Fatty acid elongase mutants and their use in synthetic oil production

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to application of a fatty acid elongase (Xanthoceras sorbifolia-derived fatty acid elongase 3-ketoacyl-CoA reductase (KCR) and enoyl-CoA reductase (ECR)) mutant and a recombinant strain in synthesis of nervonic acid. The mutant is a key amino acid in one or several of Xanthoceras sorbifolia-derived fatty acid elongase 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD) and enoyl-CoA reductase (ECR) located at a substrate binding pocket, a substrate entry channel and an enzyme-substrate interaction interface. The mutant can effectively improve the synthesis efficiency of nervonic acid, and provides a key enzyme element for construction of a high-yield nervonic acid engineering strain and industrialized biological manufacturing of nervonic acid, and has a wide application prospect in the fields of medicine, health products and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a fatty acid elongation enzyme mutant (3-ketoacyl-CoA reductase (KCR) and enoyl-CoA reductase (ECR) from Xanthocerassorbifolia) and a recombinant strain in the synthesis of nervonic acid. Background Technology

[0002] Nervonic acid (C24:1, cis-15-tetracosenoic acid) is an ultra-long chain monounsaturated fatty acid and a core component of the myelin sheath in mammalian nerve tissue. It plays an important role in repairing damaged brain nerve fibers and promoting nerve cell regeneration. It has significant therapeutic effects on demyelinating diseases such as multiple sclerosis and adrenoleukodystrophy, and therefore has broad market prospects in the fields of medicine and functional foods.

[0003] Currently, nervonic acid mainly comes from the seed oils of specific plants, such as *Sapindus mukorossi* (*Sapindus spp.*). Xanthoceras sorbifolia ), garlic fruit ( Malania oleifera However, these plants generally suffer from long growth cycles, strong environmental dependence, limited planting scale, and large fluctuations in nervonic acid content in their oils, resulting in high nervonic acid production costs and difficulties in achieving large-scale, stable supply. Therefore, the synthesis of nervonic acid using microbial cell factories has become a current research hotspot.

[0004] Yarrowia lipolytica ( Yarrowia lipolytica *Nervate* is a recognized safe oil-producing yeast with advantages such as high fatty acid synthesis throughput, mature genetic manipulation tools, and suitability for industrial-scale fermentation. It is an important chassis strain for constructing nervate microbial synthesis systems. In organisms, fatty acid biosynthesis includes two stages: de novo fatty acid synthesis and fatty acid elongation. Long-chain fatty acids with a carbon chain length ≥ C20 are mainly generated in the endoplasmic reticulum via the catalysis of a fatty acid elongation enzyme complex (FAE complex). This complex consists of four functional enzymes: 3-ketoacyl-CoA condenserase (KCS), 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), and enoyl-CoA reductase (ECR). In each elongation cycle, KCS, KCR, HCD, and ECR in the fatty acid elongation enzyme complex catalyze condensation, reduction, dehydration, and further reduction reactions, respectively, extending the fatty acid carbon chain by two carbon atoms.

[0005] Existing research generally considers KCS to be a key rate-limiting enzyme determining the chain length and synthesis rate of very long-chain fatty acids. Therefore, most existing technologies focus on screening KCS genes from different plant sources and expressing them in yeast to increase nervonic acid production. For example, CN114517164A discloses the integration and expression of plant-derived KCS genes in yeast to increase nervonic acid production. European patent application EP4249583A1 discloses the integration and expression of KCS genes derived from *Xanthoceras sorbifolium* in *Yarrowia lipolytica*. Xanthoceras sorbifolia KCS genes in various plants, including those mentioned above, are used to produce nervonic acid. Previous studies have primarily focused on the screening and functional optimization of single KCS enzymes, with limited research on other enzymes in fatty acid elongation enzyme complexes. The laboratory of the inventors of this application previously successfully obtained a recombinant strain with high nervonic acid production through systematic metabolic engineering of the oleaginous yeast Yarrowia lipolytica G3 strain. The relevant research results have been published (Su H, Shi PH, Shen ZS, et al. High-level production of nervonic acid in the oleaginous yeast Yarrowia lipolytica by systematic metabolic engineering[J]. Communications Biology, 2023, 6:1125.). In this study, the recombinant strain was verified in a 5 L fermenter, showing that its nervonic acid content accounted for 17.9% of the total cellular fatty acids, with a yield of 17.3 g / L.

[0006] The four enzymes mentioned above play different and interconnected catalytic functions in the fatty acid elongation process. KCS primarily catalyzes the condensation reaction between acyl-CoA substrates and malonyl-CoA, and to some extent influences the substrate selectivity, target carbon chain length, and reaction rate of the fatty acid elongation reaction. KCR, HCD, and ECR sequentially complete the reduction, dehydration, and re-reduction of subsequent intermediates. Therefore, the synthesis efficiency of nervonic acid is not determined by a single enzyme, but is influenced by a combination of factors, including the catalytic activity, expression level, subcellular localization, substrate adaptability, and synergistic effects between enzymes. Insufficient catalytic efficiency in any reaction step can affect the continuous progress of the elongation cycle and limit the synthesis and accumulation of the target ultra-long-chain fatty acid.

[0007] Meanwhile, current optimizations of fatty acid elongation enzymes such as KCS mainly focus on increasing gene copy number, enhancing expression levels, and screening homologous enzymes from different species. Overall, these efforts remain at the level of gene replacement or overexpression, lacking fine-tuning of key structures such as catalytic sites, substrate-binding regions, transmembrane domains, protein stability, and interaction interfaces. Since fatty acid elongation depends on the coordinated action of various enzyme components, simply enhancing the expression of one enzyme may not simultaneously improve overall elongation efficiency; instead, it may lead to catalytic imbalances, accumulation of intermediate products, increased cellular metabolic burden, or the formation of bypass products. Therefore, it is necessary to employ protein engineering techniques such as rational design, semi-rational design, and directed evolution to modify these enzymes to improve their catalytic activity, substrate adaptability, and target chain length selectivity.

[0008] Therefore, developing a highly efficient fatty acid elongation enzyme derived from the nervonic acid-rich plant *Xanthoceras sorbifolium*, elucidating its function in nervonic acid biosynthesis, and utilizing modern protein engineering technology to obtain key enzyme mutants with superior catalytic performance are of great significance for overcoming the bottleneck in nervonic acid biosynthesis, constructing high-yield nervonic acid engineered strains, and promoting the green biomanufacturing of nervonic acid. Summary of the Invention

[0009] The purpose of this invention is to address the problems of mismatched sources of components, insufficient synergistic effects between proteins, and limited efficiency in nervonic acid synthesis in existing technologies for ultra-long chain fatty acid elongase complexes (FAE complexes). This invention provides an application of fatty acid elongases (3-ketoacyl-CoA reductase (KCR) and enoyl-CoA reductase (ECR) from Xanthoceras sorbifolia), mutants, and recombinant strains in the synthesis of nervonic acid.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A fatty acid elongation enzyme mutant, wherein the mutant is one or more of the key amino acids located in the substrate binding pocket, substrate entry channel, and enzyme-substrate interaction interface of fatty acid elongation enzymes 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), and enoyl-CoA reductase (ECR) derived from *Sapindus mukorossi*; wherein the amino acid sequences of 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), and enoyl-CoA reductase (ECR) are as shown in SEQ ID NO: 24-26.

[0011] The mutant is a mutation at one or more of the following positions: 66, 143, 241, 244, and 246 of 3-ketoacyl-CoA reductase (KCR); specifically, XsKCR / I66L (SEQ ID NO:8), XsKCR / V143I (SEQ ID NO:4), XsKCR / Y241F (SEQ ID NO:5), XsKCR / T244S (SEQ ID NO:9), and XsKCR / M246L (SEQ ID NO:10). Mutations at one or more sites at positions 92, 152, 271, 274, and 281 of enoyl-CoA reductase (ECR); specifically, mutations at the following sites: XsECR / Y92W (SEQ ID NO:6), XsECR / V152I (SEQ ID NO:11), XsECR / A271G (SEQ ID NO:12), XsECR / I274Q (SEQ ID NO:7), and XsECR / A281Q (SEQ ID NO:13).

[0012] A recombinant plasmid containing the fatty acid elongation enzyme mutant or fatty acid elongation enzyme; wherein the fatty acid elongation enzyme is one of the following fatty acid elongation enzymes derived from *Sapindus mukorossi*: 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), and enoyl-CoA reductase (ECR).

[0013] A recombinant yeast strain, wherein the recombinant yeast strain contains the recombinant plasmid in a host strain.

[0014] The host strain is an oil-producing yeast; wherein, the oil-producing yeast is a species of Yersinia (Yersinia). Yarrowia ), Rhodotorula genus ( Rhodotorula ) and Candida genus ( Candida )wait.

[0015] The host strain is *Yersinia lipophila* (… Yarrowia lipolytica ), Rhodotorula buergerianum ( Rhodosporidium toruloides ), Stirryl oil yeast ( Lipomyces starkeyi ), Oil-producing mycorrhizal yeast ( Cutaneotrichosporon oleaginosus ), dermal filamentous yeast ( Trichosporon cutaneum ) or red yeast ( Phaffia rhodozyma ).

[0016] A method for constructing the recombinant yeast strain involves constructing a homologous recombinant plasmid, linearizing it using a site-directed integration method, transforming it into a host chassis, and then screening for positive transformants using uracil auxotrophic plates to obtain the strain.

[0017] The host is *Yersinia lipophila* (Yarrowia lipolytica The recombinant yeast strain YLNA20-ΔURA, a uracil auxotrophic strain, was obtained by linearizing the homologous recombinant plasmid using a site-directed integration method and then transforming it into the host IntF17 site.

[0018] One application of the mutant or recombinant yeast strain is the use of it in the synthesis of nervonic acid.

[0019] A method for synthesizing nervonic acid involves culturing the recombinant yeast strain in 30 mL of fermentation medium (28°C, 220 rpm) for 168 h to obtain nervonic acid.

[0020] The present invention has the following advantages: This invention utilizes fatty acid elongation enzymes derived from *Xanthoceras sorbifolium* and their efficient mutants to construct recombinant strains that effectively enhance the elongation efficiency of ultra-long-chain fatty acids, demonstrating promising prospects for industrial application. Specifically: This invention is the first to discover key enzymes KCR, HCD, and ECR in the fatty acid elongation pathway from the nervonic acid-rich plant *Xanthoceras sorbifolium*, thereby enriching the resources of enzymes related to the synthesis of ultra-long chain fatty acids.

