yersinia lipolyticis: 1) Using SD-Ura medium, at 30 o C. Activate the bacterial cells at 200 rpm for 2 days; 2) Transfer to 25 mL of SD-Ura medium at an inoculum of 2% (v / v) and incubate at 30°C for 15 h; 3) 4 o C. Collect bacterial cells by centrifugation at 12000 rpm, wash twice with ultrapure water, and then divide into two equal portions; 4) 50 o Under C conditions, one sample was dried by forced air drying, and the cell dry weight was measured; 5) 110 o Another sample was acidified with 500 μL of 12 M HCl for 14 h under C conditions, and then at 80°C. oAfter drying under C conditions for 1 day, dissolve the sample powder in 500 μL of pure water; 6) Take 400 μL of the above solution, place it in a centrifuge tube, add 200 μL of 1 mol / L triethylamine-acetonitrile solution and 200 μL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution, shake to mix, and incubate for 20-30 minutes. o The derivatization reaction was carried out by placing the sample in the dark for 1 h. 800 μL of n-hexane was added to terminate the reaction. The mixture was vortexed and allowed to stand for 10 min. After centrifugation at 5000 r / min for 5 min, the lower layer was collected to obtain the sample derivatization solution. Simultaneously, amino acid standard derivatization solutions were obtained. 7) The amino acid standard derivatization solutions and sample derivatization solutions were detected using liquid chromatography at a wavelength of 254 nm. The amino acid content in the sample was calculated using the standard curves of valine, isoleucine, and leucine standards, and their proportion of cell dry weight was also calculated.

[0045] (9) Method for detecting amino acid derivatized solutions using liquid chromatography: Detection was performed using an Agilent 1260 liquid chromatograph loaded with C 18 Chromatographic column (250 mm × 4.6 mm, 5 μm), column temperature set at 43 °C. o C. The detection wavelength was set to 254 nm, the injection volume to 10 μL, and the flow rate to 1.0 mL / min. Mobile phase A was 0.1 mol / L sodium acetate-acetonitrile (93:7, pH 6.5) buffer, and mobile phase B was acetonitrile-water (4:1). The duration of a single detection was 50 min. The percentage of mobile phase A changed as follows: from 100% at 0 min, it gradually changed to 97% in the first 17 min, and then rapidly decreased to 79% in the 17-18 min, where it remained for 22 min; in the 40-41 min, it rapidly decreased to 0% and remained for 4 min; in the 45-46 min, it rapidly increased to 100% and remained for 4 min.

[0046] Example 1: Construction of the ILV6 protein mutant in Yersinia lipophila 1. DNA extraction: Yersinia lipolytica Po1f was streaked onto YPD solid medium. Yarrowia lipolytica Po1f was selected as a single clone and inoculated into YPD liquid medium. After culturing for 24 hours, DNA was extracted from Po1f of Yeast lipolytica using the Yeast Genomic DNA Extraction Kit from Tiangen Biotech (Beijing) Co., Ltd.

[0047] Saccharomyces cerevisiae S288C was streaked onto YPD solid medium. Saccharomyces cerevisiaeS288C), single clones were picked and inoculated into YPD liquid medium. After culturing for 24 hours, the DNA of Saccharomyces cerevisiae was extracted using the yeast genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0048] 2. Primer design and PCR amplification of the target gene fragment: Based on the whole genome data of *Yersinia lipolyticis* strain Po1f from the National Center for Biotechnology Information (NCBI) database, a method for amplifying *Yersinia lipolyticis* Po1f was designed. YlIlv6 Gene( YlIlv6 The gene's name in GeneBank is YALI1_C13355g The primers YLILV6 pYLXP'2 F (5'-cagcactttttgcagtacatgcttggaaaacgatttgtgggtc-3') and YLILV6 pYLXP'2 R (5'-tagcacgcgtgtagatacctaaccaggaggcagagaggtctgg-3') were used (corresponding Gene ID 2909788). Using the *Yeast Rice* Po1f genomic DNA extracted in step 1 as a template, PCR amplification was performed using primers YLILV6pYLXP'2 F / YLILV6 pYLXP'2 R to obtain the encoding gene of the YlILV6 protein (i.e., the wild-type YlILV6 protein, whose protein sequence number in the NCBI database is XM_501650.3). YlIlv6 (903 bp in size), the amplified sequence was recovered using a gel extraction kit after agarose gel electrophoresis, and the amplified sequence was also analyzed. YlIlv6 Homologous arms of the vector SnaBI restriction site are introduced at both ends of the gene fragment.

[0049] Based on the whole genome data of Saccharomyces cerevisiae S288C strain from the NCBI database, a method for amplifying Saccharomyces cerevisiae S288C was designed. ScIlv6 Gene( ScIlv6Primers Sc ILV6pYLXP'2 F (5'-cagcactttttgcagtacatgctgagatcgttattgcaaagcg-3') and Sc ILV6 pYLXP'2 R (5'-tagcacgcgtgtagatacctaaccaggtggtagttgggaaatg-3') were used to amplify the ScILV6 protein (i.e., wild-type ScILV6 protein, whose protein sequence number in the NCBI database is NM_001178658.1) using the *Saccharomyces cerevisiae* genomic DNA extracted in step 1 as a template. Using these primers, PCR was performed to amplify the ScILV6 protein using primers Sc ILV6 pYLXP'2 F / Sc ILV6 pYLXP'2 R, obtaining the encoding gene for the ScILV6 protein (i.e., the wild-type ScILV6 protein, whose protein sequence number in the NCBI database is NM_001178658.1). ScIlv6 (966 bp in size), the amplified sequence was recovered using a gel extraction kit after agarose gel electrophoresis, and simultaneously... ScIlv6 Homologous arms of the vector SnaBI restriction site are introduced at both ends of the gene fragment.

