Yarrowia lipolytica genetically engineered bacteria and application thereof

Abstract

This invention provides a genetically engineered *Yersinia lipolytica* strain and its applications. The genetically engineered strain is a *Yersinia lipolytica* Po1f strain with the PEX10 gene knocked out, expressing the C16 / 18 elongase gene MaELO3 from *Morchella alpina*, the elongase gene AtKCS from *Arabidopsis thaliana*, and the elongase gene CraKCS from *Brassica oleracea*. The strain also expresses the elongase gene CgKCS from *Capsella*. Further expression of the desaturase gene MaD15D from *Morchella alpina*, and overexpression of the strain's own diacylglycerol transferase gene DGA1 and its own Δ9 desaturase gene OLE1, resulted in a nervonic acid yield of 111.6 mg / L in shake-flask fermentation. By adding 1% rapeseed oil exogenously, the nervonic acid yield reached 185.1 mg / L. The *Yersinia lipolytica* genetically engineered strain constructed in this invention is simple to operate, has stable and reliable performance, and can be applied to large-scale commercial production.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically, it relates to a genetically engineered *Yarrowia lipophila* strain and its applications. Background Technology

[0002] Nervonic acid (C 24 H 46 O2) is an ultra-long-chain monounsaturated fatty acid, named cis-15-tetracoecanoic acid or ω-9-tetracoecanoic acid based on the position of the unsaturated bond and the length of the carbon chain. Nervonic acid is a core component of nerve fibers and nerve cells, and is also an essential fatty acid for brain growth and development. Nervonic acid was first isolated from the brain of mammalian sharks, hence it is also known as shark oleic acid or shark bile oleic acid. The ability of sharks to repair themselves quickly after severe brain injury is attributed to the promoting effect of nervonic acid on the repair and regeneration of nerve fibers in damaged brain tissue. The high-value biological functions of nervonic acid make it play an important role in pharmacological and nutritional applications.

[0003] Nervonic acid is obtained through extraction from animal and plant tissues or chemical synthesis. Animal-derived nervonic acid primarily comes from marine organisms. Due to its market and commercial value, developed countries historically relied heavily on shark hunting to obtain nervonic acid. However, shark fishing has been banned to protect sharks, leading to a shortage of nervonic acid resources. Chemical synthesis using cis-13-dodecyl methyl ester as a precursor has resulted in low yields and numerous byproducts. Therefore, most commercially available nervonic acid is derived from plants. Studies have shown that nervonic acid exists in the seed oils of some wild plants, but the extraction of nervonic acid from natural plant sources is limited by factors such as growth cycle, habitat, and quantity.

[0004] With the development of genetic engineering, the large-scale production of nervonic acid through the engineered modification of plants, microalgae, and microorganisms has become a potential possibility. Compared to Saccharomyces cerevisiae, Yersinia lipolytica, as an oil-producing yeast, can produce fats and oils at a high level and synthesize fatty acids, providing sufficient fatty acid precursors for nervonic acid. It has a wide range of substrate utilization capabilities, is a recognized safe strain, and is a superior genetically engineered bacterium for nervonic acid synthesis.

[0005] Currently, the engineering modification of nervonic acid synthesis pathway-related genes is generally achieved by introducing them exogenously. However, the nervonic acid synthesis pathway is very complex, involving numerous related genes, and the effects of nervonic acid production vary depending on the species from which these genes are derived, when combined with different fungal strains. As mentioned earlier, nervonic acid is widely present in various animals, plants, and microorganisms. Therefore, there is an urgent need to screen for genes suitable for improving nervonic acid production levels in *Yarrowia lipolytica* from the vast number of existing nervonic acid synthesis pathway-related genes from different species. Summary of the Invention

[0006] To address the problem of low yield of nervonic acid produced by Yersinia lipolyticis in existing technologies, this invention constructs a Yersinia lipolyticis strain capable of expressing fatty acid elongase genes AtKCS, CraKCS, MaELO3, and CgKCS, enabling it to effectively produce very long-chain fatty acids (VLCFAs).

[0007] Based on this, by overexpressing two copies of the elongase gene (CgKCS) and the desaturase gene (MaD15D) in the fusion expression form CgKCS-L-MaD15D, the Δ9 desaturase (OLE1) and the diacylglycerol transferase gene (DGA1) in the fusion expression form OLE1-L-DGA1, and the diacylglycerol transferase gene (DGA1), a nervonic acid-producing Yersinia lipolytica strain was successfully obtained.

[0008] To solve the above-mentioned technical problems, one of the technical solutions provided by the present invention is: a genetically engineered strain of Yarrowia lipolytica, wherein the genetically engineered strain is a strain in which the PEX10 gene is knocked out in Yarrowia lipolytica Po1f, and expresses the C16 / 18 elongase gene MaELO3 from Mortierella alpine, the elongase gene AtKCS from Arabidopsis thaliana, and the elongase gene CraKCS from Crambeabyssinica. The genetically engineered strain also expresses the elongase gene CgKCS from Cardamine graeca.

[0009] The *Yarrowia lipolyticis* Po1f described in this invention is a known genetically modified yeast with the genotype MatA, leu2-270, ura3-302, xpr2-322, axp-2 and the phenotype Leu-, Ura-, DAEP, DAXP, Suc+.

[0010] The gene PEX10 described in this invention is a gene related to β-oxidation in Yersinia lipolytica Po1f.

[0011] As described in one of the technical solutions, the genetically engineered bacteria contains one copy of the elongation enzyme gene CgKCS; or, the genetically engineered bacteria contains one copy of the elongation enzyme gene CgKCS and two copies of the elongation enzyme gene CgKCS and a fusion gene CgKCS-L-MaD15D derived from the desaturase gene MaD15D of *Morchella alpina*.

[0012] In a specific embodiment of the present invention, the fusion gene CgKCS-L-MaD15D is formed by inserting a linker into the elongase gene CgKCS and the desaturase gene MaD15D using NEB ligase, so that they use the same set of promoters and terminators. The linker is conventional in the art.

[0013] Fusion enzymes can reduce the accumulation of intermediates in the synthetic pathway. This invention fuses the elongation enzyme CgKCS and the desaturation enzyme MaD15D to promote nervonic acid production, thereby reducing the generation of other ultra-long chain fatty acids. Although this invention fuses the elongation enzyme gene CgKCS and the desaturation enzyme gene MaD15D, those skilled in the art can reasonably expect that even if these two genes were separated and expressed individually in the strain's genome, the strain's nervonic acid production would still be increased.