[0021] Furthermore, XsKCR or XsECR derived from *Xanthoceras sorbifolium* significantly increased the accumulation level of nervonic acid in *Yarrowia lipolytica*. Under unoptimized shake-flask fermentation conditions, the nervonic acid content of recombinant strains expressing XsKCR or XsECR reached 31.3% and 30.9% of total fatty acids, respectively, which were 9.7% and 8.2% higher than those of the control strain.

[0022] Using the results obtained above, mutations were performed on the relevant enzymes to obtain highly efficient mutants such as XsKCR / V143I, XsKCR / Y241F, XsECR / Y92W, and XsECR / I274Q. Furthermore, mutant recombinant strains were constructed using these mutants. These strains further improved the nervonic acid synthesis capacity without affecting the growth of the strain or the accumulation of lipids. Among them, the mutant recombinant strain containing XsECR / Y92W increased the nervonic acid content by 10.1% (compared to YLNA20), exhibiting excellent catalytic performance. Attached Figure Description

[0023] Figure 1 This is a comparison of the conserved domain / motif arrangements of 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), and enoyl-CoA reductase (ECR) proteins from different plant and yeast sources; Figure 2 This is a map of the recombinant plasmid IntF17-pFAS2-XsKCR; Figure 3This is the diagnostic PCR result of the recombinant strain in Example 1; where A: design of homologous recombination target sequence and PCR primer design; B: diagnostic PCR electrophoresis diagrams of different transformants of recombinant strains YLNA20-YlKCR, YLNA20-YlHCD, YLNA20-YlECR, YLNA20-XsKCR, YLNA20-XsHCD and YLNA20-XsECR; Figure 4 The results are the end-point biomass and oil yield of the recombinant strains YlKCR, YlHCD, YlECR, XsKCR, XsHCD, and XsHCD during fermentation. Figure 5 The results show the analysis of the fatty acid components of recombinant strains YlKCR, YlHCD, YlECR, XsKCR, XsHCD, and XsHCD. Figure 6 These are the results of molecular docking and molecular dynamics simulations of XsKCR, XsECR, and ligands; Figure 7 This is a map of the mutant recombinant plasmid IntF17-pFAS2-XsKCR / I66L; Figure 8 These are diagnostic PCR results for recombinant strains with single-point mutants of XsKCR and XsECR. A: Design of homologous recombination target sequences and PCR primers; B: Diagnostic PCR electrophoresis images of different transformants of recombinant strains YLNA20-XsKCR / I66L, YLNA20-XsKCR / V143I, YLNA20-XsKCR / Y241F, YLNA20-XsKCR / T244S, YLNA20-XsKCR / M246L, YLNA20-XsECR / Y92W, YLNA20-XsECR / V152I, YLNA20-XsECR / A271G, YLNA20-XsECR / I274Q, and YLNA20-XsECR / A281Q. Figure 9 These are the results of the fermentation endpoint biomass and oil yield of the XsKCR and XsECR single-point mutant recombinant strains; Figure 10 These are the results of the analysis of the content of each fatty acid component in the XsKCR and XsECR single-point mutant recombinant strains. Detailed Implementation

[0024] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0025] The fatty acid elongation enzyme and its mutants derived from *Sapindus mukorossi* provided by this invention can significantly improve the synthesis capacity of nervonic acid in yeast; and the engineered strains of related ultra-long chain fatty acids can be applied in the fields of medicine, food, health products, and cosmetics.

[0026] The Yarrowia lipolytica strain used in the following examples was strain YLNA20, which was obtained by using conventional genetic engineering techniques. Based on strain YLNA10 (Su H, Shi PH, Shen ZS, et al. High-level production of nervonic acid in the oleaginous yeast Yarrowia lipolytica by systematic metabolic engineering[J]. Communications Biology,2023, 6: 1125. DOI: 10.1038 / s42003-023-05502-w.) published in the laboratory, the strain was obtained by knocking out SCD (GenBank: QNP96445.1) and overexpressing RtOLE1 (GenBank: EMS19868.1, derived from...) at the IntC3 site. Rhodotorula toruloides ) and gElovl6 (from goat The gElovl6 gene (documented in GenBank: XP_005681364.1) was used to overexpress the RtOLE1 and gElovl6 genes at the IntD1 site, resulting in strain YLNA20 with a nervonic acid content of approximately 28%. The IntC3 and IntD1 sites were referenced in (Holkenbrink C., et al. EasyCloneYALI: CRISPR / Cas9-based synthetic toolbox for engineering of the yeast Yarrowia lipolytica. Biotechnology Journal, 2018, 13:1700543. DOI: 10.1002 / biot.201700543). The uracil auxotrophic strain YLNA20-ΔURA was obtained by reverse screening of strain YLNA20 using the 5-FOA method. The screening method can be found in In-Seung J, Jo BY, Yeon JJ, et al. Improving the efficiency of homologous recombination by chemical and biological approaches in Yarrowialipolytica.[J]. PloS one, 2018, 13(3):e0194954.DOI:10.1371 / journal.pone.0194954. Furthermore, the strain obtained by the screening method only recovered the ura3 screening marker gene.

[0027] The culture medium used in the examples can be obtained commercially or through formulations and methods known in the art.

[0028] Example 1: Construction of recombinant yeast strains expressing endogenous *Yarrowia lipolytica* and *Xanthoceras sorbifolium* KCR, HCD, and ECR genes. 1. Screening of KCR, HCD and ECR enzymes in *Sapindus mukorossi* Sequences of 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), and enoyl-CoA reductase (ECR) were retrieved from the NCBI database. The sequence AtKCR (GenBank: AEE34688.1, sourced from...) was obtained. Arabidopsis thaliana ), BnKCR (GenBank: AAO43448.1, sourced from Brassica napus ), MoKCR (GenBank: XP_057949429.1, sourced from Malania oleifera YlKCR (GenBank: YALI1_A06453g, sourced from...) Yarrowia lipolytica ), RtKCR (GenBank:CDR49328.1, sourced from Rhodotorula toruloides ),ScKCR (GenBank: P38286.1, sourced from Saccharomyces cerevisiae PpKCR (GenBank: XP_002493790.1, sourced from...) Pichia pastoris ); AtHCD (GenBank: AAP13377.1, sourced from Arabidopsis thaliana BnHCD (GenBank: XP_048598389.1, sourced from...) Brassica napus MoHCD (GenBank: XP_057967704.1, sourced from...)Malania oleifera YlHCD (GenBank: YALI1_F15781g, sourced from...) Yarrowia lipolytica RtHCD (GenBank: CDR48689.1, sourced from...) Rhodosporidium toruloides ),ScHCD (GenBank: KQC43059.1, sourced from Saccharomyces cerevisiae PpHCD (GenBank:CAY70228.1, sourced from...) Pichia pastoris ) and AtECR (GenBank:AEE79372.1, sourced from Arabidopsis thaliana ), BnECR (GenBank: ACR56737.1, sourced from Brassica napus MoECR (GenBank: XP_057979752.1, sourced from...) Malania oleifera YlECR (GenBank: YALI1_A05116g, sourced from...) Yarrowia lipolytica RtECR (GenBank: XP_016273525.1, sourced from...) Rhodosporidium toruloides ), ScECR (GenBank: GFP63997.1, derived from...) Saccharomyces cerevisiae PpECR (GenBank: XP_002494225.1, sourced from...) Pichia pastoris ).

[0029] Based on existing KCR, HCD, and ECR protein sequences from yeast and plants, bioinformatics identification and functional prediction of the KCR, HCD, and ECR protein sequences of *Xanthoceras sorbifolium* were performed through homology sequence searching, multiple sequence alignment, and conserved site analysis. Figure 1 It is evident that the candidate enzyme proteins of *Sapindus mukorossi* KCR, HCD, and ECR have the highest homology with the KCR, HCD, and ECR of the plants mentioned above, and their amino acid sequences are shown in SEQ ID NO:1-3.

[0030] 2. Preparation of recombinant yeast strains expressing endogenous *Yarrowia lipolytica* and *Xanthoceras sorbifolium* KCR, HCD, and ECR. The KCR, HCD, and ECR genes obtained from the above screening were codon-optimized for Yersinia lipolytica. The optimized KCR gene of X. spp. was XsKCR, the HCD gene was XsHCD, and the ECR gene was XsECR. Meanwhile, the endogenous KCR, HCD, and ECR genes of Yersinia lipolytica were YlKCR, YlHCD, and YlECR, respectively. All of the above genes were synthesized by Qingke Biotechnology Co., Ltd., and their sequences are shown in SEQ ID NO:14-19, respectively.

[0031] Based on the completion of gene synthesis, Qingke Biotechnology Co., Ltd. further constructed and delivered shuttle plasmids containing the above-mentioned target genes and ampicillin resistance genes, respectively. The base vector of each shuttle plasmid is pUC57. Finally, the shuttle plasmids that simultaneously express each gene and the ampicillin resistance gene were named pUC57-YlKCR-AMP, pUC57-YlHCD-AMP, pUC57-YlECR-AMP, pUC57-XsKCR-AMP, pUC57-sHCD-AMP, and pUC57-XsECR-AMP, respectively. Using the six shuttle plasmids obtained above as templates, the gene fragments YlKCR, YlKCR-F, YlHCD-F / YlHCD-R, YlECR-F / YlECR-R, XsKCR-F / XsKCR-R, XsHCD-F / XsHCD-R, and XsECR-F / XsECR-R were amplified using the corresponding primer pairs YlKCR-F / YlKCR-F, YlHCD-F / YlHCD-R, XlECR-F / YlECR-R, respectively.

[0032] Using pYL-SNF1-CgKCS plasmid (Su H, Shi PH, Shen ZS, et al. High-level production of nervonic acid in the oleaginous yeast Yarrowia lipolytica by systematic metabolic engineering[J]. Communications Biology, 2023, 6: 1125.DOI: 10.1038 / s42003-023-05502-w.) as a template, primer pairs CgKCS-F / Ter-R, F17UP-F / F17UP-R, F17DN-F / F17DN-R, Amp-F / Amp-R, and FSA2-F / FA2-R were used for amplification. The resulting fragments included the backbone DNA with the CgKCS expression cassette and URA, the upstream and downstream homologous arm sequences of the F17 neutral site, the Amp selection marker, and the fragments of the endogenous strong promoter FAS2 of Yarrowia lipolytica. The fragments were assembled using the Gibson assembly method to obtain the homologous recombinant plasmid IntF17-pFAS2-CgKCS-TPRC1t, which was preserved in our laboratory and may be publicly used for research purposes only.

