CN116396875A - A kind of Yarrowia lipolytica genetically engineered bacterium and its application - Google Patents

Abstract

The invention provides a Yarrowia lipolytica genetic engineering bacterium and application thereof. The genetically engineered bacterium is to knock out the gene PEX10 in Yarrowia lipolytica Po1f, and express the C16/18 elongase gene MaELO3 derived from Mortierella alpina, the elongase gene AtKCS derived from Arabidopsis thaliana, and the The bacterial strain of the elongase gene CraKCS of said genetic engineering bacterium also expresses and derives from the elongase gene CgKCS of the genus Michelina. After further expressing the desaturase gene MaD15D from Mortierella alpina, and overexpressing the strain's own diacylglycerol transferase gene DGA1 and its own Δ9 desaturase gene OLE1, the yield of neural acid in shake flask fermentation was 111.6mg/L, by adding 1% rapeseed oil from exogenous sources, the production of nervonic acid can reach 185.1mg/L. The Yarrowia lipolytica genetically engineered bacterium constructed by the invention has simple operation, stable and reliable performance, and can be applied to large-scale commercial production.

Description

A kind of Yarrowia lipolytica genetically engineered bacterium and its application

technical field

The invention belongs to the field of genetic engineering, and in particular relates to a Yarrowia lipolytica genetically engineered bacterium and its application.

Background technique

Nervous acid (C ₂₄ H ₄₆ O ₂ ) is an ultra-long chain monounsaturated fatty acid, named cis-15-tetradecenoic acid or omega-9 tetracosanoic acid according to the position of the unsaturated bond and the length of the carbon chain Enenoic acid. Nervous acid is the core component of nerve fibers and nerve cells, and it is also an essential fatty acid for brain growth and development. Nervonic acid was first isolated in the brain of mammalian sharks, so it is also known as shark oleic acid or squaloleic acid. The brain of sharks can repair itself in a short period of time after being severely damaged, also because nervous acid can promote 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.

Nervous acid is obtained by extraction from animal and plant tissues or by chemical synthesis, and nervonic acid from animal sources mainly comes from marine organisms. Due to the market value and commercial value of nervonic acid, for a long time in the past, developed countries obtained nervonic acid by killing a large number of sharks. Due to the prohibition of fishing for the protection of sharks, there is a shortage of nerve acid resources. Using chemical synthesis method to synthesize nervonic acid with cis-13-dodecyl methyl ester as precursor, the yield of nervonic acid is low and there are many by-products. Therefore, commercial nervonic acids are mostly derived from plants. Studies have shown that nervonic acid exists in the seed oil of some wild plants, but due to the limitation of growth cycle, living environment and quantity, the extraction of nervonic acid from natural plant sources is limited.

With the development of genetic engineering, it is possible to produce nervonic acid through engineering transformation of plants, microalgae and microorganisms, making the large-scale production of nervonic acid potentially possible. Compared with Saccharomyces cerevisiae, Yarrowia lipolytica, as an oleaginous yeast, can produce fat and oil at a high level, and synthesize fatty acids, can provide sufficient fatty acid precursors for nervonic acid, and has the ability to use substrates extensively. Recognized as a safe strain, it is an advantageous genetically engineered bacterium for nervonic acid synthesis.

At present, engineering transformation is generally carried out by introducing genes related to the synthesis pathway of nervonic acid from exogenous sources, but the synthesis pathway of nervonic acid is very complicated and involves many related genes, and the genes related to the synthesis pathway of nervonic acid from different species are matched with different chassis bacteria to produce Nervous acid also works differently. As mentioned above, nervonic acid widely exists in various animals, plants and microorganisms. Therefore, there is an urgent need to screen the genes related to the synthesis pathway of nervonic acid from a large number of different species sources to find the suitable ones for Yarrowia lipolytica to improve neural activity. Genes for acid production levels.

Contents of the invention

In order to solve the problem of low yield of nervonic acid produced by Yarrowia lipolytica in the prior art, the present invention constructs Yarrowia lipolytica capable of expressing fatty acid elongase genes AtKCS, CraKCS, MaELO3 and CgKCS, so that it can effectively produce Very long chain fatty acids (VLCFAs).