[0014] As described in one of the technical solutions, the genetically engineered bacteria also overexpress its own diacylglycerol transferase gene DGA1.

[0015] In a preferred embodiment of the present invention, the genetically engineered bacteria contain a total of two copies of the diacylglycerol transferase gene DGA1. That is, the strain itself contains one copy of DGA1, and an additional copy of DGA1 is introduced.

[0016] As described in one of the technical solutions, the genetically engineered bacteria contains two copies of its own diacylglycerol transferase gene DGA1, and also contains one copy of the diacylglycerol transferase gene DGA1 and its own Δ9 desaturase gene OLE1 fusion gene OLE1-L-DGA1. That is, the strain itself contains one copy of DGA1, and then an additional one copy of DGA1 and one copy of the fusion gene OLE1-L-DGA1 are introduced.

[0017] Diacylglycerol transferase DGA1 can promote the accumulation of C22:0 and C24:0 fatty acids, and Δ9 desaturase OLE1 is beneficial to the formation of C24:1. After multiple experiments by the inventors, it was found that copying one more DGA1 and one more OLE1-L-DGA1 is most beneficial to the production of nervonic acid (C24:1).

[0018] In a specific embodiment of the present invention, the fusion gene OLE1-L-DGA1 is formed by inserting a linker into the diacylglycerol transferase gene DGA1 and the Δ9 desaturase gene OLE1 using NEB ligase, so that they use the same set of promoters and terminators. The linker is conventional in the art.

[0019] Fusion enzymes can reduce the accumulation of intermediates in the synthetic pathway. This invention fuses the diacylglycerol transferase DGA1 and the Δ9 desaturase OLE1 to promote nervonic acid production, thereby reducing the generation of other long-chain fatty acids. Although this invention fuses the diacylglycerol transferase gene DGA1 and the Δ9 desaturase gene OLE1, those skilled in the art can reasonably expect that even if these two genes were separated and expressed individually in the strain's genome, the strain's nervonic acid production would still be increased.

[0020] As described in one of the technical solutions, the nucleotide sequence of the C16 / 18 elongase gene MaELO3 is shown in SEQ ID NO:37; and / or, the nucleotide sequence of the elongase gene AtKCS is shown in SEQ ID NO:34; and / or, the nucleotide sequence of the elongase gene CraKCS is shown in SEQ ID NO:35; and / or, the nucleotide sequence of the elongase gene CgKCS is shown in SEQ ID NO:33 or SEQ ID NO:36; and / or, the nucleotide sequence of the diacylglycerol transferase gene DGA1 is shown in SEQ ID NO:38; and / or, the nucleotide sequence of the fusion gene CgKCS-L-MaD15D is shown in SEQ ID NO:39; and / or, the nucleotide sequence of the fusion gene OLE1-L-DGA1 is shown in SEQ ID NO:40.

[0021] To solve the above-mentioned technical problems, the second technical solution provided by the present invention is: a method for constructing a genetically engineered bacterium as described in the first technical solution, wherein the construction method includes integrating the gene to be expressed into the genome of Yersinia lipolytica, wherein the integration method is to transfer an integration expression plasmid containing the gene to be expressed into Yersinia lipolytica; and / or, the construction method is based on the CRISPR / Cas9 operating system.

[0022] The construction method described in technical solution two, when based on the CRISPR / Cas9 operating system, includes the following steps:

[0023] (1) Construct a plasmid pair containing a donor plasmid and an sgRNA plasmid, wherein the donor plasmid contains the gene to be expressed and the sgRNA plasmid contains an sgRNA sequence targeting the integration site on the genome.

[0024] (2) The plasmid pair was simultaneously transformed into Yersinia lipophila and the target strain was obtained by screening.

[0025] As described in the second technical solution, the promoter of the gene to be expressed in the donor plasmid or the integrated expression plasmid is UAS4B+TEF; and / or, the backbone of the donor plasmid is pHR_hrGFP; and / or, the backbone of the sgRNA plasmid is pCRISPRyl; and / or, the backbone of the integrated expression plasmid is pINA1269 or p3204.

[0026] In a preferred embodiment of the present invention, the donor plasmid is a pHR_hrGFP plasmid containing the elongase gene CgKCS or the fusion gene CgKCS-L-MaD15D, and the integration expression plasmid is a pINA1269 plasmid containing the diacylglycerol transferase gene DGA1 or a p3204 plasmid containing the fusion gene OLE1-L-DGA1.

[0027] In this invention, the promoter UAS4B+TEF is a promoter composed of the enhancer portion UAS4B from the promoter hp4d derived from plasmid PINA1312 and the strong promoter TEF from DNA derived from Yersinia lipolyticis PLF. For the specific construction method, please refer to patent CN 107815425 A.

[0028] In this invention, pHR_hrGFP refers to the collective term for plasmids including pHR_A3_hrGFP, pHR_F1-3_hrGFP, pHR_AXP_hrGFP, pHR_F1_hrGFP, pHR_A1-2_hrGFP, and pHR_E1-3_hrGFP; pCRISPRyl refers to the collective term for plasmids including pCRISPRyl_A3, pCRISPRyl_F1-3, pCRISPRyl_AXP, pCRISPRyl_F1, pCRISPRyl_A1-2, and pCRISPRyl_E1-3; wherein A3, F1-3, AXP, F1, A1-2, and E1-3 correspond to specific gene loci in the Yersinia lipolytica genome.

[0029] To solve the above-mentioned technical problems, the third technical solution provided by the present invention is: a method for producing nervonic acid, the method comprising fermenting genetically engineered bacteria as described in one of the technical solutions in a culture medium and isolating nervonic acid therefrom; the culture medium is preferably YPD culture medium.

[0030] In a preferred embodiment of the present invention, the culture medium is further supplemented with oil or oleic acid; the oil is preferably rapeseed oil.

[0031] In a preferred embodiment of the invention, the rapeseed oil added is 1% by volume.

[0032] To solve the above-mentioned technical problems, the fourth technical solution provided by the present invention is: the application of genetically engineered bacteria as described in the first technical solution in the production of nervonic acid.