[0033] Using the homologous recombinant plasmid IntF17-pFAS2-CgKCS-TPRC1t constructed above as a template, a backbone DNA fragment 1 containing upstream and downstream sequences of the expression cassette (see SEQ ID NO: 30-31), the FAS promoter (see SEQ ID NO: 32), the PRC1t terminator (see SEQ ID NO: 33), and URA and AMP selection markers was amplified using primer pair PRC1t-F1 / PFAS2-R1.

[0034] The obtained backbone 1 was assembled with the amplified YlKCR, YlHCD, YlECR, XsKCR, XsHCD, and XsECR gene fragments obtained above using the Gibson assembly method. The recombinant expression plasmids expressing each gene were named IntF17-pFAS2-YlKCR, IntF17-pFAS2-YlHCD, IntF17-pFAS2-YlECR, IntF17-pFAS2-XsKCR, IntF17-pFAS2-XsHCD, and IntF17-pFAS2-XsECR. Then, each of the recombinant expression plasmids obtained above was transformed into E. coli competent cells Trans-T1, and colony PCR was performed using primers F17-F / F17-R. Sequencing confirmed the correctness of the colonies. The design diagram of the recombinant plasmids is shown below. Figure 2 As shown, the specific primer sequences are shown in Table 1: Table 1 Primers used for plasmid construction

[0035] All PCR amplifications were performed using PFU high-fidelity DNA polymerase. The amplification system was 50 µL (2 × PFU mix, 25 µL; 1.5 µL each of 10 µM primers; 1 ng / 1 µL template; water added to 50 µL). The amplification conditions were: 95 °C pre-denaturation for 3 min; 98 °C denaturation for 20 s, 60-72 °C annealing for 15 s, 72 °C extension (30 s / kb), cycle number 30; 72 °C extension for 10 min.

[0036] use NotRestriction endonucleases were used to digest the recombinant expression plasmids IntF17-pFAS2-YlKCR, IntF17-pFAS2-YlHCD, IntF17-pFAS2-YlECR, IntF17-pFAS2-XsKCR, IntF17-pFAS2-XsHCD, and IntF17-pFAS2-XsECR to obtain linear plasmids. The plasmids were confirmed by agarose gel electrophoresis. The fragments used for transformation were recovered from the gel and transformed into Yersinia lipolytica YLNA20-ΔURA using the LiAc transformation method. The transformation system is shown in Table 2.

[0037] Table 2. Transformation system of Yersinia lipophila

[0038] The transformation system was vortexed after being bathed in a 30 ℃ water bath for 1 h, and then heat-shocked at 39 ℃ for 10 min. The mixtures of each transformation system were then spread on screening plates of YNB-Δura medium and cultured at 28 ℃ for 2-4 days to obtain recombinant strains YLNA20-YlKCR, YLNA20-YlHCD, YLNA20-YlECR, YLNA20-XsKCR, YLNA20-XsHCD and YLNA20-XsECR.

[0039] Diagnostic PCR verification was performed on the different transformants of the recombinant strains obtained above: single colonies of the recombinant strains were picked from the screening plates and placed on YNB-Δura plates. After growth for about 24 h, bacterial cells were picked, and genomic DNA of the recombinant strains was extracted using a commercial fungal genome rapid extraction kit. Using the genomic DNA as a template, full-length PCR verification was performed using primers F17-dF and F17-dR. The specific primer sequences are shown in Table 3. Table 3 Primers used for PCR diagnosis of bacterial strains

[0040] PCR amplification was performed using EasyTaq DNA polymerase. The amplification volume was 20 µL (2 × EasyTaq Mix, 10 µL; 0.8 µL each of 10 µM primers; 1 ng / µL template; water added to 20 µL). Amplified fragments were identified by agarose gel electrophoresis. The amplification conditions were: 94 °C pre-denaturation for 3 min; 94 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension (60 s / kb), 30 cycles; and 72 °C extension for 10 min.

[0041] Agarose gel electrophoresis image of PCR products is shown below. Figure 3As shown: PCR amplification bands of the same theoretical size were obtained after transformation of each target gene, proving that the target expression cassette has been correctly integrated into the IntF17 site.

[0042] Example 2: Fermentation of recombinant Yersinia lipolytica to produce nervonic acid Three recombinant strains obtained in Example 1 were selected and inoculated into 5 mL of YNB-Δura liquid medium, and cultured at 28°C and 220 rpm for approximately 24 h to obtain fermentation seed culture. The seed culture was then inoculated into 30 mL of fermentation medium (15 wt% glucose, 0.6 wt% yeast extract, 1.2 wt% (NH4)2SO4, with the remainder being water), and cultured at 28°C and 220 rpm for 168 h. YLNA20 strain was used as a control, and three replicates were set up for each group.

[0043] After fermentation, fatty acids were extracted from the fermentation broth of each strain: 3 mL of each strain's fermentation broth was placed in a glass centrifuge tube and centrifuged at 4000 rpm for 10 min to collect the cells; 3 mL of 4 M HCl was added, vortexed to mix, and the mixture was shaken on a shaker at 28 ℃ for 1.5-2 h; the mixture was then boiled in a water bath for 10 min and cooled at -20 ℃ for 30 min; 6 mL of chloroform / methanol (1:1 v / v) solution was added, vortexed to mix, and centrifuged at 4000 rpm for 10 min; the lower chloroform layer was transferred to a new centrifuge tube, 3 mL of 0.15% NaCl was added, and the mixture was centrifuged at 4000 rpm for 10 min; the lower chloroform layer was transferred to a new, pre-dried and weighed glass centrifuge tube, and the chloroform was evaporated by nitrogen blowing and heating in a fume hood to obtain the oil.

[0044] The extracted oil was methylated: 2.6 mL of methanol:sulfuric acid = 98: 2 (v / v) solution was added to a glass tube, mixed well, and then reacted at 85 ℃ for 3 h, shaking once every 30 min during the reaction until the oil droplets disappeared; then 1 mL of saturated NaCl solution and 1 mL of n-hexane were added, shaken well, centrifuged at 4000 rpm for 5 min, and 800 µL of the supernatant was aspirated into a clean 1.5 mL EP tube, centrifuged at 12000 rpm for 5 min, and 400 µL of the supernatant was gently aspirated, sealed and stored for the next step of gas chromatography analysis.

[0045] The methylated fatty acids were analyzed by gas chromatography (Agilent 7890B-GC). Chromatographic conditions: HP-INOWAX column (30 m × 0.32 mm × 0.5 μm); injection temperature: 250 ℃; detector temperature: 250 ℃; injection volume: 1 μL; initial column temperature: 140 ℃, held for 1 min, increased to 180 ℃ at 10 ℃ / min, held for 2 min, increased to 210 ℃ at 5 ℃ / min, held for 4 min, then increased to 250 ℃ at 5 ℃ / min, held for 4 min; the relative content of each fatty acid component was obtained by area normalization.

[0046] The biomass and oil yield at the fermentation endpoint of the recombinant strain are as follows: Figure 4 As shown, compared to the control strain YLNA20 (OD 600 =74.9, 9.2 g / L), YLNA20-XsKCR, YLNA20-XsHCD, and YLNA20-XsECR to some extent increased the oil yield of the strains (10.1-10.8 g / L). Figure 5 As shown, in the control strain YLNA20, nervonic acid accounted for only 27.2% of the total fatty acids. The proportions of nervonic acid (C24:1) in the total fatty acids of recombinant strains YLNA20-YlKCR, YLNA20-YlHCD, YLNA20-YlECR, YLNA20-XsKCR, YLNA20-XsHCD, and YLNA20-XsECR were 25.4%, 27.4%, 27.3%, 31.5%, 27.9%, and 31.0%, respectively. Compared with the control group (28.6%; 2.6 g / L), the nervonic acid content of YLNA20-XsKCR and YLNA20-XsECR was significantly increased, by 15.5% and 13.7%, respectively, with nervonic acid yields of 3.2 g / L and 3.1 g / L.

[0047] Example 3: Construction of recombinant yeast strains expressing XsKCR and XsECR single-point mutants 1. Design of XsKCR and XsECR mutation sites As can be seen from the above examples, 3-ketoacyl-CoA reductase XsKCR and enoyl-CoA reductase XsECR from *Sapindus mukorossi* can significantly enhance the ability of *Yersinia lipolytica* to synthesize nervonic acid. To achieve higher yields, further mutations are performed, such as... Figure 6As shown in Table 4, the performance of XsKCR and XsECR was modified by selecting five mutation sites for each: XsKCR: I66L, V143I, Y241F, T244S, M246L; and XsECR: I274Q, Y92W, A271G, V152I, A281Q. The coding genes for XsKCR and XsECR were codon optimized and mutants designed according to the preferences of *Yarrowia lipolytica*, as shown in Table 4.

[0048] Table 4 XsKCR and XsECR and their mutation sites

[0049] 2. Preparation of recombinant yeast strains expressing XsKCR and XsECR single-point mutants Taking a recombinant yeast strain with a single-point mutant of XsKCR I66L as an example, using the recombinant expression plasmid IntF17-pFAS2-XsKCR as a template, a backbone DNA fragment with URA and AMP selection markers was amplified using primer pair PRC1t-F1 / PFAS2-R1. The mutation site Ile was amplified using primer pairs XsKCR-F / XsKCR-I66L-R and XsKCR-I66L-F / XsKCR-R (Table 1). 66 The upstream and downstream target gene fragments were assembled using the Gibson assembly method. The assembly product was transformed into E. coli competent cells DH5α and plated on LB plates containing 100 μg / mL ampicillin. The obtained single clones were identified by PCR using primer pair F17-F / F17-R, and finally verified by sequencing to obtain the positive recombinant expression plasmid IntF17-pFAS2-XsKCR / I66L. Figure 7 ).

[0050] The same method was used to obtain the backbone DNA fragments, and the XsKCR mutation site Val, as recorded in Table 5, was amplified using the primer pairs listed in Table 1. 143 Tyr 241 Thr 244 Met 246 and XsECR mutation site Tyr 92 Val 152 Ala 271 Ile 274 Ala 281The upstream and downstream target fragments were assembled using the Gibson assembly method, and positive mutant recombinant expression plasmids were obtained and screened. They were named IntF17-pFAS2-XsKCR / V143I, IntF17-pFAS2-XsKCR / Y241F, IntF17-pFAS2-XsKCR / T244S, IntF17-pFAS2-XsKCR / M246L, IntF17-pFAS2-XsECR / Y92W, IntF17-pFAS2-XsECR / V152I, IntF17-pFAS2-XsECR / A271G, IntF17-pFAS2-XsECR / I274Q, and IntF17-pFAS2-XsECR / A281Q, respectively.