[0050] 3. Construction of recombinant plasmids: The vector pYLXP'2 (purchased from Baosai Biotechnology) was linearized using the SnaBI enzyme and purified by a gel extraction kit for use as the backbone for recombinant plasmid construction. The linearized vector pYLXP'2 was then combined with the plasmid obtained in step 2. YlIlv6 Gene fragments were assembled using the ClonExpress Ultra One Step Cloning Kit V3 (Nanjing Novizan Biotechnology Co., Ltd.) to obtain the recombinant plasmid pYLXP'2-YlILV6 (structural diagram shown in Figure 1). Figure 2 (As shown).

[0051] The vector pYLXP'2 (purchased from Baosai Biotechnology) was linearized using the SnaBI enzyme and purified by column chromatography for use as the backbone for recombinant plasmid construction. The linearized vector pYLXP'2 was then combined with the plasmid obtained in step 2. ScIlv6 The gene fragments were assembled using the ClonExpress UltraOne Step Cloning Kit V3 (Nanjing Novizan Biotechnology Co., Ltd.) to obtain the recombinant plasmid pYLXP'2-ScILV6.

[0052] 4. Amplification of the coding gene sequence of the ILV6 mutant: Using pYLXP'2-YlILV6 as a template, site-directed mutagenesis was performed on 78_81YLILV6F / 78YLILV6R using primers (the resulting mutant protein, compared to the wild-type YlILV6 protein, only underwent a site-directed mutation of asparagine at position 78 of the wild-type YlILV6 protein sequence, mutating it to Ala, while other amino acids remained unchanged), resulting in linearized L-pYLXP'2-YlILV6 Asn78Ala. Similarly, site-directed mutagenesis was performed on 78_81YLILV6F / 81YLILV6R using primers (the resulting mutant protein, compared to the wild-type YlILV6 protein, only underwent a site-directed mutation of glycine at position 81 of the wild-type YlILV6 protein sequence, mutating it to Asp, while other amino acids remained unchanged), resulting in linearized L-pYLXP'2-YlILV6. Using Gly81Asp, site-directed mutagenesis was performed on 96YLILV6F / 96YLILV6R primers (the resulting mutant protein, compared to the wild-type YlILV6 protein, only mutated the asparagine at position 96 of the wild-type YlILV6 protein amino acid sequence, changing it to His, while other amino acid positions remained unchanged) to obtain linearized L-pYLXP'2-YlILV6Asn96His. The PCR amplification products of linearized L-pYLXP'2-YlILV6Asn78Ala, linearized L-pYLXP'2-YlILV6Gly81Asp, and linearized L-pYLXP'2-YlILV6Asn96His were digested with DpnI, then their sizes were checked by agarose gel extraction, and the products were recovered using a gel extraction kit to obtain linearized L-pYLXP'2-YlILV6. The primer pairs consist of the Asn78Ala fragment, the linearized L-pYLXP'2-YlILV6 Gly81Asp fragment, and the linearized L-pYLXP'2-YlILV6 Asn96His fragment. The sequences of each primer pair are as follows: 78_81YLILV6F: 5'-gttctgtcgcgagttgccggcactc-3'; 78YLILV6R: 5'-gcaactcgcgacagaacaccgggttcggcctgaaccagacagttgaacacgtgt-3'; 81YLILV6R: 5'-gcaactcgcgacagaacatcgggttcgttctgaaccagacagttgaacacgtgt-3'; 96YLILV6F: 5'-gattcgctggtagtgtgcaacacgg-3'; 96YLILV6R: 5'-gcacactaccagcgaatcaatgtggaagcctcgagacgccagagtgccg-3'.

[0053] Using pYLXP'2-ScILV6 as a template, site-directed mutagenesis was performed on ScILV6 Asn86Ala F / ScILV6 86 89 R using primers (the resulting mutant protein, compared to the wild-type ScILV6 protein, only mutated the asparagine at position 86 of the wild-type ScILV6 protein sequence, changing it to Ala, while other amino acids remained unchanged), resulting in linearized L-pYLXP'2-ScILV6Asn86Ala. Then, site-directed mutagenesis was performed on ScILV6 Gly89Asp F / ScILV6 8689 R using primers (the resulting mutant protein, compared to the wild-type ScILV6 protein, only mutated the glycine at position 89 of the wild-type ScILV6 protein sequence, changing it to Asp, while other amino acids remained unchanged), resulting in linearized L-pYLXP'2-ScILV6 Gly89Asp. Site-directed mutagenesis was performed on Asn104His F / ScILV6 Asn104His R (resulting in a protein mutant where, compared to wild-type ScILV6, the mutant only mutates asparagine at position 104 of the wild-type ScILV6 amino acid sequence, replacing it with His, while other amino acids remain unchanged) to obtain linearized L-pYLXP'2-ScILV6Asn104His. The PCR amplification products of linearized L-pYLXP'2-ScILV6 Asn86Ala, L-pYLXP'2-ScILV6Gly89Asp, and L-pYLXP'2-ScILV6Asn104His were digested with DpnI, then examined by agarose gel electrophoresis and recovered using a gel extraction kit to obtain linearized L-pYLXP'2-ScILV6. The primer pairs consist of the Asn86Ala fragment, the linearized L-pYLXP'2-ScILV6 Gly89Asp fragment, and the linearized L-pYLXP'2-ScILV6 Asn104His fragment. The sequences of each primer pair are as follows: ScILV6 Asn86Ala F: 5'-gaactgtttggtgcaagccgaaccc-3'; ScILV6 86 89 R: 5'-ttgcaccaaacagttcaagacatgc-3'; ScILV6 Gly89Asp F: 5'-gaactgtttggtgcaaaacgaacccgatgtct-3'; ScILV6 Asn104His F: 5'-gctgccagaggctttcacattgattcg-3'; ScILV6 Asn104His R: 5'-aaagcctctggcagctaacgtacccg-3'.