[0033] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0034] The positive and progressive effects of this invention are as follows: The beneficial effects of the nervonic acid-producing Yersinia lipolytica genetically engineered strain of this invention are: (1) High nervonic acid yield: The construction method of the nervonic acid-producing Yersinia lipolytica genetically engineered strain NA06 of this invention is simple. Through fermentation, it can produce high levels of nervonic acid, achieving a yield of 111.6 mg / L in a 50 mL shake flask, which has good application prospects. (2) By adding its synthesis precursor, when adding 1% rapeseed oil, the nervonic acid-producing Yersinia lipolytica genetically engineered strain NA06 of this invention can achieve a yield of 185.1 mg / L of nervonic acid in a 50 mL shake flask. (3) Its construction method is simple. Only 4 genes (CgKCS, CgKCS-L-MaD15D, DGA1, DGA1-L-OLE1) are introduced through CRISPR / Cas9 and integrative plasmid complementation screening markers. The obtained strain has good stability after continuous subculturing and can be applied to large-scale commercial production, with good prospects. Attached Figure Description

[0035] Figure 1 To investigate the metabolic pathway of nervonic acid production in Yersinia lipolytica after the introduction of elongase and nervonic acid synthase genes.

[0036] Figure 2 This is a diagram showing the results of shake-flask fermentation of a nervonic acid-producing strain.

[0037] Figure 3A The effect of adding different types of oil / oleic acid on the yield of nervonic acid (i.e., C24:1).

[0038] Figure 3B The effect of different rapeseed oil addition concentrations on the yield of nervonic acid (i.e., C24:1).

[0039] Figure 3C The results show the fermentation of strain NA06 to produce nervonic acid (i.e., C24:1) with or without the addition of 1% rapeseed oil.

[0040] Figure 4A comparison diagram showing the production of ultra-long chain fatty acids by the CgKCS gene without codon optimization and the CgKCS-opt gene with codon optimization, where C24:1 refers to nervonic acid. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are performed according to conventional methods and conditions, or as selected according to the product instructions.

[0042] The *Yersinia lipolyticis* used in this application is based on patent CN 111979135. In section A, strain GQ07, which can effectively produce LCFAs (corresponding to NA02 of this invention, which is based on the knockout of the PEX10 gene in *Yersinia lipolytica* Po1f and the expression of the elongase gene AtKCS from *Arabidopsis thaliana*, the elongase gene CraKCS from *Brassica napus*, and the C16 / 18 elongase gene MaELO3 from *Morchella alpina*), further overexpressed the elongase gene CgKCS and CgKCS-opt from *Cyperus spp.* to explore the effect of codon optimization on the gene CgKCS; further overexpression of the fusion expression forms of the elongase gene (CgKCS-opt) and desaturase gene (MaD15D) CgKCS-opt-L-MaD15D, the fusion expression form of the Δ9 desaturase (OLE1) and diacylglycerol transferase gene (DGA1) OLE1-L-DGA1, and the diacylglycerol transferase gene (DGA1) finally yielded an engineered *Yersinia lipolytica* strain producing nervonic acid (see [link to original text]). Figure 1 ).

[0043] The reagents and raw materials used in this invention are all commercially available.

[0044] The strains and plasmids involved in this invention are from the following sources:

[0045] 1. Yarrowia lipolytica Po1f, plasmid pINA1269: prepared according to the method described in Madzak, C., Treton, B., Blanchin-Roland, S. 2000. Strong hybrid promoters and integrative expression / secretion vectors for quasi-constitutive expression of heterologous proteins in theireast Yarrowia lipolytica. J Mol Microbiol Biotechnol, 2(2), 207-216.

[0046] 2. Plasmid pINA1312: Prepared by the method described in Nicant, JM, Madzak, C., van den Broek, P., Gysler, C., Duboc, P., Niederberger, P., Gaillardin, C. 2002. Protein expression and secretion in the yeast Yarrowia lipolytica. FEMS Yeast Res, 2(3), 371-379.

[0047] 3. Plasmids pCRISPRyl_F1 and pHR_F1_hrGFP, pCRISPRyl_A3 and pHR_A3_hrGFP, pCRISPRyl_F1-3 and pHR_F1-3_hrGFP, pCRISPRyl_AXP and pHR_AXP_hrGFP, pCRISPRyl_A1-2 and pHR_A1-2_hrGFP, pCRISPRyl_E1-3 and pHR_E1-3_hrGFP: See the preparation method described in Zhang, XK, Wang, DN, Chen, J., Liu, ZJ, Wei, LJ, Hua, Q. 2020. Metabolic engineering of beta-carotene biosynthesis in Yarrowia lipolytica. Biotechnol Lett, 42(6), 945-956.

[0048] 4. Plasmid p3204: The promoter of pINA1312 was replaced with UAS4B+TEF. Yeast lipolytica Po1f-ΔPEX10: The PEX10 gene was knocked out from Yeast lipolytica Po1f. See the preparation method described in Gao, Q., Cao, X., Huang, YY, Yang, JL, Chen, J., Wei, LJ, and Hua, Q. 2018. Overproduction of FattyAcid Ethyl Esters by the Oleaginous Yeast Yarrowia lipolytica through Metabolic Engineering and Process Optimization, ACS Synthetic Biology 7, 1371-1380.

[0049] 5. Plasmids pHR_F1_MaELO3 and p32UTMaELO3: Contain the C16 / 18 elongase gene MaELO3 from *Morchella alpina*. Plasmids pHR_A3_AtKCS and p32UTAtKCS: Contain the elongase gene AtKCS from *Arabidopsis thaliana*. Plasmids pHR_F1-3_CraKCS and p32UTCraKCS: Contain the elongase gene CraKCS from *Brassica napus*. Plasmids pHR_AXP_CgKCS and p32UTCgKCS: Contain the elongase gene CgKCS from *Capsella*. Plasmids pHR_AXP_CgKCS-opt and p32UTCgKCS-opt: Contain the codon-optimized elongase gene CgKCS-opt from *Capsella*. Plasmids pHR_A1-2_CgKCS-opt-L-MaD15D, pHR_E1-3_CgKCS-opt-L-MaD15D, and p32UTCgKCS-opt-L-MaD15D contain the elongase gene CgKCS-opt from the genus *Cephalotaxus* and the desaturase gene MaD15D from *Morchella alpina*. Plasmid p32UTOLE1-L-DGA1 contains the endogenous Δ9 desaturase gene OLE1 and the diacylglycerol transferase gene DGA1 from *Yarrowia lipolytica*. Plasmids p69DGA1 and p32DGA1 contain the endogenous diacylglycerol transferase gene DGA1 from *Yarrowia lipolytica*.

[0050] The primer sequences for constructing the above plasmids are shown in Table 1.