[0051] The recombinant strain was then prepared and verified according to the method in Example 1. The agarose gel electrophoresis image of the PCR product is shown below. Figure 8 As shown: PCR amplification bands of the same theoretical size were obtained after transformation of each target gene, proving that the target expression cassette has been correctly integrated into the IntF17 site.

[0052] Example 4: Fermentation of XsKCR and XsECR and single-point mutant recombinant Yersinia lipolytica to produce nervonic acid Three recombinant strains obtained in Example 3 were selected and inoculated into 5 mL of YNB-Δura liquid medium, and cultured at 28°C and 220 rpm for approximately 24 h to obtain fermentation seed culture. Each seed culture was then inoculated into 30 mL of fermentation medium (15 wt% glucose, 0.6 wt% yeast extract, 1.2 wt% (NH4)2SO4, with the remainder being water), and cultured at 28°C and 220 rpm for 168 h. YLNA20 strain was used as a control, and three replicates were set up for each group. YLNA20-XsKCR and YLNA20-XsECR were also used as controls.

[0053] After fermentation, total cell lipids were extracted using the acid-heat method as described in Example 2, and the total lipid yield was calculated by weighing. The extracted lipids were then methylated, and the relative content of each fatty acid component was determined by gas chromatography (see [link to example]). Figure 9 and 10 ).

[0054] The biomass and oil yield of each recombinant strain were measured at the fermentation endpoint (168 h), and the results are as follows: Figure 9As shown in the figure. Compared with the starting strain YLNA20 and the control strains that overexpressed wild-type XsKCR and XsECR respectively, all single-point mutant strains showed no significant differences in biomass accumulation and lipid synthesis. The OD600 values ​​of each mutant strain remained in the range of 70–80, and the lipid yield was between 9 and 11 g / L, which was basically equivalent to the control strain.

[0055] The fatty acid composition of single-point mutant recombinant strains is as follows: Figure 10 As shown, compared to the original strain YLNA20 (nervonic acid content 28.7%; nervonic acid yield 2.7 g / L), the strains YLNA20-XsKCR / WT and YLNA20-XsECR / WT, which overexpress XsKCR and XsECR, showed significantly increased nervonic acid synthesis, reaching 31.7% and 31.2% of total fatty acids, respectively, with nervonic acid yields of 3.2 g / L and 3.3 g / L. g / L; Furthermore, compared with the control strains YLNA20-XsKCR / WT and YLNA20-XsECR / WT, the NA content of strains YLNA20-XsKCR / V143I, YLNA20-XsKCR / Y241F, YLNA20-XsECR / Y92W and YLNA20-XsECR / I274Q was significantly increased, reaching 33.1%, 33.7%, 34.4% and 33.6% of total fatty acids, respectively, representing increases of 4.5%, 6.3%, 10.1% and 7.5%, and the shake-flask level nervonic acid yield reached 3.5 g / L, 3.4 g / L, 3.5 g / L and 3.6 g / L. The NA content of strains YLNA20-XsKCR / I66L, YLNA20-XsKCR / T244S, YLNA20-XsKCR / M246L, YLNA20-XsECR / V152I, YLNA20-XsECR / A271G, and YLNA20-XsECR / A281Q was not significantly increased. This indicates that mutants V143I, Y241F, Y92W, and I274Q significantly improved the catalytic performance of 3-ketoacyl-CoA reductase XsKCR and enoyl-CoA reductase XsECR, respectively, and the method of this invention is also suitable for industrial production.