[0054] 5. Construction of ILV6 mutant recombinant plasmid: The recombinant plasmids pYLXP'2-YlILV6 Asn78Ala, pYLXP'2-YlILV6 Gly81Asp, pYLXP'2-YlILV6 Asn96His, pYLXP'2-ScILV6 Asn86Ala, pYLXP'2-ScILV6 Gly89Asp, and pYLXP'2-ScILV6 Asn104His were obtained using the ClonExpress Ultra One Step Cloning Kit V3 (Nanjing Novizan Biotechnology Co., Ltd.). The recombinant plasmids pYLXP'2-ScILV6Asn86Ala, pYLXP'2-ScILV6Gly89Asp, and pYLXP'2-ScILV6Asn104His were used to express the YlILV6Asn78Ala protein mutant (compared to the wild-type YlILV6 protein, the YlILV6 Asn78Ala protein mutant has only the 78th amino acid position Asn mutated to Ala, while other amino acid positions remain unchanged) and the YlILV6Gly81Asp protein mutant (compared to the wild-type YlILV6 protein, the YlILV6 Gly81Asp protein mutant has only the 81st amino acid position Gly mutated to Asp, while other amino acid positions remain unchanged, as shown in Sequence 1) in *YlILV6*. The following are mutant variants of the ScILV6 protein: Asn96His (compared to wild-type YlILV6 protein, the Asn96His protein mutant has only the 96th amino acid position of the wild-type YlILV6 protein mutated from Asn to His, while other amino acid positions remain unchanged); ScILV6 Asn86Ala (compared to wild-type ScILV6 protein, the Asn86Ala protein mutant has only the 86th amino acid position of the wild-type ScILV6 protein mutated from Asn to Ala, while other amino acid positions remain unchanged); ScILV6 Gly89Asp (compared to wild-type ScILV6 protein, the Gly89Asp protein mutant has the 89th amino acid position of the wild-type ScILV6 protein mutated from Gly to Asp, while other amino acid positions remain unchanged); and ScILV6 Asn104His (compared to wild-type ScILV6 protein...).The ScILV6 Asn104His protein mutant is formed by mutating Asn to His at position 104 of the wild-type ScILV6 protein amino acid sequence, while keeping other amino acid positions unchanged.

[0055] Example 2: Construction of Yersinia lipophila Po1f ΔKu70 YLT3::Ilvs strain 1. Genomic DNA extraction from Yersinia lipophila strain Po1f The method for extracting genomic DNA from the Yersinia lipophila Po1f strain is the same as step 1 in Example 1.

[0056] 2. Construction of the recombinant plasmid pYLXP'2-YlILV3-YlILV5-YlILV2*: The construction of the pYLXP'2-YlILV3-YlILV5-YlILV2* recombinant plasmid can be achieved through direct artificial synthesis. YlIlv3 Gene, YlIlv5 Gene, YlIlv2 *Gene( YlIlv2* Genes are... YlIlv2 (The gene obtained by deleting the Cla I restriction site in the coding region), and YlIlv3 Gene, YlIlv5 Gene, YlIlv2 The gene was tandemly linked in a monocistronic form to obtain the YlILV3-YlILV5-YlILV2* gene fragment, which was then linked with the linearized pYLXP'2 vector to construct the gene. Alternatively, it can be constructed using the following steps: (1) Primer design and PCR amplification of the target gene fragment: Based on the whole genome data of *Yersinia lipolyticis* strain Po1f from the NCBI database, primer pairs YL ILV2pYLXP'2 F1 / YL ILV2 pYLXP'2 R1, YL ILV2 pYLXP'2 F2 / YL ILV2 pYLXP'2 R2, YLILV3 pYLXP'2 F / YL ILV3 pYLXP'2 R, and YLILV5 pYLXP'2 F / YL ILV5 pYLXP'2 R were designed. Using the *Yersinia lipolyticis* Po1f genomic DNA extracted in step 1 as a template, primer pair YL ILV2 pYLXP'2 F1 / YLILV2 pYLXP'2 R1 was used to amplify the *Yersinia lipolyticis* Po1f genome. YlIlv2-1The gene (see NCBI database for specific nucleotide sequence: NC_090772.1:c29952-29830) was amplified using primer pair YL ILV2 pYLXP'2 F2 / YL ILV2 pYLXP'2R2 to amplify the *Yersinia lipophila* Po1f gene. YlIlv2-2 The gene (see NCBI database for specific nucleotide sequence: NC_090772.1:c29365-27442) was amplified using primer pair YL ILV3 pYLXP'2 F / YL ILV3 pYLXP'2 R. YlIlv3 The gene (SEQ ID NO:48) was amplified using primer pair YLILV5 pYLXP'2 F / YLILV5 pYLXP'2 R, representing the *Yersinia lipophilia* Po1f gene. YlIlv5 Gene (SEQ ID NO:51), and simultaneously obtained during amplification YlIlv2-1 Gene fragments, YlIlv2-2 Gene fragments, YlIlv3 Gene fragments, YlIlv5 Homologous arms of the vector SnaBI restriction site were introduced at both ends of the gene fragment. The sequences of each primer are as follows: YlILV2-pYLXP'2 F1:5'-cagcactttttgcagtactaatgcttcatttggaaatatgccgata-3'; YlILV2-pYLXP'2 R1: 5'-tcagcagttgccggctgggcagcag-3'; YlILV2-pYLXP'2 F2: 5'-ccagccggcaactgctgaacgatcccagcccgctccccacttc-3'; YlILV2-pYLXP'2 R2: 5'-tagcacgcgtgtagatacttagttctcaaaggtggtctggtgg-3'; YlILV3-pYLXP'2 F: 5'-aatgattcgagcacgaaactacgctactaaggcgcacactttg-3'; YlILV3-pYLXP'2 R: 5'-tagcacgcgtgtagatacttaggcgtcagtgatacagccagtg-3'; YlILV5-pYLXP'2 F: 5'-cagcactttttgcagtactaacgacgctgaaagtccgccgattttt-3'; YlILV5-pYLXP'2 R: 5'-tagcacgcgtgtagatacttagttgttctcgggtcgcagcttt-3'.