[0051] Table 1 Primer sequences of plasmid pairs

[0052]

[0053] In the following examples, the elongase gene CgKCS is the uncodon-optimized gene (nucleotide sequence as shown in SEQ ID NO:33). The elongase genes AtKCS, CraKCS, CgKCS-opt, and MaELO3 all refer to the optimized AtKCS, CraKCS, CgKCS, and MaELO3 genes, with nucleotide sequences as shown in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37, respectively. The optimized nucleotide sequence of the diacylglycerol transferase gene DGA1 is shown in SEQ ID NO:38, the nucleotide sequence of the fusion gene CgKCS-opt-L-MaD15D is shown in SEQ ID NO:39, and the nucleotide sequence of the fusion gene OLE1-L-DGA1 is shown in SEQ ID NO:40.

[0054] Example 1: Construction of a genetically engineered *Yarrowia lipolyticis* strain that produces long-chain fatty acids

[0055] (1) Construction of expression plasmids p32UTAtKCS, p32UTCraKCS, p32UTCgKCS-opt and p32UTMaELO3 for the elongase genes AtKCS, CraKCS, CgKCS-opt and MaELO3. Primers 32UTAtKCS-f and 32UTAtKCS-r (nucleotide sequences shown in SEQ ID NO:1-2), 32UTCraKCS-f and 32UTCraKCS-r (nucleotide sequences shown in SEQ ID NO:3-4), 32UTCgKCS-opt-f and 32UTCgKCS-opt-r (nucleotide sequences shown in SEQ ID NO:5-6), and 32UTMaELO3-f and 32UTMaELO3-r (nucleotide sequences shown in SEQ ID NO:7-8) were used sequentially to extract the optimized sequence of the elongation enzyme gene AtKCS from Arabidopsis thaliana (nucleotide sequence shown in SEQ ID NO:34), the optimized sequence of the elongation enzyme gene CraKCS from Crambe abyssinica (nucleotide sequence shown in SEQ ID NO:35), and the nucleotide sequence of Cardamine spp. The optimized sequences of the elongase gene CgKCS-opt from *Gnaphalium graeca* (its nucleotide sequence is shown in SEQ ID NO:36) and the optimized sequence of the C16 / 18 elongase gene MaELO3 from *Mortierella alpine* (its nucleotide sequence is shown in SEQ ID NO:37) were constructed into plasmid p3204 with the promoter UAS4B+TEF via the restriction sites PmlI and BamHI, resulting in plasmids p32UTAtKCS, p32UTCraKCS, p32UTCgKCS-opt, and p32UTMaELO3. Among them, the codon-optimized AtKCS, CraKCS, CgKCS-opt and MaELO3 genes were obtained by optimizing the nucleotide sequences of AtKCS, CraKCS, CgKCS-opt and MaELO3 from Arabidopsis thaliana, Crambe abyssinica, Cardamine graeca and Mortierella alpine, respectively.

[0056] (2) Based on the existing CRISPR / Cas9 operating system, the donor plasmid pHR_F1_MaELO3 containing the optimized MaELO3 gene promoter UAS4B+TEF, the donor plasmid pHR_A3_AtKCS containing the optimized AtKCS gene promoter UAS8B+TEF, the donor plasmid pHR_F1-3_CraKCS containing the optimized CraKCS gene promoter, and the donor plasmid pHR_AXP_CgKCS-opt containing the optimized CgKCS-opt gene promoter were constructed. These plasmids can be combined with the sgRNA plasmids pCRISPRyl_F1, pCRISPRyl_A3, pCRISPRyl_F1-3, and pCRISPRyl_AXP to form a knock-in plasmid pair, respectively.

[0057] The knock-in plasmid pair in this embodiment is a conventional recombinant vector in the art. The sgRNA plasmid contains a leucine selection marker, and the donor plasmid contains a uracil selection marker. It can transform uracil and leucine auxotrophic yeast lipophilic yeast. The knock-in plasmid pair can sequentially knock in the above-mentioned optimized MaELO3 gene, AtKCS gene, CraKCS gene, and CgKCS-opt gene.

[0058] The specific steps are as follows:

[0059] (1) First, it is necessary to obtain the expression cassette of the target gene. Primers are used in sequence: 32UTAtKCS-f and 32UTAtKCS-r (nucleotide sequences are shown in SEQ ID NO: 1-2), 32UTCraKCS-f and 32UTCraKCS-r (nucleotide sequences are shown in SEQ ID NO: 3-4), 32UTCgKCS-opt-f and 32UTCgKCS-opt-r (nucleotide sequences are shown in SEQ ID NO: 4-6), and 32UTMaELO3-f and 32UTMaELO3-r (nucleotide sequences are shown in SEQ ID NO: 6). (As shown in NO:7-8) The corresponding “AtKCS”, “CraKCS”, and “CgKCS-opt” of the p32UTAtKCS, p32UTCraKCS, p32UTCgKCS-opt, and p32UTMaELO3 plasmids were obtained and sequentially constructed into plasmids pHR_A3_hrGFP, pHR_F1-3_hrGFP, pHR_AXP_hrGFP, and pHR_F1_hrGFP using the NheI and pteI restriction sites, respectively, to obtain plasmids pHR_A3_AtKCS, pHR_F1-3_CraKCS, and pHR_AXP_CgKCS-opt. The “UAS4B+TEF-MaELO3” fragment was then digested using the NheI and BssHII restriction sites to obtain plasmid pHR_F1_MaELO3.

[0060] (4) The plasmid pHR_F1_MaELO3 and the sgRNA plasmid pCRISPRyl_F1 obtained in step (3) were simultaneously transformed into Yersinia lipolytica Po1f-ΔPEX10. After recovering the selection marker, strain GQ06 was obtained. It was verified that the MaELO3 gene was knocked into strain GQ06. The transformation was performed using the Frozen EZ Yeast Transformation II kit. TM (Purchased from Zymo Research) Follow the instructions in the kit's manual.

[0061] (5) The plasmid pHR_A3_AtKCS and the sgRNA plasmid pCRISPRyl_A3 obtained in step (3) were simultaneously transformed into Yersinia lipolytica Po1f-ΔPEX10-F1MaELO3. After recovering the selection marker, strain NA01 was obtained. It was verified that the AtKCS gene was knocked into strain NA01. The transformation was performed using the Frozen EZ Yeast Transformation II kit. TM (Purchased from Zymo Research) Follow the instructions in the kit's manual.

[0062] (6) The plasmid pHR_F1-3_CraKCS and the sgRNA plasmid pCRISPRyl_F1-3 obtained in step (3) were simultaneously transformed into *Yarrowia lipolytica* Po1f-ΔPEX10-F1MaELO3-A3AtKCS. After recovering the selection marker, strain NA02 (i.e., strain GQ07 in patent application CN 111979135 A) was obtained. It was verified that the CraKCS gene was knocked into strain NA02. The transformation was performed using the Frozen EZ Yeast Transformation II kit. TM (Purchased from Zymo Research) Follow the instructions in the kit's manual.