[0056] >SEQ ID NO.1 XsHCD amino acid sequence MAGFLSLLRRLYLSVYNWTIFLGWSQVLYLALKTLNESGHQHVYNAVQKPLLLAQTAAVLEILHGLVGLVRSPITSTLPQISSRLYVTWGILWSFPQIQTHLLVSSLVISWSVTEIIRYSFFGMKEAFGFTPSWFLWLRYSTFLLLYPTGITSEVGLIYFALPYMKDSDKYCIRMPNKWNFSFDYFYNAILVLGFYVPGSPHLYGYMLRQRKKALSKSKRE >SEQ ID NO.2 XsKCR amino acid sequence MMEHCFLDTLKAQPIWVLVLFTVGSLSVLRCALSILNWVYVNFLRPAKNLKKYGSWALVTGPTDGIGKGFAFELARKGLNLVLVGRNPDKLKDVSDSIQAKYGKTQIRNVVVDFSGDLDEGVKRIKETIEGLDVGVLINNVGVSYPYARFFHEVDEELLKNLIKINVEGTTKVTQAVLPGMLKRKKGAIVNIGSGAAIVIPSDPLYSVYAATKAYIDQFSRCLYVEYKKSGIHVQCQVPLYVATKMASIKRSSFIVPSTDGYARAAMRWIGYEPRCTPYWPHTLLWGLAYKLPESAIDAWRLRFCLGIRKRGQLKDSRKME >SEQ ID NO.3 XsECR amino acid sequence MKVTVVSRSGRELIKGGLELNDSATVADLQEAIHKRTKKFYPSRQRLTLPLSPESKERPTVLNYKKSLKEYCDGNSGDLTVVFKDLGPQVSYRTLFFFEYIGPLLLYPVFYYFPVYKYFGYKGERVIHPVQTYALYYWCFHYFKRIMETFFVHRFSHATSPVSNVFRNCAYYWTFGSYIAYYVNHPLYTPVSDLQMKIGFGFGLLCQITNFYCHILLKNLRSPDGAGGYQIPRGFLFNIVTCANYTTEIYQWLGFNIATQTVAGYIFLVVAAFIMTNWALAKHRRLKKLFDGKEGRPKYPRRWVILPPFL >SEQ ID NO.4 XsKCR / V143I amino acid sequence MMEHCFLDTLKAQPIWVLVLFTVGSLSVLRCALSILNWVYVNFLRPAKNLKKYGSWALVTGPTDGIGKGFAFELARKGLNLVLVGRNPDKLKDVSDSIQAKYGKTQIRNVVVDFSGDLDEGVKRIKETIEGLDVGVLINNVGISYPYARFFHEVDEELLKNLIKINVEGTTKVTQAVLPGMLKRKKGAIVNIGSGAAIVIPSDPLYSVYAATKAYIDQFSRCLYVEYKKSGIHVQCQVPLYVATKMASIKRSSFIVPSTDGYARAAMRWIGYEPRCTPYWPHTLLWGLAYKLPESAIDAWRLRFCLGIRKRGQLKDSRKME >SEQ ID NO.5 XsKCR / Y241F amino acid sequence MMEHCFLDTLKAQPIWVLVLFTVGSLSVLRCALSILNWVYVNFLRPAKNLKKYGSWALVTGPTDGIGKGFAFELARKGLNLVLVGRNPDKLKDVSDSIQAKYGKTQIRNVVVDFSGDLDEGVKRIKETIEGLDVGVLINNVGVSYPYARFFHEVDEELLKNLIKINVEGTTKVTQAVLPGMLKRKKGAIVNIGSGAAIVIPSDPLYSVYAATKAYIDQFSRCLYVEYKKSGIHVQCQVPLFVATKMASIKRSSFIVPSTDGYARAAMRWIGYEPRCTPYWPHTLLWGLAYKLPESAIDAWRLRFCLGIRKRGQLKDSRKME >SEQ ID NO.6 XsECR / Y92W amino acid sequence MKVTVVSRSGRELIKGGLELNDSATVADLQEAIHKRTKKFYPSRQRLTLPLSPESKERPTVLNYKKSLKEYCDGNSGDLTVVFKDLGPQVSWRTLFFFEYIGPLLLYPVFYYFPVYKYFGYKGERVIHPVQTYALYYWCFHYFKRIMETFFVHRFSHATSPVSNVFRNCAYYWTFGSYIAYYVNHPLYTPVSDLQMKIGFGFGLLCQITNFYCHILLKNLRSPDGAGGYQIPRGFLFNIVTCANYTTEIYQWLGFNIATQTVAGYIFLVVAAFIMTNWALAKHRRLKKLFDGKEGRPKYPRRWVILPPFL >SEQ ID NO.7 XsECR / I274Q amino acid sequence MKVTVVSRSGRELIKGGLELNDSATVADLQEAIHKRTKKFYPSRQRLTLPLSPESKERPTVLNYKKSLKEYCDGNSGDLTVVFKDLGPQVSYRTLFFFEYIGPLLLYPVFYYFPVYKYFGYKGERVIHPVQTYALYYWCFHYFKRIMETFFVHRFSHATSPVSNVFRNCAYYWTFGSYIAYYVNHPLYTPVSDLQMKIGFGFGLLCQITNFYCHILLKNLRSPDGAGGYQIPRGFLFNIVTCANYTTEIYQWLGFNIATQTVAGYIFLVVAAFQMTNWALAKHRRLKKLFDGKEGRPKYPRRWVILPPFL >SEQ ID NO.8 XsKCR / I66L amino acid sequence MMEHCFLDTLKAQPIWVLVLFTVGSLSVLRCALSILNWVYVNFLRPAKNLKKYGSWALVTGPTDGLGKGFAFELARKGLNLVLVGRNPDKLKDVSDSIQAKYGKTQIRNVVVDFSGDLDEGVKRIKETIEGLDVGVLINNVGVSYPYARFFHEVDEELLKNLIKINVEGTTKVTQAVLPGMLKRKKGAIVNIGSGAAIVIPSDPLYSVYAATKAYIDQFSRCLYVEYKKSGIHVQCQVPLYVATKMASIKRSSFIVPSTDGYARAAMRWIGYEPRCTPYWPHTLLWGLAYKLPESAIDAWRLRFCLGIRKRGQLKDSRKME >SEQ ID NO.9 XsKCR / T244S amino acid sequence MMEHCFLDTLKAQPIWVLVLFTVGSLSVLRCALSILNWVYVNFLRPAKNLKKYGSWALVTGPTDGIGKGFAFELARKGLNLVLVGRNPDKLKDVSDSIQAKYGKTQIRNVVVDFSGDLDEGVKRIKETIEGLDVGVLINNVGVSYPYARFFHEVDEELLKNLIKINVEGTTKVTQAVLPGMLKRKKGAIVNIGSGAAIVIPSDPLYSVYAATKAYIDQFSRCLYVEYKKSGIHVQCQVPLYVASKMASIKRSSFIVPSTDGYARAAMRWIGYEPRCTPYWPHTLLWGLAYKLPESAIDAWRLRFCLGIRKRGQLKDSRKME >SEQ ID NO.10 XsKCR / M246L amino acid sequence MMEHCFLDTLKAQPIWVLVLFTVGSLSVLRCALSILNWVYVNFLRPAKNLKKYGSWALVTGPTDGIGKGFAFELARKGLNLVLVGRNPDKLKDVSDSIQAKYGKTQIRNVVVDFSGDLDEGVKRIKETIEGLDVGVLINNVGVSYPYARFFHEVDEELLKNLIKINVEGTTKVTQAVLPGMLKRKKGAIVNIGSGAAIVIPSDPLYSVYAATKAYIDQFSRCLYVEYKKSGIHVQCQVPLYVATKLASIKRSSFIVPSTDGYARAAMRWIGYEPRCTPYWPHTLLWGLAYKLPESAIDAWRLRFCLGIRKRGQLKDSRKME >SEQ ID NO.11 XsECR / V152I amino acid sequence MKVTVVSRSGRELIKGGLELNDSATVADLQEAIHKRTKKFYPSRQRLTLPLSPESKERPTVLNYKKSLKEYCDGNSGDLTVVFKDLGPQVSYRTLFFFEYIGPLLLYPVFYYFPVYKYFGYKGERVIHPVQTYALYYWCFHYFKRIMETFFIHRFSHATSPVSNVFRNCAYYWTFGSYIAYYVNHPLYTPVSDLQMKIGFGFGLLCQITNFYCHILLKNLRSPDGAGGYQIPRGFLFNIVTCANYTTEIYQWLGFNIATQTVAGYIFLVVAAFIMTNWALAKHRRLKKLFDGKEGRPKYPRRWVILPPFL >SEQ ID NO.12 XsECR / A271G amino acid sequence MKVTVVSRSGRELIKGGLELNDSATVADLQEAIHKRTKKFYPSRQRLTLPLSPESKERPTVLNYKKSLKEYCDGNSGDLTVVFKDLGPQVSYRTLFFFEYIGPLLLYPVFYYFPVYKYFGYKGERVIHPVQTYALYYWCFHYFKRIMETFFVHRFSHATSPVSNVFRNCAYYWTFGSYIAYYVNHPLYTPVSDLQMKIGFGFGLLCQITNFYCHILLKNLRSPDGAGGYQIPRGFLFNIVTCANYTTEIYQWLGFNIATQTVAGYIFLVVGAFIMTNWALAKHRRLKKLFDGKEGRPKYPRRWVILPPFL >SEQ ID NO.13 XsECR / A281Q amino acid sequence MKVTVVSRSGRELIKGGLELNDSATVADLQEAIHKRTKKFYPSRQRLTLPLSPESKERPTVLNYKKSLKEYCDGNSGDLTVVFKDLGPQVSYRTLFFFEYIGPLLLYPVFYYFPVYKYFGYKGERVIHPVQTYALYYWCFHYFKRIMETFFVHRFSHATSPVSNVFRNCAYYWTFGSYIAYYVNHPLYTPVSDLQMKIGFGFGLLCQITNFYCHILLKNLRSPDGAGGYQIPRGFLFNIVTCANYTTEIYQWLGFNIATQTVAGYIFLVVAAFIMTNWALQKHRRLKKLFDGKEGRPKYPRRWVILPPFL >SEQ ID NO.14 YlKCR nucleotide sequence >SEQ ID NO.15 YlHCD nucleotide sequence ATGGCATCGGCTCTGGTAACCAAGTACCTCACGCTGTACAACAGCATTTCGACCTTTCTGTGGTTTGCCGTTCTGGCACGGCTTCTGATTCTGCTGCCTCTGGTGGGATACGATTACGTGTCTGGGGGTCTGCGGGACTTCCTCATTGGAGTCCAGACTCTGGCAGGCCTGGAGATCTTCCACTCGCTGTTCGGCCTGGTGCGATCGCCCCTGCAGACCACCGTGATGCAAATCTTCTCGCGATTTGCCCTCGTGTGGGGCGTGCTGTACCAGTACCCCCAGACCGGCACTGACATCTTCTTCACCTTCATGGTGATTGCGTGGTCCGTGACCGAGGTGATCCGATACTCCTTCTACGCGCTTAACCTAGCTGGCTGGGGCGTCCCCGACTGGCACCAGTGGATCCGATACAACTTCTTCCTGGTGCTGTACCCTCTGGGTGTGTCTGGCGAGATGAAGCTCATGTGGAACGCCATTGCCTACGCCCGAACCGAGCAGCCCTACTTTGCTCTCTTCCTCAAGGCCGCCATGGTCATCTGGCCCATTGGACTCTACATTCTCTATTCCCACATGCTCAAGCAGCGATCCAAGCATTACCGACAGGTTATCAAGAAGGACAGATAA >SEQ ID NO.16 YlECR nucleotide sequence ATGGTCAATCTGTCTCTGCGTCCCCGTCCTGCCAAGGCCAAGTTCAAGGGTCTGCCCGCCAACCTCGAGGTGTCTCCTGAGGACACGGTGGCCTCTGTGGTGGCGAAATTGAGCGCTGCCACCAAACTGTCCAAGTCCCGAATTCGGCTCACTGTGGCCGATGAGGAGAATGGAGGCGCTCCCGGAGCCAAGAAGAAGCACATTGTGCTCAAGCCCGAGCACGCTGTCGGCGATTACCTCTTCTCCGACTCTCCTGTGGTGTTTGTGAAGGACTTGGGCCCCCAGATCCCCTGGAGAACCGTCTTCATTCTCGAGTACCTGGGACCTCTTCTGGCCCACCCCATCATCTTCTTTGGCCAGAAGTTCTTTTACCGACAGAGCTTTGAGTACACGTTTGCTCAGAAGCTGGTCTTCACTCTATGCATGCTGCATTTCCTCAAGCGAGAGATTGAGACCATCTACATCCACAAGTTCTCCTCCGCCACCATGCCTCTGTTCAACCTCTTTAAGAACTCCGGCTACTACTGGTTCATTGCCGGTTTCAACCTTGCCTTTTTCGTTTATGCCCCCGCCTCCTTTTCTTCTCCCCAGGCTCCTCTGTGGAAGCGTTTCCTCTTTTCCACTGGCTTCTTTGAGCGAACCCCTCTGTTCCTCAACCTCATGGCCGCCCTGTGGCTCTGGGGAGAGACCTCCAACTTCTGGACCCACTTCAACTTGGCTTCTCTGAGAAACGATGGCTCCAAGGACCACAAGATCCCCTTTGGCTACGGCTTCAACCTTGTTTCCTGCCCCAACTACTTCTTTGAGGTTGTCAGCTGGATCGCAATTGCCCTCATGTGTGGCAACTGGTCCGCTTATGTCTTTACTGCCATTGGCTTCGGTCAGATGTACGTCTGGGCCGTCCAGAAGCACCGACGATACAAGCGGGAGTTTGGAGACCGGTACCCTCGAAACAGAAAGGTCATGGTTCCTTTCTTGCTTTAG >SEQ ID NO.17 XsHCD nucleotide sequence ATGGCTGGCTTTCTCTCTCTGCTTCGACGACTGTACCTGTCCGTCTACAACTGGACCATCTTTCTGGGTTGGTCACAGGTGCTGTACCTGGCACTCAAGACTCTGAACGAGTCTGGACACCAGCACGTCTATAACGCTGTCCAGAAGCCTCTGCTTCTGGCACAGACTGCTGCTGTTCTTGAGATTCTGCACGGACTGGTGGGTCTGGTGCGATCCCCTATCACCTCCACTCTGCCTCAGATCTCTTCGCGACTGTACGTGACCTGGGGCATTCTGTGGAGCTTCCCTCAGATCCAGACTCACCTGCTGGTTTCTTCTCTGGTCATCTCTTGGTCTGTGACCGAGATCATCCGATACTCTTTCTTCGGCATGAAAGAGGCCTTCGGTTTCACACCTTCTTGGTTTCTGTGGCTGCGATACTCCACCTTTCTGCTGCTCTACCCTACCGGTATCACCTCTGAGGTCGGCCTGATCTACTTCGCACTGCCTTACATGAAAGACTCCGACAAGTACTGCATCCGAATGCCTAACAAGTGGAACTTCTCTTTCGACTACTTCTACAACGCCATTCTGGTGCTCGGTTTCTACGTGCCTGGTTCGCCTCACCTGTACGGTTACATGCTGCGACAGCGAAAGAAGGCCTTATCCAAGTCTAAGCGAGAGTAG >SEQ ID NO.18 XsKCR nucleotide sequence