[0057] (2) Construction of recombinant plasmids: The vector pYLXP'2 was linearized using SnaBI enzyme, purified by a gel extraction kit column, and then compared with the sample obtained in step (1). YlIlv2-1 gene fragments ,lIlv2-2 The gene fragment was obtained and assembled into the recombinant plasmid pYLXP'2-YlILV2 using the ClonExpress Ultra One Step Cloning Kit V3. The vector pYLXP'2 was linearized using SnaBI enzyme, purified by a gel extraction kit, and then compared with the gene fragment obtained in step (1). YlIlv3 Gene fragments, YlIlv5 The gene fragments were assembled using the ClonExpress Ultra One Step Cloning Kit V3 to construct recombinant plasmids pYLXP'2-YlILV3 and pYLXP'2-YlILV5.

[0058] (3) Construction of pYLXP'2-YlILV3-YlILV5 recombinant plasmid: The pYLXP'2-YlILV3 recombinant plasmid was digested with ClaI and AvrII to obtain 232 bp and 7872 bp fragments, and the 7872 bp plasmid backbone was recovered. The pYLXP'2-YlILV5 recombinant plasmid was double-digested with ClaI and NheI to obtain 2527 bp and 5130 bp fragments, and the 2527 bp target gene fragment was recovered. The recovered 7872 bp plasmid backbone was integrated with the 2527 bp target gene fragment using T4 ligase to form a linear pYLXP'2-YlILV3-YlILV5 fragment. Enzyme digestion with NdeI was used for verification. The control bands were 5792 bp, 2742 bp, and 1865 bp, and the positive bands were 6563 bp, 2742 bp, 2295 bp, and 1865 bp.

[0059] (4) Construction of pYLXP'2-YlILV2* recombinant plasmid: Using the primer pair YlILV2 ClaI F (5'-cattgacattgatggtgacgcttct-3') and YlILV2 ClaI R (5'-cgtcaccatcaatgtcaatgacgtc-3'), the pYLXP'2-YlILV2 recombinant plasmid was linearized via PCR to delete the pYLXP'2-YlILV2 recombinant plasmid as a template. YlIlv2 The ClaI restriction site in the gene coding region was used to obtain a product containing the YlILV2* fragment. The product containing the YlILV2* fragment was digested with DpnI enzyme, and then the 8428 bp fragment was recovered by gel extraction. The recovered 8428 bp fragment was self-circulated using the ClonExpress Ultra One Step Cloning Kit V3 (Nanjing Novizan Biotechnology Co., Ltd.) to construct the pYLXP'2-YlILV2* recombinant plasmid.

[0060] (5) Construction of pYLXP'2-YlILV3-YlILV5-YlILV2* recombinant plasmid: The linear pYLXP'2-YlILV3-YlILV5 fragment obtained in step (3) was digested with ClaI and AvrII to obtain 232 bp and 10167 bp fragments, and the 10167 bp plasmid backbone was recovered. The pYLXP'2-YlILV2* recombinant plasmid was double-digested with ClaI and NheI to obtain 3298 bp and 5130 bp fragments, and the 3298 bp target gene fragment was recovered. The recovered 10167 bp plasmid backbone was ligated with the 3298 bp target gene fragment using T4 ligase to integrate into the pYLXP'2-YlILV3-YlILV5-YlILV2* recombinant plasmid. Enzyme digestion with NdeI was used for verification. The control bands were 6239 bp and 1865 bp, and the positive bands were 5792 bp, 2742 bp and 1865 bp.

[0061] 3. Construction of the recombinant plasmid pUC57-YLT3-URA3-loxP-YlILV3-YlILV5-YlILV2*: (1) Construction of pUC57-YLT3 recombinant plasmid: Based on the whole genome data of *Yersinia lipolyticis* strain Po1f from the NCBI database, primer pair YLT3-BamHI-F / YLT3-HindIII-R was designed. Using the *Yersinia lipolyticis* Po1f genomic DNA extracted in step 1 as a template, primer pair YLT3-BamHI-F / YLT3-HindIII-R was used to amplify the genomic integration homologous arm sequence. YLT3(370bp). The sequences of each primer are as follows: YLT3-BamHI-F: 5'-atcggatcccatgctatattctatgaattcaggc-3'; YLT3-HindIII-R: 5'-caagctttacattagtattgcaattttaatga-3'.

[0062] The 2674bp backbone of the vector pUC57 (Baosai Biotechnology) was recovered after digestion with BamHI / HindIII and gel extraction. The amplified... YLT3 The target gene fragment of 376 bp was recovered after digestion with BamHI / HindIII using a gel extraction kit. The recovered 2674 bp backbone was ligated with the 376 bp target gene fragment using T4 ligase to construct the pUC57-YLT3 recombinant plasmid.

[0063] (2) Construction of pYLXP'-URA3-loxP recombinant plasmid: The URA3 fragment on the pYLXP'2 vector was amplified using primer pairs YlURA3F / YlURA3R, and then integrated into the SnaBI-linearized pYLXP' vector (Baosai Biotechnology) in one step to construct the pYLXP'-URA3 recombinant vector. The lox71 sequence was integrated into the AvrII site of the pYLXP'-URA3 recombinant vector using primer pairs lox71-AvrII F and lox71-AvrII R, and the lox66 sequence was integrated into the NheI site of the pYLXP'-URA3 recombinant vector using primer pairs lox66-NheI F and lox66-NheI R, thus constructing the pYLXP'-URA3-loxP recombinant vector. The sequences of each primer are as follows: YlURA3F: 5'-ccagcactttttgcagtactaaccgcagccctcctacgaagctcgagc-3'; YlURA3R: 5'-catagcacgcgtgtagatacctaacagttaatcttctggtaagcc-3'; lox71-AvrII F: 5'-tccctaaatttgatgaaagcctaggtaccgttcgtatagcatacattatacgaagttat-3'; lox71-AvrII R: 5'-ccaacccggtctctgtcgtcataacttcgtataatgtatgctatacgaacggta-3'; lox66-NheI F: 5'-cgttatcaaatctagttaataacttcgtatagcatacattatacgaacgtta-3'; lox66-NheI R: 5'-actcctccgttatttgtctcgctagctaacgttcgtataatgtatgctatacgaagttat-3'.