[0063] (7) The plasmid pHR_AXP_CgKCS-opt and the sgRNA plasmid pCRISPRyl_AXP obtained in step (3) were simultaneously transformed into *Yarrowia lipolytica* Po1f-ΔPEX10-F1MaELO3-A3AtKCS-F1-3CraKCS. After recovering the selection marker, strain NA03 was obtained. Verification confirmed that the CgKCS-opt gene was knocked into strain NA03. Transformation was performed using the FrozenEZ Yeast Transformation II kit. TM (Purchased from Zymo Research) Follow the instructions in the kit's manual.

[0064] Example 2: Construction of a genetically engineered lecithin-producing Yersinia lipolytica strain

[0065] (1) Construction of the fusion expression forms of the elongase gene and the desaturase gene, CgKCS-opt-L-MaD15D, the fusion expression forms of the endogenous Δ9 desaturase OLE1 and DGA1, OLE1-L-DGA1, and the expression plasmids of the diacylglycerol transferase gene DGA1, p32UTCgKCS-opt-L-MaD15D, p32UTOLE1-L-DGA1, p32DGA1, and p69DGA1. Primers 32UTCgKCS-opt-L-MaD15D-f and 32UTCgKCS-opt-L-MaD15D-r (nucleotide sequences shown in SEQ ID NO:21-22), 32UTOLE1-L-DGA1-f and 32UTOLE1-L-DGA1-r (nucleotide sequences shown in SEQ ID NO:23-24), 69-DGA1-f and 69-DGA1-r (nucleotide sequences shown in SEQ ID NO:25-26), and 32DGA1-f and 32DGA1-r (nucleotide sequences shown in SEQ ID NO:27-28) were used sequentially to introduce the elongase gene CgKCS-opt from *Cardamine graeca* and the gene from *Mortierella*. The optimized sequence of the desaturase gene MaD15D (as shown in SEQ ID NO:39) of alpine, the optimized sequence of the fusion gene CgKCS-opt-L-MaD15D (as shown in SEQ ID NO:40), the optimized sequence of the endogenous Δ9 desaturase OLE1 and the diacylglycerol transferase gene DGA1, the optimized sequence of DGA1 (as shown in SEQ ID NO:38), were constructed into the plasmid p3204 with the promoter UAS4B+TEF through the restriction sites PmlI and BamHI, resulting in plasmids p32UTCgKCS-opt-L-MaD15D, p32UTOLE1-L-DGA1, p32DGA1, and pINA69. The codon-optimized CgKCS-opt and MaD15D genes were obtained by optimizing the nucleotide sequences of CgKCS-opt and MaD15D from the genera *Cardamine graeca* and *Mortierella alpine*, respectively.

[0066] (2) Based on the existing CRISPR / Cas9 operating system, donor plasmids pHR_A1-2_CgKCS-opt-L-MaD15D and pHR_E1-3_CgKCS-opt-L-MaD15D containing the optimized CgKCS-opt-L-MaD15D gene were constructed. These plasmids were then combined with sgRNA plasmids pCRISPRyl_A1-2 and pCRISPRyl_E1-3 to form knock-in plasmid pairs, respectively.

[0067] The knock-in plasmid pair in this embodiment is a conventional recombinant vector in the art. The sgRNA plasmid contains a leucine selection marker, and the donor plasmid contains a uracil selection marker. It can transform uracil and leucine auxotrophic yeast lipophilic yeast. This knock-in plasmid pair can sequentially knock in the above-mentioned optimized CgKCS-opt-L-MaD15D gene.

[0068] The specific steps are as follows:

[0069] (1) First, the target gene needs to be obtained. Primers 32UTCgKCS-opt-L-MaD15D-f and 32UTCgKCS-opt-L-MaD15D-r (nucleotide sequences are shown in SEQ ID NO:1-2 of the sequence listing, respectively) are used to obtain the “CgKCS-opt-L-MaD15D” fragment of the p32UTCgKCS-opt-L-MaD15D plasmid. The fragment is then constructed into plasmids pHR_A1-2_hrGFP and pHR_E1-3_hrGFP by using the restriction enzyme sites NheI and BssHII, respectively, to obtain plasmids pHR_A1-2_CgKCS-opt-L-MaD15D and pHR_E1-3_CgKCS-opt-L-MaD15D.

[0070] (2) The plasmid pHR_A1-2_CgKCS-opt-L-MaD15D and the sgRNA plasmid pCRISPRyl_A1-2 obtained in step (1) were simultaneously transformed into *Yarrowia lipolytica* Po1f-ΔPEX10-F1MaELO3-A3AtKCS-F1-3CraKCS-AXPCgKCS-opt. After recovering the selection marker, strain NA04 was obtained. Verification confirmed that the CgKCS-opt-L-MaD15D gene was knocked into strain NA04. Transformation was performed using the Frozen EZ Yeast Transformation II kit. TM (Purchased from Zymo Research) Follow the instructions in the kit's manual.

[0071] (3) The plasmid pHR_E1-3_CgKCS-opt-L-MaD15D and the sgRNA plasmid pCRISPRyl_E1-3 obtained in step (1) were simultaneously transformed into *Yarrowia lipolytica* Po1f-ΔPEX10-F1MaELO3-A3AtKCS-F1-3CraKCS-AXPCgKCS-opt-A1-2CgKCS-opt-L-MaD15D. After recovering the selection marker, strain NA05 was obtained. Verification confirmed that the CgKCS-opt-L-MaD15D gene was knocked into strain NA05. Transformation was performed using the Frozen EZ YeastTransformation II kit. TM (Purchased from Zymo Research) Follow the instructions in the kit's manual.

[0072] (4) Transform the plasmids p32UTOLE1-L-DGA1 and p69DGA1 obtained in step (1) into Yersinia lipolytica NA05 to obtain Yersinia lipolytica strain NA06 that produces nervonic acid.

[0073] (5) Transform the plasmid p32DGA1 obtained in step (1) into Yersinia lipolytica NA03 to obtain Yersinia lipolytica strain NA07 that produces nervonic acid.

[0074] The construction steps of plasmid p32DGA1 are as follows: the DGA1 gene derived from Yersinia lipolytica is constructed into plasmid p3204 through the restriction sites PmlI and BamHI to obtain plasmid p32DGA1. The nucleotide sequence of the DGA1 gene is shown in SEQ ID NO:38.