ATGATGGAGCACTGCTTTCTGGACACCTTAAAGGCTCAGCCTATCTGGGTGCTTGTGCTGTTCACCGTGGGTTCTCTGTCCGTTCTGCGATGTGCTCTGTCCATCTTAAACTGGGTGTACGTCAACTTTCTGCGACCTGCTAAGAACCTGAAGAAGTACGGTTCTTGGGCTCTGGTCACTGGTCCAACCGACGGTATCGGTAAGGGCTTTGCCTTCGAGCTTGCTCGAAAGGGTCTGAACCTGGTGCTTGTCGGTCGAAACCCTGACAAGCTGAAAGACGTCTCCGACTCCATCCAGGCCAAGTACGGCAAGACACAGATCCGAAACGTCGTCGTGGACTTCTCTGGCGACCTCGATGAGGGTGTCAAGCGAATCAAAGAGACGATCGAAGGCCTTGATGTCGGTGTTCTGATCAACAACGTCGGTGTCTCTTATCCATACGCTCGATTCTTCCACGAGGTGGACGAGGAGCTGCTGAAGAACCTCATCAAGATCAACGTCGAGGGTACTACCAAGGTCACTCAGGCCGTTCTGCCTGGTATGCTGAAGCGAAAGAAGGGTGCCATCGTCAACATCGGCTCTGGTGCCGCAATCGTCATTCCATCTGACCCTCTGTACTCCGTCTACGCTGCTACCAAGGCCTACATCGACCAGTTCTCCAGATGTCTCTACGTCGAGTACAAGAAGTCTGGCATTCACGTCCAGTGTCAGGTGCCTCTGTACGTCGCCACCAAGATGGCCTCCATCAAGCGATCTTCGTTCATCGTGCCTTCTACCGACGGTTACGCACGAGCTGCTATGCGATGGATTGGCTACGAACCTCGATGCACTCCATACTGGCCTCACACTCTTCTGTGGGGTCTGGCCTACAAGCTGCCTGAGTCTGCCATCGACGCCTGGAGACTGCGATTCTGTCTGGGTATCCGAAAGCGAGGTCAGCTGAAGGACTCTCGAAAGATGGAGTAG >SEQ ID NO.19 XsECR nucleotide sequence ATGAAGGTCACTGTCGTCTCTCGATCTGGTCGAGAGCTGATCAAGGGTGGTCTGGAGCTGAACGACTCTGCTACCGTCGCTGATCTGCAAGAGGCTATCCACAAGCGAACCAAGAAGTTCTACCCTTCTCGACAGCGACTGACTCTGCCTCTGTCTCCAGAGTCCAAAGAGCGACCTACTGTGCTGAACTACAAGAAGTCTCTGAAAGAGTACTGTGACGGTAACTCTGGTGACCTTACCGTGGTGTTCAAAGACCTTGGTCCACAGGTGTCTTACCGAACTCTGTTCTTCTTCGAGTACATCGGTCCACTTCTGCTGTACCCTGTGTTCTACTACTTCCCTGTCTACAAGTACTTCGGCTACAAGGGTGAACGAGTCATCCATCCAGTTCAGACCTACGCTCTGTACTACTGGTGCTTCCACTACTTCAAGCGAATCATGGAAACCTTCTTCGTCCACCGATTCTCTCACGCTACTTCGCCTGTCTCCAACGTGTTCCGAAACTGTGCCTACTACTGGACCTTCGGCTCTTACATCGCCTACTACGTCAACCACCCTCTGTACACACCTGTCTCTGACCTGCAGATGAAGATCGGCTTCGGTTTCGGACTTCTGTGTCAGATCACCAACTTCTACTGCCACATTCTTCTGAAGAACCTGCGATCTCCAGATGGTGCTGGTGGTTACCAGATTCCACGAGGCTTTCTGTTCAACATCGTCACTTGCGCCAACTACACCACCGAGATCTACCAGTGGCTTGGCTTTAACATCGCCACACAGACCGTTGCTGGCTACATCTTTCTGGTCGTTGCTGCCTTCATCATGACCAACTGGGCTCTTGCTAAGCATCGACGACTGAAGAAGCTGTTCGACGGTAAGGAGGGACGACCTAAGTATCCACGACGATGGGTCATTCTGCCTCCATTTCTGTGA >SEQ ID NO.20 XsKCR / V143I nucleotide sequence ATGATGGAGCACTGCTTTCTGGACACCTTAAAGGCTCAGCCTATCTGGGTGCTTGTGCTGTTCACCGTGGGTTCTCTGTCCGTTCTGCGATGTGCTCTGTCCATCTTAAACTGGGTGTACGTCAACTTTCTGCGACCTGCTAAGAACCTGAAGAAGTACGGTTCTTGGGCTCTGGTCACTGGTCCAACCGACGGTATCGGTAAGGGCTTTGCCTTCGAGCTTGCTCGAAAGGGTCTGAACCTGGTGCTTGTCGGTCGAAACCCTGACAAGCTGAAAGACGTCTCCGACTCCATCCAGGCCAAGTACGGCAAGACACAGATCCGAAACGTCGTCGTGGACTTCTCTGGCGACCTCGATGAGGGTGTCAAGCGAATCAAAGAGACGATCGAAGGCCTTGATGTCGGTGTTCTGATCAACAACGTCGGTATCTCTTATCCATACGCTCGATTCTTCCACGAGGTGGACGAGGAGCTGCTGAAGAACCTCATCAAGATCAACGTCGAGGGTACTACCAAGGTCACTCAGGCCGTTCTGCCTGGTATGCTGAAGCGAAAGAAGGGTGCCATCGTCAACATCGGCTCTGGTGCCGCAATCGTCATTCCATCTGACCCTCTGTACTCCGTCTACGCTGCTACCAAGGCCTACATCGACCAGTTCTCCAGATGTCTCTACGTCGAGTACAAGAAGTCTGGCATTCACGTCCAGTGTCAGGTGCCTCTGTACGTCGCCACCAAGATGGCCTCCATCAAGCGATCTTCGTTCATCGTGCCTTCTACCGACGGTTACGCACGAGCTGCTATGCGATGGATTGGCTACGAACCTCGATGCACTCCATACTGGCCTCACACTCTTCTGTGGGGTCTGGCCTACAAGCTGCCTGAGTCTGCCATCGACGCCTGGAGACTGCGATTCTGTCTGGGTATCCGAAAGCGAGGTCAGCTGAAGGACTCTCGAAAGATGGAGTAG >SEQ ID NO.21 XsKCR / Y241F nucleotide sequence ATGATGGAGCACTGCTTTCTGGACACCTTAAAGGCTCAGCCTATCTGGGTGCTTGTGCTGTTCACCGTGGGTTCTCTGTCCGTTCTGCGATGTGCTCTGTCCATCTTAAACTGGGTGTACGTCAACTTTCTGCGACCTGCTAAGAACCTGAAGAAGTACGGTTCTTGGGCTCTGGTCACTGGTCCAACCGACGGTATCGGTAAGGGCTTTGCCTTCGAGCTTGCTCGAAAGGGTCTGAACCTGGTGCTTGTCGGTCGAAACCCTGACAAGCTGAAAGACGTCTCCGACTCCATCCAGGCCAAGTACGGCAAGACACAGATCCGAAACGTCGTCGTGGACTTCTCTGGCGACCTCGATGAGGGTGTCAAGCGAATCAAAGAGACGATCGAAGGCCTTGATGTCGGTGTTCTGATCAACAACGTCGGTATCTCTTATCCATACGCTCGATTCTTCCACGAGGTGGACGAGGAGCTGCTGAAGAACCTCATCAAGATCAACGTCGAGGGTACTACCAAGGTCACTCAGGCCGTTCTGCCTGGTATGCTGAAGCGAAAGAAGGGTGCCATCGTCAACATCGGCTCTGGTGCCGCAATCGTCATTCCATCTGACCCTCTGTACTCCGTCTACGCTGCTACCAAGGCCTACATCGACCAGTTCTCCAGATGTCTCTACGTCGAGTACAAGAAGTCTGGCATTCACGTCCAGTGTCAGGTGCCTCTGTTCGTCGCCACCAAGATGGCCTCCATCAAGCGATCTTCGTTCATCGTGCCTTCTACCGACGGTTACGCACGAGCTGCTATGCGATGGATTGGCTACGAACCTCGATGCACTCCATACTGGCCTCACACTCTTCTGTGGGGTCTGGCCTACAAGCTGCCTGAGTCTGCCATCGACGCCTGGAGACTGCGATTCTGTCTGGGTATCCGAAAGCGAGGTCAGCTGAAGGACTCTCGAAAGATGGAGTAG >SEQ ID NO.22 XsECR / Y92W nucleotide sequence ATGAAGGTCACTGTCGTCTCTCGATCTGGTCGAGAGCTGATCAAGGGTGGTCTGGAGCTGAACGACTCTGCTACCGTCGCTGATCTGCAAGAGGCTATCCACAAGCGAACCAAGAAGTTCTACCCTTCTCGACAGCGACTGACTCTGCCTCTGTCTCCAGAGTCCAAAGAGCGACCTACTGTGCTGAACTACAAGAAGTCTCTGAAAGAGTACTGTGACGGTAACTCTGGTGACCTTACCGTGGTGTTCAAAGACCTTGGTCCACAGGTGTCTTGGCGAACTCTGTTCTTCTTCGAGTACATCGGTCCACTTCTGCTGTACCCTGTGTTCTACTACTTCCCTGTCTACAAGTACTTCGGCTACAAGGGTGAACGAGTCATCCATCCAGTTCAGACCTACGCTCTGTACTACTGGTGCTTCCACTACTTCAAGCGAATCATGGAAACCTTCTTCGTCCACCGATTCTCTCACGCTACTTCGCCTGTCTCCAACGTGTTCCGAAACTGTGCCTACTACTGGACCTTCGGCTCTTACATCGCCTACTACGTCAACCACCCTCTGTACACACCTGTCTCTGACCTGCAGATGAAGATCGGCTTCGGTTTCGGACTTCTGTGTCAGATCACCAACTTCTACTGCCACATTCTTCTGAAGAACCTGCGATCTCCAGATGGTGCTGGTGGTTACCAGATTCCACGAGGCTTTCTGTTCAACATCGTCACTTGCGCCAACTACACCACCGAGATCTACCAGTGGCTTGGCTTTAACATCGCCACACAGACCGTTGCTGGCTACATCTTTCTGGTCGTTGCTGCCTTCATCATGACCAACTGGGCTCTTGCTAAGCATCGACGACTGAAGAAGCTGTTCGACGGTAAGGAGGGACGACCTAAGTATCCACGACGATGGGTCATTCTGCCTCCATTTCTGTGA >SEQ ID NO.23 XsECR / I274Q nucleotide sequence ATGAAGGTCACTGTCGTCTCTCGATCTGGTCGAGAGCTGATCAAGGGTGGTCTGGAGCTGAACGACTCTGCTACCGTCGCTGATCTGCAAGAGGCTATCCACAAGCGAACCAAGAAGTTCTACCCTTCTCGACAGCGACTGACTCTGCCTCTGTCTCCAGAGTCCAAAGAGCGACCTACTGTGCTGAACTACAAGAAGTCTCTGAAAGAGTACTGTGACGGTAACTCTGGTGACCTTACCGTGGTGTTCAAAGACCTTGGTCCACAGGTGTCTTACCGAACTCTGTTCTTCTTCGAGTACATCGGTCCACTTCTGCTGTACCCTGTGTTCTACTACTTCCCTGTCTACAAGTACTTCGGCTACAAGGGTGAACGAGTCATCCATCCAGTTCAGACCTACGCTCTGTACTACTGGTGCTTCCACTACTTCAAGCGAATCATGGAAACCTTCTTCGTCCACCGATTCTCTCACGCTACTTCGCCTGTCTCCAACGTGTTCCGAAACTGTGCCTACTACTGGACCTTCGGCTCTTACATCGCCTACTACGTCAACCACCCTCTGTACACACCTGTCTCTGACCTGCAGATGAAGATCGGCTTCGGTTTCGGACTTCTGTGTCAGATCACCAACTTCTACTGCCACATTCTTCTGAAGAACCTGCGATCTCCAGATGGTGCTGGTGGTTACCAGATTCCACGAGGCTTTCTGTTCAACATCGTCACTTGCGCCAACTACACCACCGAGATCTACCAGTGGCTTGGCTTTAACATCGCCACACAGACCGTTGCTGGCTACATCTTTCTGGTCGTTGCTGCCTTCCAGATGACCAACTGGGCTCTTGCTAAGCATCGACGACTGAAGAAGCTGTTCGACGGTAAGGAGGGACGACCTAAGTATCCACGACGATGGGTCATTCTGCCTCCATTTCTGTGA >SEQ ID NO.24 XsKCR / I66L nucleotide sequence ATGATGGAGCACTGCTTTCTGGACACCTTAAAGGCTCAGCCTATCTGGGTGCTTGTGCTGTTCACCGTGGGTTCTCTGTCCGTTCTGCGATGTGCTCTGTCCATCTTAAACTGGGTGTACGTCAACTTTCTGCGACCTGCTAAGAACCTGAAGAAGTACGGTTCTTGGGCTCTGGTCACTGGTCCAACCGACGGTCTGGGTAAGGGCTTTGCCTTCGAGCTTGCTCGAAAGGGTCTGAACCTGGTGCTTGTCGGTCGAAACCCTGACAAGCTGAAAGACGTCTCCGACTCCATCCAGGCCAAGTACGGCAAGACACAGATCCGAAACGTCGTCGTGGACTTCTCTGGCGACCTCGATGAGGGTGTCAAGCGAATCAAAGAGACGATCGAAGGCCTTGATGTCGGTGTTCTGATCAACAACGTCGGTGTCTCTTATCCATACGCTCGATTCTTCCACGAGGTGGACGAGGAGCTGCTGAAGAACCTCATCAAGATCAACGTCGAGGGTACTACCAAGGTCACTCAGGCCGTTCTGCCTGGTATGCTGAAGCGAAAGAAGGGTGCCATCGTCAACATCGGCTCTGGTGCCGCAATCGTCATTCCATCTGACCCTCTGTACTCCGTCTACGCTGCTACCAAGGCCTACATCGACCAGTTCTCCAGATGTCTCTACGTCGAGTACAAGAAGTCTGGCATTCACGTCCAGTGTCAGGTGCCTCTGTACGTCGCCACCAAGATGGCCTCCATCAAGCGATCTTCGTTCATCGTGCCTTCTACCGACGGTTACGCACGAGCTGCTATGCGATGGATTGGCTACGAACCTCGATGCACTCCATACTGGCCTCACACTCTTCTGTGGGGTCTGGCCTACAAGCTGCCTGAGTCTGCCATCGACGCCTGGAGACTGCGATTCTGTCTGGGTATCCGAAAGCGAGGTCAGCTGAAGGACTCTCGAAAGATGGAGTAG >SEQ ID NO.25 XsKCR / T244S nucleotide sequence ATGATGGAGCACTGCTTTCTGGACACCTTAAAGGCTCAGCCTATCTGGGTGCTTGTGCTGTTCACCGTGGGTTCTCTGTCCGTTCTGCGATGTGCTCTGTCCATCTTAAACTGGGTGTACGTCAACTTTCTGCGACCTGCTAAGAACCTGAAGAAGTACGGTTCTTGGGCTCTGGTCACTGGTCCAACCGACGGTATCGGTAAGGGCTTTGCCTTCGAGCTTGCTCGAAAGGGTCTGAACCTGGTGCTTGTCGGTCGAAACCCTGACAAGCTGAAAGACGTCTCCGACTCCATCCAGGCCAAGTACGGCAAGACACAGATCCGAAACGTCGTCGTGGACTTCTCTGGCGACCTCGATGAGGGTGTCAAGCGAATCAAAGAGACGATCGAAGGCCTTGATGTCGGTGTTCTGATCAACAACGTCGGTATCTCTTATCCATACGCTCGATTCTTCCACGAGGTGGACGAGGAGCTGCTGAAGAACCTCATCAAGATCAACGTCGAGGGTACTACCAAGGTCACTCAGGCCGTTCTGCCTGGTATGCTGAAGCGAAAGAAGGGTGCCATCGTCAACATCGGCTCTGGTGCCGCAATCGTCATTCCATCTGACCCTCTGTACTCCGTCTACGCTGCTACCAAGGCCTACATCGACCAGTTCTCCAGATGTCTCTACGTCGAGTACAAGAAGTCTGGCATTCACGTCCAGTGTCAGGTGCCTCTGTACGTCGCCTCCAAGATGGCCTCCATCAAGCGATCTTCGTTCATCGTGCCTTCTACCGACGGTTACGCACGAGCTGCTATGCGATGGATTGGCTACGAACCTCGATGCACTCCATACTGGCCTCACACTCTTCTGTGGGGTCTGGCCTACAAGCTGCCTGAGTCTGCCATCGACGCCTGGAGACTGCGATTCTGTCTGGGTATCCGAAAGCGAGGTCAGCTGAAGGACTCTCGAAAGATGGAGTAG >SEQ ID NO.26 XsKCR / M246L nucleotide sequence ATGATGGAGCACTGCTTTCTGGACACCTTAAAGGCTCAGCCTATCTGGGTGCTTGTGCTGTTCACCGTGGGTTCTCTGTCCGTTCTGCGATGTGCTCTGTCCATCTTAAACTGGGTGTACGTCAACTTTCTGCGACCTGCTAAGAACCTGAAGAAGTACGGTTCTTGGGCTCTGGTCACTGGTCCAACCGACGGTATCGGTAAGGGCTTTGCCTTCGAGCTTGCTCGAAAGGGTCTGAACCTGGTGCTTGTCGGTCGAAACCCTGACAAGCTGAAAGACGTCTCCGACTCCATCCAGGCCAAGTACGGCAAGACACAGATCCGAAACGTCGTCGTGGACTTCTCTGGCGACCTCGATGAGGGTGTCAAGCGAATCAAAGAGACGATCGAAGGCCTTGATGTCGGTGTTCTGATCAACAACGTCGGTATCTCTTATCCATACGCTCGATTCTTCCACGAGGTGGACGAGGAGCTGCTGAAGAACCTCATCAAGATCAACGTCGAGGGTACTACCAAGGTCACTCAGGCCGTTCTGCCTGGTATGCTGAAGCGAAAGAAGGGTGCCATCGTCAACATCGGCTCTGGTGCCGCAATCGTCATTCCATCTGACCCTCTGTACTCCGTCTACGCTGCTACCAAGGCCTACATCGACCAGTTCTCCAGATGTCTCTACGTCGAGTACAAGAAGTCTGGCATTCACGTCCAGTGTCAGGTGCCTCTGTACGTCGCCACCAAGCTGGCCTCCATCAAGCGATCTTCGTTCATCGTGCCTTCTACCGACGGTTACGCACGAGCTGCTATGCGATGGATTGGCTACGAACCTCGATGCACTCCATACTGGCCTCACACTCTTCTGTGGGGTCTGGCCTACAAGCTGCCTGAGTCTGCCATCGACGCCTGGAGACTGCGATTCTGTCTGGGTATCCGAAAGCGAGGTCAGCTGAAGGACTCTCGAAAGATGGAGTAG >SEQ ID NO.27 XsECR / V152I nucleotide sequence ATGAAGGTCACTGTCGTCTCTCGATCTGGTCGAGAGCTGATCAAGGGTGGTCTGGAGCTGAACGACTCTGCTACCGTCGCTGATCTGCAAGAGGCTATCCACAAGCGAACCAAGAAGTTCTACCCTTCTCGACAGCGACTGACTCTGCCTCTGTCTCCAGAGTCCAAAGAGCGACCTACTGTGCTGAACTACAAGAAGTCTCTGAAAGAGTACTGTGACGGTAACTCTGGTGACCTTACCGTGGTGTTCAAAGACCTTGGTCCACAGGTGTCTTACCGAACTCTGTTCTTCTTCGAGTACATCGGTCCACTTCTGCTGTACCCTGTGTTCTACTACTTCCCTGTCTACAAGTACTTCGGCTACAAGGGTGAACGAGTCATCCATCCAGTTCAGACCTACGCTCTGTACTACTGGTGCTTCCACTACTTCAAGCGAATCATGGAAACCTTCTTCATCCACCGATTCTCTCACGCTACTTCGCCTGTCTCCAACGTGTTCCGAAACTGTGCCTACTACTGGACCTTCGGCTCTTACATCGCCTACTACGTCAACCACCCTCTGTACACACCTGTCTCTGACCTGCAGATGAAGATCGGCTTCGGTTTCGGACTTCTGTGTCAGATCACCAACTTCTACTGCCACATTCTTCTGAAGAACCTGCGATCTCCAGATGGTGCTGGTGGTTACCAGATTCCACGAGGCTTTCTGTTCAACATCGTCACTTGCGCCAACTACACCACCGAGATCTACCAGTGGCTTGGCTTTAACATCGCCACACAGACCGTTGCTGGCTACATCTTTCTGGTCGTTGCTGCCTTCATCATGACCAACTGGGCTCTTGCTAAGCATCGACGACTGAAGAAGCTGTTCGACGGTAAGGAGGGACGACCTAAGTATCCACGACGATGGGTCATTCTGCCTCCATTTCTGTGA >SEQ ID NO.28 XsECR / A271G nucleotide sequence ATGAAGGTCACTGTCGTCTCTCGATCTGGTCGAGAGCTGATCAAGGGTGGTCTGGAGCTGAACGACTCTGCTACCGTCGCTGATCTGCAAGAGGCTATCCACAAGCGAACCAAGAAGTTCTACCCTTCTCGACAGCGACTGACTCTGCCTCTGTCTCCAGAGTCCAAAGAGCGACCTACTGTGCTGAACTACAAGAAGTCTCTGAAAGAGTACTGTGACGGTAACTCTGGTGACCTTACCGTGGTGTTCAAAGACCTTGGTCCACAGGTGTCTTACCGAACTCTGTTCTTCTTCGAGTACATCGGTCCACTTCTGCTGTACCCTGTGTTCTACTACTTCCCTGTCTACAAGTACTTCGGCTACAAGGGTGAACGAGTCATCCATCCAGTTCAGACCTACGCTCTGTACTACTGGTGCTTCCACTACTTCAAGCGAATCATGGAAACCTTCTTCGTCCACCGATTCTCTCACGCTACTTCGCCTGTCTCCAACGTGTTCCGAAACTGTGCCTACTACTGGACCTTCGGCTCTTACATCGCCTACTACGTCAACCACCCTCTGTACACACCTGTCTCTGACCTGCAGATGAAGATCGGCTTCGGTTTCGGACTTCTGTGTCAGATCACCAACTTCTACTGCCACATTCTTCTGAAGAACCTGCGATCTCCAGATGGTGCTGGTGGTTACCAGATTCCACGAGGCTTTCTGTTCAACATCGTCACTTGCGCCAACTACACCACCGAGATCTACCAGTGGCTTGGCTTTAACATCGCCACACAGACCGTTGCTGGCTACATCTTTCTGGTCGTTGGCGCCTTCATCATGACCAACTGGGCTCTTGCTAAGCATCGACGACTGAAGAAGCTGTTCGACGGTAAGGAGGGACGACCTAAGTATCCACGACGATGGGTCATTCTGCCTCCATTTCTGTGA >SEQ ID NO.29 XsECR / A281Q nucleotide sequence ATGAAGGTCACTGTCGTCTCTCGATCTGGTCGAGAGCTGATCAAGGGTGGTCTGGAGCTGAACGACTCTGCTACCGTCGCTGATCTGCAAGAGGCTATCCACAAGCGAACCAAGAAGTTCTACCCTTCTCGACAGCGACTGACTCTGCCTCTGTCTCCAGAGTCCAAAGAGCGACCTACTGTGCTGAACTACAAGAAGTCTCTGAAAGAGTACTGTGACGGTAACTCTGGTGACCTTACCGTGGTGTTCAAAGACCTTGGTCCACAGGTGTCTTACCGAACTCTGTTCTTCTTCGAGTACATCGGTCCACTTCTGCTGTACCCTGTGTTCTACTACTTCCCTGTCTACAAGTACTTCGGCTACAAGGGTGAACGAGTCATCCATCCAGTTCAGACCTACGCTCTGTACTACTGGTGCTTCCACTACTTCAAGCGAATCATGGAAACCTTCTTCGTCCACCGATTCTCTCACGCTACTTCGCCTGTCTCCAACGTGTTCCGAAACTGTGCCTACTACTGGACCTTCGGCTCTTACATCGCCTACTACGTCAACCACCCTCTGTACACACCTGTCTCTGACCTGCAGATGAAGATCGGCTTCGGTTTCGGACTTCTGTGTCAGATCACCAACTTCTACTGCCACATTCTTCTGAAGAACCTGCGATCTCCAGATGGTGCTGGTGGTTACCAGATTCCACGAGGCTTTCTGTTCAACATCGTCACTTGCGCCAACTACACCACCGAGATCTACCAGTGGCTTGGCTTTAACATCGCCACACAGACCGTTGCTGGCTACATCTTTCTGGTCGTTGCTGCCTTCATCATGACCAACTGGGCTCTTCAGAAGCATCGACGACTGAAGAAGCTGTTCGACGGTAAGGAGGGACGACCTAAGTATCCACGACGATGGGTCATTCTGCCTCCATTTCTGTGA >SEQ ID NO.30 Upstream homology arm sequence required for integrating the donor fragment at the F17 locus tccttgagcttgggtcgtgtgattcggggggccatgttagatgtggaagacgtaaatagtcgtttggtggtaggtgtatgggccgacagagtagagacacctttaatagaaagattcagagatcgtaacatcatgggctgatataagagtgttagagttacctcggcaggtgagtagggtggagatggggaaatacacacagacaaacaagactctagagacgctgtcacaattacgagactactacaatgccatttattctgcttcagattatcaaaattcagaccatcattactacatcccacccttgcagttgaaagacctcggtaaaaacactaattcatgtgatggcggccattaccccgtggggaaggttaataaatatggtgggggaggggtgataattacaattgtcgagatgtgacgctgaaagagtagaacttcttgaaaattgtgtacaattgcatcccacttaaaacttagagtgtgatttgtacaaaaatgataccgtttgtcatttcccacctctttgcttactaagcaacataaaggctttgcatcaccccaattaatccgatcgaccgaagtataatatccagcgcaagtttcagcgctccggaacaattgttgccctgtaacagattgaagatcacatgacccatcttccgactcctaccctgtctttgaggtctcttggctgcattattgtgggttattgtggtgtgatgtttcggaaaaagtggtgcgaccattgggagtgtacgagacttggattaaccaagtgaacaagcaccaacagtaggatggtgttggaaagtgcccatgg >SEQ ID NO.31 Downstream homology arm sequence required for integrating the donor fragment at the F17 locus cagacccaccgactgacacaaatgccgtacgatccaaccacagcacgtccttctgtttatttgcactcttgagacgcacgtctacacggcgtttggcactaaaccccgtacatattgcgcaggcgttggggtacatgaggcaaactttcgggctggggtcgggatatgatgatggtgagaatgtagtttcggggatgagtcggtgttgtagaagggaaatacgtggctgtgctttaaataaaagaatgggacagattacgtagcacgtagataggatggccacgttccatactcgtaatgtattcgcatacactgttaaggatcagctgggttatactctgctgcctggctttcatgtatgttggagaggaggcgtattgtgtggttgtacttgaggttcgttagggtcttgtatatacagcgcgaaatgaggcagcccttgtaccggtagctaataggcaagcgctttttttggtctacatattcaccccatgccattcttaagggcactgggatttgtattgttactggtcggaaagaatcggcagcactgtgtggggagactgggatggccgaatatggcgcagctgaaacagggtagttctcccattaattaaagatagaagttctttgcacagccagagcccgccagccgggtagcatacccaatgactcgttcacatgactataaaacggacccggaaaaatccaacatttcgctctacatgcagatgcaggttggcccggaaatatccggatgggcggtcaaactagcaagtggcgtgtcgattaaatacgggcaagaacttg >SEQ ID NO.32 FAS2 promoter sequence Gttgatccaaatgataccagaaaatacaccaaaatatggccgcaatggggcatgaatagcactcatattgcgctagaccagcttcccaattcactatattgcatatacaagtagtagtgctcgttcaaaacgcgtcccgaaaacctgtccaccagttgcactgttgtctcctttctgcttggcagatatttacaagcctcggttgacgatcacttatttatccgaaatgccgcagtgagagagtgcaccttagatcgcgtgttttcaggtgtagtttcagcagctgactacacatacatgtcacccactcatcaacacaagcccaactatcacccgatacaagactacagtattgttttactgtagcagccctgttgggttccggctgtccttttctcaatctcaaccgtctcccaacgttgggagcagcataaggtttggaaacaaacgacgcaggaaaaaaaagttggtttatcagttgggggagggcgttcaagtagggagaggatatatggtaaatgggcgcgtggaatgaagtagagaatggtttgcttgagaaaacgacccttaaattaatcatggccattttttccccactcaaaatccccccacaacaccctgcaacatacacttcccagcactcaacaaagttgatcctccctcaacgtatgccactgcatgcatgatacgggggtcgcgattctaaataaaacgactgtcgtgcctcaattgaaacgtcacgagaaatctgtgaactacacacacaaacagttcattacg >SEQ ID NO.33 PRC1t terminator sequence gatgtcggtaatggacagtttcgtgagaggctggaccgacaacttgtagccatcagtagcttagcacgagctattttattagctagattattattgaatatatatgccattgaatgaagagaatctgtagatgagccgtaggtgtgctgtgggacgtgtatggaccggtgatataggaggaatggttcgtttatgctacagtagccagatgtatcaggaaggatctcatatcaaaaggtccttcgcatatcatcacagactgaacaatccacagcacacgtagccttcacacaatcttctatgaagtggtgttattgttagtactgggtttcacttgatcgatagggcaagttga。