[0064] (3) Construction of the pUC57-YLT3-URA3-loxP recombinant plasmid: Using the pUC57-YLT3 plasmid constructed in step (1) as a template, PCR amplification was performed using primers L-YLT3 F and L-YLT3 R to obtain a linearized L-pUC57-YLT3 with a size of 3050 bp. Using the pYLXP'-URA3-loxP constructed in step (2) as a template, PCR amplification was performed using primers URA3-LoxP_YLT3 F and URA3-LoxP_YLT3 R to obtain a L-URA3-LoxP gene fragment with a size of 2273 bp. The linearized L-pUC57-YLT3 (3050 bp) and the L-URA3-LoxP gene fragment (2273 bp) were circularized using the ClonExpressUltraOneStepCloning Kit V3 (Nanjing Novizan Biotechnology Co., Ltd.) to obtain the pUC57-YLT3-URA3-loxP recombinant plasmid. The sequences of each primer are as follows: L-YLT3 F: 5'-tcattcaccagaaaagagattctag-3'; L-YLT3 R: 5'-gcaagcacttctggtgtgtgctact-3'; URA3-LoxP_YLT3 F: 5'-gaatccaaagattcctctccctaaatttgatgaaagcctagg-3'; URA3-LoxP_YLT3 R: 5'-gtcgactcctccgttattgtctcgc-3'.

[0065] (4) Construction of the recombinant plasmid pUC57-YLT3-URA3-loxP-YlILV3-YlILV5-YlILV2*: The pUC57-YLT3-URA3-loxP recombinant plasmid obtained in step (3) was digested with ClaI / AvrII to obtain 232bp and 5055bp fragments. The 5055bp vector backbone fragment (i.e., the pUC57-YLT3-URA3 fragment) was recovered by gel extraction. The pYLXP'2-YlILV3-YlILV5-YlILV2* recombinant plasmid obtained in step 2 was digested with ClaI / NheI to obtain 8335bp and 5130bp fragments. The fragment with a band size of 8335bp was recovered by gel extraction, which is YlILV3-YlILV5-YlILV2*. The pUC57-YLT3-URA3 fragment (5055 bp) and the YlILV3-YlILV5-YlILV2* fragment were ligated using T4 ligase to obtain the recombinant plasmid pUC57-YLT3-URA3-loxP-YlILV3-YlILV5-YlILV2*.

[0066] 4. Construction of the *Yarrowia lipophila* Po1f ΔKu70 YLT3::Ilvs strain: The recombinant plasmid pUC57-YLT3-URA3-loxP-YlILV3-YlILV5-YlILV2* constructed in step 3 was digested with EcoRI and HindIII to obtain 2695 bp and 10695 bp fragments. The 10695 bp fragment was recovered by gel electrophoresis and became the YLT3-URA3-YlILV3-YlILV5-YlILV2* fragment. The YLT3-URA3-YlILV3-YlILV5-YlILV2* fragment was transformed into *Y. lipolyticis* Po1f ΔKu70 using the lithium acetate method. Recombinant engineered *Y. lipolyticis* Po1f ΔKu70 YLT3::URA3-Ilvs strain was obtained through auxotrophic plate selection. The pYLXP'-Cre plasmid (from Baosai Biotechnology; the pYLXP'-Cre plasmid is used to recover the URA3 fragment from the integrated fragment, located between lox71 and lox66 sequences. The Cre protein can target and cleave the URA3 fragment tandemly between lox71 and lox66) was then transformed into *Y. lipolyticis* Po1f ΔKu70 YLT3::URA3-Ilvs strain. *Y. lipolyticis* Po1f was obtained through selection on SD-Leu-FOA medium. The ΔKu70YLT3::Ilvs / pYLXP'-Cre strain was cultured and passaged in YPD, resulting in the loss of the pYLXP'-Cre plasmid, thus obtaining the YK70YLT3::Ilvs strain (denoted as YKI strain).

[0067] Example 3: Preliminary screening of engineered bacteria containing ILV6 mutant The recombinant plasmids pYLXP'2-YlILV6 Asn78Ala, pYLXP'2-YlILV6 Gly81Asp, pYLXP'2-YlILV6 Asn96His, pYLXP'2-ScILV6 Asn86Ala, pYLXP'2-ScILV6 Gly89Asp, and pYLXP'2-ScILV6 Asn104His constructed in Example 2 were transformed into the *Yarrowia lipolyticis* Po1f ΔKu70 YLT3::Ilvs strain (i.e., the YKI strain) constructed in Example 2 using the lithium acetate transformation method, resulting in six ILV6 mutant engineered strains, which were subsequently designated as YKI / pYLXP'2-YlILV6 Asn78Ala, YKI / pYLXP'2-YlILV6, and YKI / pYLXP'2-YlILV6 strains. The following strains were used: Gly81Asp, YKI / pYLXP'2-YlILV6 Asn96His, YKI / pYLXP'2-ScILV6Asn86Ala, YKI / pYLXP'2-ScILV6 Gly89Asp, and YKI / pYLXP'2-ScILV6Asn104His. Simultaneously, the pYLXP'2 plasmid was transformed into YKI strains using the lithium acetate method to construct the YKI / pYLXP'2 strain, which served as a blank control. The recombinant plasmids pYLXP'2-YlILV6 and pYLXP'2-ScILV6 were transformed into YKI strains using the lithium acetate method, respectively, to construct the YKI / pYLXP'2-YlILV6 and YKI / pYLXP'2-ScILV6 strains, which served as control strains. In this embodiment, the expression of all genes is controlled by the TEF promoter and XPR2 terminator elements on the pYLXP'2 vector.