[0075] According to the present invention, the construction steps of plasmid p32UTOLE1-L-DGA1 in step (4) are as follows: the OLE1 gene and DGA1 gene from Yersinia lipolyticis are ligated into plasmid p3204 by NEB ligase through the restriction sites PmlI and BamHI to obtain plasmid p32UTOLE1-L-DGA1, and the nucleotide sequence of the OLE1-L-DGA1 gene is shown in SEQ ID NO:40.

[0076] The construction steps of plasmid p69DGA1 are as follows: the DGA1 gene derived from Yeast lipolyticis is constructed into plasmid pINA1269 to obtain plasmid p69DGA1.

[0077] Example 3: Determination of nervonic acid production by the strain

[0078] The strains GQ06, NA01, NA02, NA03, NA04, NA05, NA06, and NA07 prepared in Examples 1 and 2 were inoculated with 100 μL of bacterial culture in test tubes containing 3 mL of YPD (the YPD medium consists of 2% glucose, 2% peptone, and 1% yeast extract, with the remainder being water, all percentages being by mass). The cultures were incubated at 37°C and 220 rpm for 24 h. Then, the strains were inoculated into Erlenmeyer flasks containing 50 mL of YPD. The inoculation volume was determined based on the OD600 of the bacterial culture in the shake flask after inoculation being 0.01. Fermentation was carried out at 37°C and 220 rpm for 3 days, with each shake flask experiment performed in duplicate.

[0079] Fatty acid extraction: After fermentation, thoroughly mix the bacterial culture in the shake flask. Transfer 20 mL of the culture to a 50 mL screw-top centrifuge tube. Centrifuge at 6400 rpm for 4 min. Discard the supernatant, add 20 mL of deionized water, mix thoroughly using a vortex mixer, centrifuge under the same conditions, discard the supernatant, and repeat the step once. Add 5 mL of 4 mol / L hydrochloric acid to the centrifuge tube, mix thoroughly, and vortex at 30°C and 220 rpm for 30 min. Remove the centrifuge tube from the shaker and place it in a boiling water bath for 5 min. After the time is up, quickly place it on ice and keep it for 5 min. Repeat this step once. Add 20 mL of a thoroughly mixed solution with a methanol:chloroform ratio of 1:2, vortex at 30°C and 220 rpm for 30 min, then centrifuge at 6400 rpm for 4 min. Clear stratification will occur. Aspirate the clear liquid at the bottom and transfer it to a stoppered glass test tube. The glass test tube should be pre-dried at 105°C to constant weight and then weighed.

[0080] Fatty acid derivatization: This experiment used GC (gas chromatography) for product detection. Since GC cannot detect fatty acids, it was necessary to derivatize them into their corresponding fatty acid methyl esters for detection. The specific procedure is as follows: Add 3 mL of freshly prepared 0.5 mol / L methanol-potassium hydroxide solution (2.8 g potassium hydroxide dissolved in 100 mL methanol) to a glass tube cooled to room temperature, stopper the tube, and dissolve the oil using sonication. After dissolution, incubate the tube in a 75°C water bath for 20 min. After 20 min, remove the glass tube and add 3 mL of 14% boron trichloride solution to the tube. Incubate the tube at 75°C for 20 min. Remove the tube and add 1 mL of saturated NaCl and 500 μL of n-hexane. Vortex the tube at 150 rpm for 1.5 min. After vortexing, the liquid in the tube will separate into layers. Transfer the upper layer to a 1.5 mL EP tube and centrifuge at 12000 rpm for 1 min. After centrifugation, transfer 50 μL of the supernatant to a new 1.5 mL EP tube, then add 150 μL of n-hexane and 40 μL of 0.5 g / L C17:0 fatty acid methyl ester internal standard. Mix thoroughly, i.e., Vinternal standard:Vsample = 1:4. Filter the mixture through a 0.22 μm organic phase filter into a gas chromatography bottle for GC analysis.

[0081] Fatty acid content determination: GC detection was used with a DB-5HT(30m)T column and a 0.1μm gas chromatograph. The injection port temperature was 280℃, the injection volume was 1μL, the detector temperature was 200℃, and the split ratio was 20:1. The specific procedure was as follows: the initial column temperature was 150℃, held for 2 min, then increased to 180℃ at a rate of 20℃ / min, followed by an increase to 215℃ at a rate of 4℃ / min, held for 1.5 min, and finally increased to 300℃ at a rate of 20℃ / min.

[0082] Each culture was inoculated into 2 mL of YPD medium (which consists of 2% glucose, 2% peptone, and 1% yeast extract, with the remainder being water, all percentages being by mass), and incubated for 24 hours. Then, the initial OD was used as the starting point. 600 The culture medium was inoculated at a rate of 0.01 μL into fresh 50 mL YPD medium. After 3 days of fermentation, nervonic acid was extracted and the content of each nervonic acid component was determined by GC (gas chromatography).

[0083] Determination of nervonic acid content: After fermentation, total oil was extracted from the engineered *Yarrowia lipolytica* broth. The extracted oil was dissolved in 1 mL of n-hexane, and 200 μl of the sample was mixed with 1 g / L internal standard C28:0 nervonic acid. The mixture was filtered through a 0.22 μm organic phase filter membrane and then analyzed by GC.

[0084] The chromatographic column was a DB-5HT. The initial column temperature was 150℃, held for 2 min, then increased to 180℃ at a rate of 20℃ / min, followed by 200℃ at a rate of 8℃ / min, then 218℃ at a rate of 1℃ / min, and finally increased to 350℃ at a rate of 3.5℃ / min and held for 10 min. The injection volume was 1 μL, the detector temperature was 200℃, the injection port temperature was 280℃, and the split ratio was 20:1.

[0085] The results of nervonic acid measurement are as follows Figure 2 As shown in the figure, the NA06 engineered strain produced 111.6 mg / L, making it the strain with the highest nervonic acid production among the nervonic acid-producing strains of Yersinia lipolytica.

[0086] Example 4: Fermentation culture of nervonic acid producing strain

[0087] This experiment used the nervonic acid-producing *Yarrowia lipolytica* strain NA07 as the fermentation strain. The optimal culture medium composition for nervonic acid synthesis by adding different oil / oleic acid ratios to 50 mL of YPD fermentation medium was investigated. Strawberries preserved in glycerol culture tubes were streaked on YPD agar plates. Single clones were picked and inoculated into 15 mL short test tubes containing 3 mL of YPD medium (ampicillin and kanamycin resistance were added to the test tubes to prevent contamination). The tubes were incubated overnight at 30°C and 220 rpm in a shaker. Cells in the exponential growth phase were then inoculated into 250 mL shake flasks. 0.25 mL of different oil / oleic acid ratios were added, including rapeseed oil (CO), soybean oil (SO), sunflower seed oil (SSO), oleic acid (OA), and waste cooking oil (WCO). Results are as follows: Figure 3A As shown, the control group indicates that no additional oil / oleic acid was added. The optimal auxiliary carbon source was rapeseed oil. When 0.25 mL of rapeseed oil was added to YPD medium, the nervonic acid content reached 95.2 mg / L.