On this basis, the fusion expression form CgKCS-L-MaD15D of two copies of elongase gene (CgKCS) and desaturase gene (MaD15D), Δ9 desaturase (OLE1) and diacylglycerol transferase gene ( DGA1) fusion expression form OLE1-L-DGA1 and diacylglycerol transferase gene (DGA1) gene, successfully obtained the production strain of Yarrowia lipolytica producing neuric acid.

In order to solve the above-mentioned technical problems, one of the technical solutions provided by the present invention is: a genetically engineered bacterium of Yarrowia lipolytica (Yarrowia lipolytica), the genetically engineered bacterium is knockout gene PEX10 in Yarrowia lipolytica Po1f , and express 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 bacterium also expresses the elongase gene CgKCS derived from Cardamine graeca.

The Yarrowia lipolytica Po1f of the present invention is a known genetically modified yeast whose genotype is MatA, leu2-270, ura3-302, xpr2-322, axp-2; phenotype is Leu-, Ura-, DAEP, DAXP, Suc+.

The gene PEX10 of the present invention is a gene related to β-oxidation in Yarrowia lipolytica Po1f.

The genetically engineered bacterium as described in one of the technical solutions, the genetically engineered bacterium contains 1 copy of the elongase gene CgKCS; or, the genetically engineered bacterium contains 1 copy of the elongase gene CgKCS and 2 copies of the Fusion gene CgKCS-L-MaD15D of elongase gene CgKCS and desaturase gene MaD15D derived from Mortierella alpina.

In a specific embodiment of the present invention, the fusion gene CgKCS-L-MaD15D is a linker inserted into the elongase gene CgKCS and the desaturase gene MaD15D by NEB ligase, so that they use the same set of promoters and a terminator, the linker is conventional in the art.

The fusion enzyme can reduce the accumulation of intermediates in the synthesis pathway. The present invention fuses the elongation enzyme CgKCS and the desaturase MaD15D to promote the production of neuramic acid and reduce the production of other ultra-long chain fatty acids. Although the present invention fuses the elongase gene CgKCS and the desaturase gene MaD15D, those skilled in the art can reasonably expect that even if these two genes are disassembled and respectively transferred to the genome of the strain and expressed separately, the Increase the production of neuramic acid produced by the strain.

The genetically engineered bacterium according to one of the technical solutions, wherein the genetically engineered bacterium also overexpresses its own diacylglycerol transferase gene DGA1.

In a preferred embodiment of the present invention, the genetically engineered bacteria contain a total of 2 copies of the diacylglycerol transferase gene DGA1. That is, the strain itself contains 1 copy of DGA1, and then an additional copy of DGA1 is transferred.

The genetically engineered bacterium as described in one of the technical solutions, the genetically engineered bacterium contains 2 copies of its own diacylglycerol transferase gene DGA1, and also contains 1 copy of the diacylglycerol transferase gene DGA1 and its own The fusion gene OLE1-L-DGA1 of the native Δ9 desaturase gene OLE1. That is, the strain itself contains 1 copy of DGA1, and then an additional copy of DGA1 and 1 copy of the fusion gene OLE1-L-DGA1 are transferred.

Diacylglycerol transferase DGA1 can promote the accumulation of C22:0 and C24:0 fatty acids, and the Δ9 desaturase OLE1 is conducive to the formation of C24:1. After many experiments by the inventors, one more copy of DGA1 and one OLE1-L-DGA1 is Most conducive to the production of nervonic acid (C24:1).

In a specific embodiment of the present invention, the fusion gene OLE1-L-DGA1 is a linker inserted into the diacylglycerol transferase gene DGA1 and the Δ9 desaturase gene OLE1 by NEB ligase, so that the same set of promoter and terminator, and the linker is routine in the art.

The fusion enzyme can reduce the accumulation of intermediates in the synthesis pathway. The present invention fuses diacylglycerol transferase DGA1 and Δ9 desaturase OLE1 to promote the production of neuramic acid and reduce the production of other ultra-long chain fatty acids. Although the present 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 are disassembled and transferred into the genome of the strain separately Expression can also increase the production of neuramic acid produced by the strain.

For the genetically engineered bacteria 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 As 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 as SEQ ID NO:35 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 fusion gene CgKCS- The nucleotide sequence of 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.