[0068] The strains constructed above were subjected to DL-valine tolerance screening experiments, and the results are shown in Table 1 and 2. Figure 3 As shown.

[0069] From Table 1 and Figure 3 It was found that overexpression of ILV6 could improve the tolerance of engineered bacteria to DL-valine. Among them, the overexpression of YlILV6 in *Yarrowia lipolytica* was less effective than that of ScILV6, but the application effect of the YlILV6 mutant was significantly better than that of the ScILV6 mutant. Furthermore, the YKI / pYLXP'2-YlILV6 Gly81Asp strain showed the best tolerance to DL-valine, with an IC50 value of [missing value]. 50 The concentration reached 982.4 mg / L, showing a significant difference compared to the blank control strain YKI / pYLXP'2.

[0070] Example 4: Screening of a library of point saturation mutants of glycine at position 81 (ILV6 Gly81) in the amino acid sequence of Yersinia lipolyticis ILV6 protein. Primer 78_81 YL ILV6 F was combined with the reverse primer. YlIlv6 Gly81Ala R、 YlIlv6 Gly81Arg R、 YlIlv6 Gly81Asn R、 YlIlv6 Gly81Cys R、 YlIlv6 Gly81Gln R、 YlIlv6 Gly81Glu R、 YlIlv6 Gly81His R、 YlIlv6 Gly81Ile R、 YlIlv6 Gly81Leu R、 YlIlv6 Gly81Lys R、 YlIlv6 Gly81Met R、 YlIlv6 Gly81Phe R、 YlIlv6 Gly81Pro R YlIlv6 Gly81Ser R、 YlIlv6 Gly81Thr R、 YlIlv6 Gly81Trp R、 YlIlv6 Gly81Tyr R、 YlIlv6 Using primer pairs composed of Gly81Val R and the pYLXP'2-YlILV6 plasmid as a template, a point-saturated mutant gene library encoding the YlILV6 protein mutant was constructed using PCR technology. YlIlv6 Gly81Ala、 YlIlv6 Gly81Arg YlIlv6 Gly81Asn、 YlIlv6 Gly81Cys YlIlv6 Gly81Gln、 YlIlv6 Gly81Glu、 YlIlv6 Gly81His、 YlIlv6 Gly81Ile、 YlIlv6 Gly81Leu、 YlIlv6 Gly81Lys、 YlIlv6 Gly81Met YlIlv6 Gly81Phe、 YlIlv6 Gly81Pro YlIlv6 Gly81Ser、 YlIlv6 Gly81Thr、 YlIlv6 Gly81Trp YlIlv6 Gly81Tyr、 YlIlv6(Gly81Val). Among them, the sequences of each reverse primer are as follows: YlIlv6 Gly81Ala R: 5’-GCAACTCGCGACAGAACGGCGGGTTCGTTCTGAACCAGACAGTT-3’; YlIlv6 Gly81Arg R: 5’-GCAACTCGCGACAGAACTCGGGGTTCGTTCTGAACCAGACAGTT-3’; YlIlv6 Gly81Asn R: 5’-GCAACTCGCGACAGAACGTTGGGTTCGTTCTGAACCAGACAGTT-3’; YlIlv6 Gly81Cys R: 5’-GCAACTCGCGACAGAACACAGGGTTCGTTCTGAACCAGACAGTT-3’; YlIlv6 Gly81Gln R: 5’-GCAACTCGCGACAGAACCTGGGGTTCGTTCTGAACCAGACAGTT-3’; YlIlv6 Gly81Glu R: 5’-GCAACTCGCGACAGAACCTCGGGTTCGTTCTGAACCAGACAGTT-3’; YlIlv6 Gly81His R: 5’-GCAACTCGCGACAGAACGTGGGGTTCGTTCTGAACCAGACAGTT-3’; YlIlv6 Gly81Ile R: 5’-GCAACTCGCGACAGAACGATGGGTTCGTTCTGAACCAGACAGTT-3’; YlIlv6 Gly81Leu R: 5’-GCAACTCGCGACAGAACCAGGGGTTCGTTCTGAACCAGACAGTT-3’; YlIlv6 Gly81Lys R: 5'-GCAACTCGCGACAGAACCTTGGGTTCGTTCTGAACCAGACAGTT-3'; YlIlv6 Gly81Met R: 5'-GCAACTCGCGACAGAACCATGGGTTCGTTCTGAACCAGACAGTT-3'; YlIlv6 Gly81Phe R: 5'-GCAACTCGCGACAGAACGAAGGGTTCGTTCTGAACCAGACAGTT-3'; YlIlv6 Gly81Pro R: 5'-GCAACTCGCGACAGAACGGGGGGTTCGTTCTGAACCAGACAGTT-3'; YlIlv6 Gly81Ser R: 5'-GCAACTCGCGACAGAACAGAGGGTTCGTTCTGAACCAGACAGTT-3'; YlIlv6 Gly81Thr R: 5'-GCAACTCGCGACAGAACGGTGGGTTCGTTCTGAACCAGACAGTT-3'; YlIlv6 Gly81Trp R: 5'-GCAACTCGCGACAGAACCCAGGGTTCGTTCTGAACCAGACAGTT-3'; YlIlv6 Gly81Tyr R: 5'-GCAACTCGCGACAGAACGTAGGGTTCGTTCTGAACCAGACAGTT-3'; YlIlv6 Gly81Val R: 5'-GCAACTCGCGACAGAACCACGGGTTCGTTCTGAACCAGACAGTT-3'。