[0088] This experiment used the nervonic acid-producing *Yarrowia lipolytica* strain NA07 as the fermentation strain. By adding different volumes of rapeseed oil to 50 mL of YPD fermentation medium, the optimal culture medium composition for nervonic acid synthesis by *Yarrowia lipolytica* was investigated. The strain preserved in glycerol culture tubes was streaked on YPD solid plates. Single clones were picked and inoculated into 15 mL short test tubes containing 3 mL of YPD medium (ampicillin and kanamycin resistance were added to the test tubes to prevent contamination). The test tubes were incubated overnight at 30℃ in a shaker at 220 rpm. Cells in the exponential growth phase were then inoculated into 250 mL shake flasks. Different rapeseed oil addition gradients were set: 0, 0.25 mL, 0.5 mL, 0.75 mL, 1 mL, and 1.25 mL (corresponding to volume percentages of 0%, 0.5%, 1%, 1.5%, 2%, and 2.5%, respectively). The results are as follows: Figure 3B As shown. Under the experimentally determined optimal rapeseed oil addition of 0.5 mL in 50 mL YPD medium, the fermentation results of strain NA06 are as follows. Figure 3C As shown, NA06 has a nervonic acid yield of 185.1 mg / L, which is the highest nervonic acid yield found in Yersinia lipolytica to date.

[0089] Figure 3B The results showed that adding rapeseed oil at different gradients (0, 0.25 mL, 0.5 mL, 0.75 mL, 1 mL, and 1.25 mL) was more beneficial to the synthesis of nervonic acid in Yersinia lipolytica. Figure 3C The results showed that in 50 mL of YPD with 0.5 mL of rapeseed oil added, the fermentation results of strain NA06 showed that the yield of nervonic acid in Yersinia lipolytica reached the highest level of 185.1 mg / L.

[0090] Example 5: The effect of codon optimization on the gene CgKCS

[0091] (1) Construction of expression plasmids p32UTAtKCS, p32UTCraKCS, p32UTCgKCS and p32UTCgKCS-opt for the elongase genes AtKCS, CraKCS, CgKCS and CgKCS-opt. The optimized sequence of the elongase gene AtKCS from Arabidopsis thaliana (its nucleotide sequence is shown in SEQ ID NO:34), the optimized sequence of the elongase gene CraKCS from Crambe abyssinica (its nucleotide sequence is shown in SEQ ID NO:35), the unoptimized sequence of the elongase gene CgKCS from Cardamine graeca (its nucleotide sequence is shown in SEQ ID NO:33), and the optimized sequence CgKCS-opt of the elongase gene CgKCS (its nucleotide sequence is shown in SEQ ID NO:36) were constructed into the plasmid p3204 with the promoter UAS4B+TEF through the restriction sites PmlI and BamHI, resulting in plasmids p32UTAtKCS, p32UTCraKCS, p32UTCgKCS, and p32UTCgKCS-opt. Among them, the uncodon-optimized CgKCS is derived from the genus Cardamine graeca; the codon-optimized AtKCS, CraKCS, and CgKCS-opt genes were obtained by optimizing the nucleotide sequences of AtKCS, CraKCS, and CgKCS derived from Arabidopsis thaliana, Crambe abyssinica, and Cardamine graeca, respectively.

[0092] (2) Based on the existing CRISPR / Cas9 operating system, optimized donor plasmids pHR_A3_AtKCS (with UAS4B+TEF promoter), pHR_F1-3_CraKCS (with optimized CraKCS promoter), pHR_AXP_CgKCS (with optimized CgKCS promoter), and pHR_AXP_CgKCS-opt (with optimized CgKCS-opt promoter) were constructed. These plasmids, pHR_AXP_CgKCS-opt and pCRISPRyl_A3, pCRISPRyl_F1-3, pCRISPRyl_AXP, and pCRISPRyl_AXP respectively, can form knock-in plasmid pairs.

[0093] The knock-in plasmid pair in this embodiment is a conventional recombinant vector in the art. The sgRNA plasmid contains a leucine selection marker, and the donor plasmid contains a uracil selection marker. It can transform uracil and leucine auxotrophic yeast lipophilic yeast. The knock-in plasmid pair can sequentially knock in the above-mentioned optimized AtKCS gene, CraKCS gene, CgKCS gene and CgKCS-opt gene.

[0094] The specific steps are as follows:

[0095] (1) First, it is necessary to obtain the expression cassette of the target gene. Primers are used in sequence: 32UTAtKCS-f and 32UTAtKCS-r (nucleotide sequences are shown in SEQ ID NO: 1-2), 32UTCraKCS-f and 32UTCraKCS-r (nucleotide sequences are shown in SEQ ID NO: 3-4), 32UTCgKCS-f and 32UTCgKCS-r (nucleotide sequences are shown in SEQ ID NO: 29-30), and 32UTCgKCS-opt-f and 32UTCgKCS-opt-r (nucleotide sequences are shown in SEQ ID NO: 29-30). (As shown in NO:5-6) Obtain the corresponding "AtKCS", "CraKCS", "CgKCS", and "CgKCS-opt" of the plasmids p32UTAtKCS, p32UTCraKCS, p32UTCgKCS, and p32UTCgKCS-opt. Construct them into the plasmids pHR_A3_hrGFP, pHR_F1-3_hrGFP, pHR_AXP_hrGFP, and pHR_AXP_hrGFP respectively through the restriction sites NheI and pteI, to obtain the plasmids pHR_A3_AtKCS, pHR_F1-3_CraKCS, pHR_AXP_CgKCS, and pHR_AXP_CgKCS-opt.

[0096] (2) The plasmid pHR_AXP_CgKCS and the sgRNA plasmid pCRISPRyl_AXP obtained in step (1) were simultaneously transformed into *Yarrowia lipolytica* Po1f-ΔPEX10-F1MaELO3. After recovering the selection marker, strain JL00 was obtained. Verification confirmed that the CgKCS gene was knocked into strain JL00. The transformation was performed using the Frozen EZ Yeast Transformation II kit. TM (Purchased from Zymo Research) Follow the instructions in the kit's manual.