In order to solve the above technical problems, the second technical solution provided by the present invention is: a method for constructing a genetically engineered bacterium as described in one of the technical solutions, the construction method comprising integrating the gene to be expressed into the Yarrowia lipolytica In the genome, the integration method is to transfer an integrated expression plasmid containing the gene to be expressed into Yarrowia lipolytica; and/or, the construction method is based on the CRISPR/Cas9 operating system.

The construction method as described in the second technical solution, when the construction method is based on the CRISPR/Cas9 operating system, comprises the following steps:

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

(2) Simultaneously transform the plasmid pair into Yarrowia lipolytica, and screen to obtain the target strain.

According to the construction method 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.

In a preferred embodiment of the present invention, the donor plasmid is a pHR_hrGFP plasmid comprising the elongase gene CgKCS or the fusion gene CgKCS-L-MaD15D, and the integrated expression plasmid is a plasmid comprising the diacylglycerol transferase gene The pINA1269 plasmid for DGA1 or the p3204 plasmid containing the fusion gene OLE1-L-DGA1.

In the present invention, the promoter UAS4B+TEF is a promoter composed of the enhancer part UAS4B derived from the promoter hp4d of the plasmid PINA1312 and the strong promoter TEF derived from DNA in Yarrowia lipolytica Polf, For its specific construction method, refer to patent CN 107815425 A.

In the present invention, the PHR_HRGFP refers to the collective referred to the plasma of PHR_A3_HRGFP, PHR_F1 -3_HRGFP, PHR_AXP_HRGFP, PHR_F1_HRGFP, PHR_A1-2_HRGFP, and pHR_E1 -HRGFP; CrisPryl refers to the plasmid PCRISPRYL_A3, PCRISPRYL_F1 -3, pCRISPRyl_AXP, pCRISPRyl_F1, pCRISPRyl_A1-2, and pCRISPRyl_E1-3 are collectively referred to as plasmids; wherein, A3, F1-3, AXP, F1, A1-2, and E1-3 correspond to Yarrowia lipolytica genome respectively specific genetic loci.

In order to solve the above-mentioned technical problems, the third technical solution provided by the present invention is: a method for producing neural acid, which includes fermenting the genetically engineered bacteria as described in one of the technical solutions in a culture medium, and isolating nerve acid from it. acid; the medium is preferably YPD medium.

In a preferred embodiment of the present invention, fat or oleic acid is additionally added to the medium; the fat is preferably rapeseed oil.

In a more preferred embodiment of the present invention, the volume percentage of the rapeseed oil added is 1%.

In order to solve the above technical problems, the fourth technical solution provided by the present invention is: the application of the genetically engineered bacteria described in the first technical solution in the production of nervonic acid.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The positive progress effect of the present invention lies in: the Yarrowia lipolytica genetic engineering bacterium of the present invention producing neuric acid, its beneficial effect is (1) the output of neural acid is high: the Yarrowia lipolytica gene of the present invention produces neuric acid The construction method of engineering strain NA06 is simple, and it can produce high-yield nervonic acid through fermentation, which can make the output of nervonic acid reach 111.6mg/L in a 50mL shake flask, and has a good application prospect. (2) By adding its synthetic precursor, when adding 1% rapeseed oil, the Yarrowia lipolytica genetically engineered bacterium NA06 of the present invention can make neural acid reach 185.1mg in a 50mL shake flask /L output. (3) The construction method is simple, and only four genes (CgKCS, CgKCS-L-MaD15D, DGA1, DGA1-L-OLE1) are introduced by means of CRISPR/Cas9 and integrated plasmid complementation screening markers. The subculture has good stability and can be applied to large-scale commercial production with good prospects.

Description of drawings

Fig. 1 is the metabolic pathway of Yarrowia lipolytica to produce nervonic acid after introducing elongase gene and nervonic acid synthase gene.

Fig. 2 is a graph showing the results of shake flask fermentation of the neuramic acid producing strain.

Figure 3A is the effect of adding different types of oil/oleic acid on the production of nervonic acid (ie C24:1).

Figure 3B is the effect of different concentrations of rapeseed oil on the production of nervonic acid (ie C24:1).

Fig. 3C is the result of fermentation of strain NA06 to produce neuramic acid (ie C24:1) with or without the addition of 1% rapeseed oil.