[0071] The coding gene sequences of each protein mutant were assembled with the SnaBI-linearized vector pYLXP'2 (purchased from Baosai Biotechnology) using the ClonExpress Ultra One Step Cloning Kit V3 (Nanjing Novizan Biotechnology Co., Ltd.) to obtain recombinant plasmids pYLXP'2-YlILV6 Gly81Ala, pYLXP'2-YlILV6Gly81Arg, pYLXP'2-YlILV6 Gly81Asn, pYLXP'2-YlILV6 Gly81Cys, pYLXP'2-YlILV6Gly81Gln, pYLXP'2-YlILV6 Gly81Glu, pYLXP'2-YlILV6 Gly81His, pYLXP'2-YlILV6Gly81Ile, and pYLXP'2-YlILV6 Gly81Leu, pYLXP'2-YlILV6 Gly81Lys, pYLXP'2-YlILV6Gly81Met, pYLXP'2-YlILV6 Gly81Phe, pYLXP'2-YlILV6 Gly81Pro, pYLXP'2-YlILV6Gly81Ser, pYLXP'2-YlILV6 Gly81Thr, pYLXP'2-YlILV6 Gly81Trp, pYLXP'2-YlILV6Gly81Tyr, pYLXP'2-YlILV6 Gly81Val;The recombinant plasmids described above were transformed into the *Yarrowia lipolyticis* Po1f ΔKu70 YLT3::Ilvs strain constructed in Example 2 using the lithium acetate conversion method, resulting in the corresponding recombinant strains (details of each recombinant strain are shown in Table 2). Each recombinant strain expressed the following protein mutants: YlILV6Gly81Ala, YlILV6Gly81Arg, YlILV6Gly81Asn, YlILV6Gly81Cys, YlILV6Gly81Gln, YlILV6Gly81Glu, YlILV6Gly81His, YlILV6Gly81Ile, YlILV6Gly81Leu, YlILV6Gly81Lys, YlILV6Gly81Met, and YlILV6... The following protein mutants were constructed: Gly81Phe, YlILV6Gly81Pro, YlILV6Gly81Ser, YlILV6Gly81Thr, YlILV6Gly81Trp, YlILV6Gly81Tyr, and YlILV6Gly81Val (compared to wild-type YlILV6 protein, each of these protein mutants only underwent a point saturation mutation at position 81 of the wild-type YlILV6 protein amino acid sequence, while other amino acid positions remained unchanged). DL-valine tolerance screening experiments were performed on the recombinant bacteria constructed above, and the results are shown in Table 2. Figure 4 As shown.

[0072] From Table 2 and Figure 4 It was found that, after 36 h of screening culture, compared with the recombinant engineered bacteria expressing wild-type YlILV6 protein, the recombinant engineered bacteria expressing YlILV6 Gly81 mutant protein all showed a certain degree of improved tolerance. Furthermore, the engineered bacteria YKI / pYLXP'2-YlILV6 Gly81Pro showed the best tolerance to DL-valine, with an IC50 value of [missing value]. 50 The concentration reached 986.6 mg / L; YKI / pYLXP'2-YlILV6Gly81Asp showed the second-lowest tolerance to DL-valine, with an IC50 concentration of 986.6 mg / L. 50 The concentration reached 982.2 mg / L; compared with the recombinant engineered strain YKI / pYLXP'2-YlILV6, the IC50 concentration was 982.2 mg / L. 50 A significant difference was observed at 506.1 mg / L. Furthermore, the IC50 concentration of the recombinant engineered strain YKI / pYLXP'2-YlILV6Gly81Tyr was [not specified]. 50The concentration reached 820.3 mg / L, which also showed a significant improvement in tolerability.

[0073] Example 5: Comparison of branched-chain amino acids in Yersinia lipolyticis fermentation with high tolerance to DL-valine. The recombinant engineered bacteria YKI / pYLXP'2-YlILV6 Gly81Asp, YKI / pYLXP'2-YlILV6 Gly81Tyr, and YKI / pYLXP'2-YlILV6 Gly81Pro, which exhibited superior DL-valine tolerance phenotypes in Examples 3 and 4, were used as controls. YKI strains containing YlILV6 and its mutants YlILV6 Asn78Ala and YlILV6 Asn96His were selected as controls. The changes in the dry weight percentage of intracellular branched-chain amino acids after fermentation of each recombinant engineered bacteria were detected according to the sample preparation method for Yersinia lipolytica amino acid detection. The detection results are as follows: Figure 5 As shown.

[0074] As Figure 5 It was found that after 14 h of inoculation and culture, compared with the YKI / pYLXP'2 strain (valine content 18.2 mg / g), the YKI / pYLXP'2-YlILV6 Gly81Asp recombinant engineered strain showed the best effect in increasing valine content, reaching 32.4 mg / g, an increase of 86.7%; the YKI / pYLXP'2-YlILV6 Gly81Pro recombinant engineered strain reached 29.2 mg / g, an increase of 68.2%; and the YKI / pYLXP'2-YlILV6 Gly81Tyr recombinant engineered strain increased valine content by 24.2%.

[0075] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may use the above technical content as inspiration to make changes or modifications. These are equivalent embodiments with variations. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical concept of the present invention still fall within the protection scope of the claims of the present invention.

Claims

1. A mutant of Yersinia lipophilia ILV6 protein, characterized in that, The ILV6 protein mutant is obtained by mutating glycine at position 81 of the amino acid sequence of the wild-type Yersinia lipolytica ILV6 protein; the ILV6 protein mutant is as follows: A1), A2), or A3). A1) The wild-type ILV6 protein was obtained by mutating glycine at position 81 to aspartic acid, and its amino acid sequence is shown in Sequence 1. A2) The ILV6 protein mutant is obtained by mutating glycine at position 81 of the wild-type ILV6 protein amino acid sequence to proline, and its amino acid sequence is shown in Sequence 2. A3) The ILV6 protein mutant is obtained by mutating glycine at position 81 of the wild-type ILV6 protein amino acid sequence to tyrosine, and its amino acid sequence is shown in Sequence 3.

2. The biological material associated with the ILV6 protein mutant of claim 1 is any one of the following: B1) A nucleic acid molecule encoding the ILV6 protein mutant of claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2).

3. The biomaterial according to claim 2, characterized in that, B1) The coding sequence of the nucleic acid molecule is shown in sequence 4, sequence 5 or sequence 6.