Claims

1. A fatty acid elongation enzyme mutant, characterized in that: The mutant is a key amino acid located in the substrate binding pocket, substrate entry channel, or enzyme-substrate interaction interface of one or more of the fatty acid elongation enzymes 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), and enoyl-CoA reductase (ECR) derived from *Sapindus mukorossi*. The amino acid sequences of 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), and enoyl-CoA reductase (ECR) are shown in SEQ ID NO:24-26, respectively.

2. The fatty acid elongation enzyme mutant according to claim 1, characterized in that: The mutant is a mutation at one or more of the following positions: 66, 143, 241, 244, and 246 of 3-ketoacyl-CoA reductase (KCR); specifically, XsKCR / I66L (SEQ ID NO:31), XsKCR / V143I (SEQ ID NO:27), XsKCR / Y241F (SEQ ID NO:28), XsKCR / T244S (SEQ ID NO:32), and XsKCR / M246L (SEQ ID NO:33). Mutations at one or more sites at positions 92, 152, 271, 274, and 281 of enoyl-CoA reductase (ECR); specifically, mutations at the following sites: XsECR / Y92W (SEQ ID NO:29), XsECR / V152I (SEQ ID NO:34), XsECR / A271G (SEQ ID NO:35), XsECR / I274Q (SEQ ID NO:30), and XsECR / A281Q (SEQ ID NO:36).

3. A recombinant plasmid, characterized in that: The recombinant plasmid contains the fatty acid elongation enzyme mutant or fatty acid elongation enzyme as described in claim 1; wherein the fatty acid elongation enzyme is one of the following fatty acid elongation enzymes derived from *Sapindus mukorossi*: 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), and enoyl-CoA reductase (ECR).

4. A recombinant yeast strain, characterized in that: The recombinant yeast strain is a host strain containing the recombinant plasmid as described in claim 1.

5. The recombinant yeast strain according to claim 4, characterized in that: The host strain is an oil-producing yeast; wherein, the oil-producing yeast is a species of Yersinia (Yersinia). Yarrowia ), Rhodotorula genus ( Rhodotorula ) and Candida genus ( Candida One or more of them.

6. The recombinant yeast strain according to claim 5, characterized in that: The host strain is *Yersinia lipophila* (… Yarrowia lipolytica ), Rhodotorula buergerianum ( Rhodosporidium toruloides ), Stirryl oil yeast ( Lipomyces starkeyi ), Oil-producing mycorrhizal yeast ( Cutaneotrichosporon oleaginosus ), dermal filamentous yeast ( Trichosporon cutaneum ) or red yeast ( Phaffia rhodozyma ).

7. A method for constructing the recombinant yeast strain according to claim 4, characterized in that: Homologous recombinant plasmids were constructed, linearized using a site-directed integration method, and transformed into the host chassis. Positive transformants were screened using uracil auxotrophic plates to obtain the recombinant yeast strain.

8. The method for constructing the recombinant yeast strain according to claim 7, characterized in that: The host is *Yersinia lipophila* ( Yarrowia lipolytica The recombinant yeast strain YLNA20-ΔURA, a uracil auxotrophic strain, was obtained by linearizing the homologous recombinant plasmid using a site-directed integration method and then transforming it into the host IntF17 site.

9. An application as described in claim 1 or 4, characterized in that: The use of the mutant of claim 1 or the recombinant yeast strain of claim 4 in the synthesis of nervonic acid.

10. A method for synthesizing nervonic acid, characterized in that: The recombinant yeast strain described in claim 4 was cultured in a fermentation medium with shaking at 28°C to obtain nervonic acid.

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