[0097] (3) The plasmid pHR_AXP_CgKCS_opt and the sgRNA plasmid pCRISPRyl_AXP obtained in step (1) were simultaneously transformed into *Yarrowia lipolyticis* Po1f-ΔPEX10-F1MaELO3. After recovering the selection marker, strain JL01 was obtained. Verification confirmed that the CgKCS_opt gene was knocked into strain JL01. Transformation was performed using the Frozen EZ YeastTransformation II kit. TM (Purchased from Zymo Research) Follow the instructions in the kit's manual.

[0098] (4) The plasmid pHR_A3_AtKCS and sgRNA plasmid pCRISPRyl_A3 obtained in step (1) were simultaneously transformed into *Yarrowia lipolytica* Po1f-ΔPEX10-F1MaELO3-AXPCgKCS or Po1f-ΔPEX10-F1MaELO3-AXPCgKCS_opt. The selection markers were recovered, and it was verified that the AtKCS gene was knocked into this strain. The transformation was performed using the FrozenEZ Yeast Transformation II kit. TM (Purchased from Zymo Research) Follow the instructions in the kit's manual.

[0099] (5) The plasmid pHR_F1-3_CraKCS and sgRNA plasmid pCRISPRyl_F1-3 obtained in step (1) were simultaneously transformed into *Yarrowia lipolytica* Po1f-ΔPEX10-F1MaELO3-A3AtKCS-AXPCgKCS or Po1f-ΔPEX10-F1MaELO3-A3AtKCS-AXPCgKCS_opt. After recovering the selection markers, strains JL02 and JL03 were obtained. Verification showed that the CgKCS gene was knocked into strain JL02, and the CgKCS_opt gene was knocked into strain JL03. The transformation was performed using the Frozen EZ Yeast Transformation II kit. TM (Purchased from Zymo Research) Follow the instructions in the kit's manual.

[0100] The constructed *Yarrowia lipolyticis* genetically engineered strain was inoculated from a glycerol preservation tube into 5 mL of YPD medium and cultured for 16-24 hours. Then, it was inoculated again into 50 mL of fresh YPD medium with an initial OD600 of 0.01. Fermentation was carried out at 30°C and 220 rpm for 3 days, after which the bacteria were harvested. After lipid extraction and methyl esterification, the content of fatty acids of various chain lengths was determined by gas chromatography (GC).

[0101] Figure 4 The results showed that the C20 and C22 fatty acid contents of the control strain GQ07 were significantly higher than those of JL02 and JL03. This indicates that overexpression of CgKCS is more conducive to the synthesis of ultra-long chain fatty acids, and the C24 fatty acid content increased significantly from a very low level. In particular, in the strains that overexpressed the codon-optimized CgKCS_opt gene, the cells were able to synthesize 4.6 mg / L of nervonic acid, which was 4.75 times higher than that of the control strain. This proves that the optimized CgKCS_opt gene, in combination with the other three fatty acid elongation enzymes, enables cells to synthesize nervonic acid more efficiently.

[0102] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention. These improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of lipophilic yeast (Yersinia spp.) The genetically engineered bacteria are those from *Yarrowia lipolyticis* Po1f strains with a gene knocked out. And expresses origin from alpine typhus ( C16 / 18 elongase gene Derived from Arabidopsis thaliana ( elongase gene and derived from sea cabbage ( elongase gene The strain is characterized by, The genetically engineered bacteria also expresses the genera *Cephalotaxus* (*Cephalotaxus*). elongase gene ; The genetically engineered bacteria contains one copy of the elongase gene. and 2 copies of the elongase gene and desaturase genes derived from alpine spores Fusion genes ; The genetically engineered bacteria contain two copies of its own diacylglycerol transferase gene. It also contains one copy of the diacylglycerol transferase gene. and its own Δ9 desaturase gene Fusion genes ; The elongase gene The nucleotide sequence is shown in SEQ ID NO: 36; The C16 / 18 elongase gene The nucleotide sequence is shown in SEQ ID NO: 37; the elongase gene The nucleotide sequence is shown in SEQ ID NO: 34; the elongase gene The nucleotide sequence is shown in SEQ ID NO: 35; the diacylglycerol transferase gene The nucleotide sequence is shown in SEQ ID NO: 38; the fusion gene The nucleotide sequence is shown in SEQ ID NO: 39; the fusion gene The nucleotide sequence is shown in SEQ ID NO:

40.

2. A method for constructing a genetically engineered bacterium as described in claim 1, characterized in that, The construction method includes integrating the gene to be expressed into the genome of Yersinia lipolytica, wherein the integration method is to transfer an integration expression plasmid containing the gene to be expressed into Yersinia lipolytica; and / or, the construction method is based on the CRISPR / Cas9 operating system.

3. The construction method as described in claim 2, characterized in that, When the construction method is based on the CRISPR / Cas9 operating system, it includes the following steps: (1) Construct a plasmid pair containing a donor plasmid and an sgRNA plasmid, wherein the donor plasmid contains the gene to be expressed and the sgRNA plasmid contains an sgRNA sequence targeting the integration site on the genome; (2) The plasmid pair was simultaneously transformed into Yersinia lipophila and the target strain was obtained by screening.

4. The construction method as described in claim 2 or 3, characterized in that, The promoter of the gene to be expressed in the donor plasmid or the integrated expression plasmid is UAS4B+TEF; and / or, the backbone of the donor plasmid is pHR_hrGFP; and / or, the backbone of the sgRNA plasmid is pCRISPRyl; and / or, the backbone of the integrated expression plasmid is pINA1269 or p3204.

5. The construction method as described in claim 4, characterized in that, The donor plasmid contains the elongase gene. or the fusion gene The pHR_hrGFP plasmid, wherein the integration expression plasmid contains the diacylglycerol transferase gene. pINA1269 plasmid or containing the fusion gene The p3204 plasmid.

6. A method for producing nervonic acid, characterized in that, The method includes fermenting the genetically engineered bacteria as described in claim 1 in a culture medium and isolating nervonic acid therefrom.

7. The method as described in claim 6, characterized in that, The culture medium is YPD medium.

8. The method as described in claim 6 or 7, characterized in that, The culture medium also contains additional oils or oleic acid.

9. The method as described in claim 8, characterized in that, The oil is rapeseed oil.

10. The method as described in claim 9, characterized in that, The rapeseed oil added is 1% by volume.

11. The application of the genetically engineered bacteria as described in claim 1 in the production of nervonic acid.

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