4. The use of the ILV6 protein mutant of claim 1 or the biological material of claim 2 or 3 in any of the following: C1) Application in the preparation of DL-valine-tolerant recombinant bacteria and / or the enhancement of DL-valine-tolerant recombinant bacteria; C2) Applications in the production of amino acids or / and in increasing amino acid yield; C3) Application in the preparation of recombinant bacteria for producing branched-chain long-chain alcohols and / or recombinant bacteria for increasing the yield of branched-chain long-chain alcohols; C4) Applications in the production of branched long-chain alcohols and / or in increasing the yield of branched long-chain alcohols; C5) Application in the preparation of recombinant bacteria for producing short-chain fatty acids or / and recombinant bacteria for increasing the yield of short-chain fatty acids; C6) Applications in the production of short-chain fatty acids or / and in increasing the yield of short-chain fatty acids.

5. A recombinant *Yarrowia lipophila* strain, characterized in that, The recombinant Yersinia lipophila contains the nucleic acid molecule described in claim 2 (B1), or the expression cassette described in claim 2 (B2), or the recombinant vector described in claim 3).

6. The recombinant bacteria according to claim 5, characterized in that, The recombinant *Yersinia lipolytica* strain is prepared by transforming the nucleic acid molecule described in claim B1), the expression cassette described in claim B2), or the recombinant vector described in claim B3) into *Yersinia lipolytica* using *Yersinia lipolytica* as the chassis cell.

7. The recombinant *Yarrowia lipophila* strain according to claim 6, characterized in that, The *Yersinia lipolyticis* strain is *Yersinia lipolyticis* Po1f strain, *Yersinia lipolyticis* Po1f ΔKu70 strain, or *Yersinia lipolyticis* Po1f ΔKu70 YLT3::Ilvs recombinant strain.

8. The recombinant *Yarrowia lipophila* strain according to claim 7, characterized in that, The recombinant *Y. lipolytica* Po1f ΔKu70 YLT3::Ilvs strain is based on the *Y. lipolytica* Po1f ΔKu70 strain as the chassis cell, and its genome is... YLT3 Random integration at the site in tandem in the form of monocistronic units YlIlv3 Gene, YlIlv5 Gene, YlIlv2* The gene was constructed to obtain the stated YlIlv2* Genes are... YlIlv2 The gene obtained by deleting the ClaI restriction site in the coding region of the gene.

9. The recombinant lipophilic Yersinia according to claim 8, characterized in that, The method for constructing the recombinant Yeast lipophilicus Po1f ΔKu70 YLT3::Ilvs strain is as follows: (1) Yeast extract from lipophilic yeast YLT3 The gene fragment was ligated into the pUC57 vector to obtain the recombinant plasmid pUC57-YLT3; the recombinant plasmid pUC57-YLT3 was amplified to obtain the linearized L-pUC57-YLT3 gene fragment. (2) The pYLXP'2 vector URA3 The gene fragment was ligated with the linearized pYLXP' vector treated with SnaBI restriction enzyme to obtain the recombinant plasmid pYLXP'-URA3; the lox71 and lox66 sequences were inserted into the AvrII and NheI sites of the recombinant plasmid pYLXP'-URA3, respectively, to obtain the recombinant plasmid pYLXP'-URA3-loxP; the recombinant plasmid pYLXP'-URA3-loxP was amplified to obtain the linearized L-URA3-LoxP gene fragment; the linearized L-pUC57-YLT3 gene fragment was ligated with the linearized L-URA3-LoxP gene fragment to obtain the recombinant plasmid pUC57-YLT3-URA3-loxP; (3) Yeast extract Po1f YlIlv3 Gene, YlIlv5 Gene, YlIlv2* The gene was tandemly linked to the vector pYLXP'2 in monocistronic form to obtain the recombinant plasmid pYLXP'2-YlILV3-YlILV5-YlILV2*; (4) The recombinant plasmid pUC57-YLT3-URA3-loxP was double-digested to obtain the pUC57-YLT3-URA3 backbone fragment; the recombinant plasmid pYLXP'2-YlILV3-YlILV5-YlILV2* was double-digested to obtain the YlILV3-YlILV5-YlILV2* gene fragment; the pUC57-YLT3-URA3 backbone fragment and the YlILV3-YlILV5-YlILV2* gene fragment were ligated to obtain the recombinant plasmid pUC57-YLT3-URA3-loxP-YlILV3-YlILV5-YlILV2*. (5) The recombinant plasmid pUC57-YLT3-URA3-loxP-YlILV3-YlILV5-YlILV2* was double-digested to obtain the YLT3-URA3-loxP-YlILV3-YlILV5-YlILV2* gene fragment. The YLT3-URA3-loxP-YlILV3-YlILV5-YlILV2* gene fragment was transformed into Yersinia lipolyticis Po1f ΔKu70. After screening with auxotrophic plates, the recombinant Yersinia lipolyticis Po1f ΔKu70 YLT3::URA3-Ilvs strain was obtained. (6) The pYLXP'-Cre vector was transformed into the recombinant strain of Yersinia lipolytica Po1f ΔKu70 YLT3::URA3-Ilvs. After screening with leucine auxotrophic medium containing 5-fluoroorotic acid, the recombinant strain of Yersinia lipolytica Po1f ΔKu70 YLT3::Ilvs / pYLXP'-Cre was obtained. The recombinant strain of Yersinia lipolytica Po1f ΔKu70 YLT3::Ilvs / pYLXP'-Cre was passaged under nutrient-free selection pressure and the pYLXP'-Cre plasmid was lost to obtain the recombinant strain of Yersinia lipolytica Po1f ΔKu70 YLT3::Ilvs.

10. The use of the recombinant *Yarrowia lipolytica* strain according to any one of claims 5-9 in any of the following: D1) Applications in the production of amino acids or / and in increasing amino acid yield; D2) Applications in the production of branched long-chain alcohols and / or in increasing the yield of branched long-chain alcohols; D3) Applications in the production of short-chain fatty acids or / and in increasing the yield of short-chain fatty acids.

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