Figure 4 is a comparison chart of the production of ultra-long-chain fatty acids between the CgKCS gene without codon optimization and the CgKCS-opt gene with codon optimization, wherein C24:1 refers to nervonic acid.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention but not to limit the scope of the present invention. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

The Yarrowia lipolytica used in the present application is the bacterial strain GQ07 (corresponding to NA02 of the present invention, which can effectively produce LCFAs in the patent CN 111979135 A, which knocks out the PEX10 gene in Yarrowia lipolytica Po1f, and expresses the On the basis of the elongase gene AtKCS of Arabidopsis thaliana, the elongase gene CraKCS of Crassula oleracea, and the C16/18 elongase gene MaELO3 of Mortierella alpina), in order to increase the output of nervonic acid, the present invention further overexpresses the genus Milia The elongase gene CgKCS and CgKCS-opt, explore the impact of codon optimization on the gene CgKCS; further overexpress the fusion expression form of elongase gene (CgKCS-opt) and desaturase gene (MaD15D) CgKCS-opt-L-MaD15D , Δ9 desaturase (OLE1) and diacylglycerol transferase gene (DGA1) fusion expression form OLE1-L-DGA1, and diacylglycerol transferase gene (DGA1) gene to finally obtain neuramic acid-producing Yarrowia lipolytica Engineering bacteria (see Figure 1).

The reagents and raw materials used in the present invention are all commercially available.

The strains and plasmid sources involved in the present invention are as follows:

1. Yarrowia lipolytica Po1f, plasmid pINA1269: According to Madzak, C., Treton, B., Blancchin-Roland, S. 2000. Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in theyast Yarrowia lipolytica. J Mol Microbiol Biotechnol, 2 (2), 207-216 recorded preparation method.

2. Plasmid pINA1312: see Nicaud, J.M., 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 preparation method recorded in the preparation.

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 Zhang, X.K., Wang, D.N., Chen, J., Liu, Z.J., Wei, L.J., Hua, Q. 2020. Metabolic engineering of beta-carotenebiosynthesis in Yarrowia lipolytica. Biotechnol Lett, 42(6), 945-956 The preparation method described in preparation.

4. Plasmid p3204: replace the promoter with UAS4B+TEF on the basis of pINA1312. Yarrowia lipolytica Po1f-ΔPEX10: The PEX10 gene was knocked out on the basis of Yarrowia lipolytica Po1f. See Gao, Q., Cao, X., Huang, Y.Y., Yang, J.L., Chen, J., Wei, L.J., 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 prepared by the preparation method recorded.

5. Plasmids pHR_F1_MaELO3 and p32UTMaELO3: containing the C16/18 elongase gene MaELO3 derived from Mortierella 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 derived from Crassula oleracea. Plasmids pHR_AXP_CgKCS and p32UTCgKCS: both contain the elongase gene CgKCS derived from the genus Myelina. Plasmids pHR_AXP_CgKCS-opt and p32UTCgKCS-opt: they also contain the codon-optimized elongase gene CgKCS-opt from the genus Myrina. Plasmids pHR_A1-2_CgKCS-opt-L-MaD15D, pHR_E1-3_CgKCS-opt-L-MaD15D, and p32UTCgKCS-opt-L-MaD15D: Containing the elongase gene CgKCS-opt derived from Myelina and derived from Mortierella alpina The desaturase gene MaD15D. Plasmid p32UTOLE1-L-DGA1: contains the endogenous Δ9 desaturase gene OLE1 and diacylglycerol transferase gene DGA1 derived from Yarrowia lipolytica. Plasmids p69DGA1 and p32DGA1: contain the endogenous diacylglycerol transferase gene DGA1 from Yarrowia lipolytica.

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

Primer sequences of table 1 plasmid pair

In the following examples, the elongase gene CgKCS is a gene without codon optimization (the nucleotide sequence is shown in SEQ ID NO: 33), and the elongase gene AtKCS, CraKCS, CgKCS-opt and MaELO3 all refer to optimized AtKCS, CraKCS, The nucleotide sequences of CgKCS and MaELO3 genes are respectively shown in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37. The nucleotide sequence of the optimized sequence of 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, the fusion gene OLE1-L - The nucleotide sequence of DGA1 is shown in SEQ ID NO:40.

Example 1 Constructing Yarrowia lipolytica Genetic Engineering Bacteria Producing Long-chain Fatty Acids

(1) Construction of elongase gene AtKCS, CraKCS, CgKCS-opt and MaELO3 expression plasmids p32UTAtKCS, p32UTCraKCS, p32UTCgKCS-opt and p32UTMaELO3. The primers 32UTAtKCS-f and 32UTAtKCS-r (the nucleotide sequences are respectively shown in the sequence listing SEQ ID NO:1-2), 32UTCraKCS-f and 32UTCraKCS-r (the nucleotide sequences are respectively shown in the sequence listing SEQ ID NO: 3～4), 32UTCgKCS-opt-f and 32UTCgKCS-opt-r (the nucleotide sequences are respectively shown in the sequence table SEQ ID NO: 5～6), 32UTMaELO3-f and 32UTMaELO3-r (the nucleotide sequences The optimized sequence of the elongase gene AtKCS derived from Arabidopsis thaliana (the nucleotide sequence is shown in SEQ ID NO: 34), sea cabbage (Crambe abyssinica) elongase gene CraKCS optimized sequence (its nucleotide sequence is shown in SEQID NO: 35), the optimized sequence of the elongase gene CgKCS-opt (its nucleotide sequence of Cardamine graeca) The sequence is shown in SEQ ID NO: 36) and the optimized sequence of the C16/18 elongase gene MaELO3 of Mortierella alpine (Mortierella alpine) (its nucleotide sequence is shown in SEQ ID NO: 37) through the restriction site PmlI and BamHI were constructed into the plasmid p3204 whose promoter was UAS4B+TEF, and the plasmids p32UTAtKCS, p32UTCraKCS, p32UTCgKCS-opt and p32UTMaELO3 were obtained in turn. Among them, the codon-optimized AtKCS, CraKCS, CgKCS-opt and MaELO3 genes were derived from Arabidopsis thaliana, Crambe abyssinica, Cardamine graeca and Mortierella alpina, respectively. The nucleotide sequences of AtKCS, CraKCS, CgKCS-opt and MaELO3 of mold (Mortierella alpine) were obtained after optimization.

(2) Based on the existing CRISPR/Cas9 operating system, construct the donor plasmid pHR_F1_MaELO3 containing the optimized MaELO3 gene with UAS4B+TEF as the promoter, and the donor plasmid pHR_A3_AtKCS and the optimized CraKCS gene as the AtKCS gene with the UAS8B+TEF optimized promoter Donor plasmid pHR_F1-3_CraKCS and optimized CgKCS-opt gene donor plasmid pHR_AXP_CgKCS-opt. A pair of knock-in plasmids can be formed with sgRNA plasmids pCRISPRyl_F1, pCRISPRyl_A3, pCRISPRyl_F1-3 and pCRISPRyl_AXP respectively.

The knock-in plasmid pair in this example is a conventional recombinant vector in the field, wherein the sgRNA plasmid contains a leucine selection marker, and the donor plasmid contains a uracil selection marker, which can transform uracil and leucine auxotrophic solutions lipolytica, the knock-in plasmid pair can knock in the above-mentioned optimized MaELO3 gene, AtKCS gene, CraKCS gene and CgKCS-opt gene in sequence.

Specific steps are as follows:

(1) First, the expression cassette of the target gene needs to be obtained, and the primers are sequentially used to use primers 32UTAtKCS-f and 32UTAtKCS-r (the nucleotide sequences are respectively shown in the sequence table SEQ ID NO: 1-2), 32UTCraKCS-f and 32UTCraKCS -r (the nucleotide sequences are respectively shown in the sequence table SEQ ID NO: 3 ~ 4), 32UTCgKCS-opt-f and 32UTCgKCS-opt-r (the nucleotide sequences are respectively shown in the sequence table SEQ ID NO: 4 ~ 6 shown), 32UTMaELO3-f and 32UTMaELO3-r (the nucleotide sequences are respectively shown in the sequence table SEQ ID NO: 7 ~ 8) to obtain the corresponding "AtKCS" and "CraKCS" corresponding to the p32UTAtKCS, p32UTCraKCS, p32UTCgKCS-opt and p32UTMaELO3 plasmids , "CgKCS-opt" were sequentially constructed into plasmids pHR_A3_hrGFP, pHR_F1-3_hrGFP, pHR_AXP_hrGFP, pHR_F1_hrGFP through restriction sites NheI and pteI, respectively, to obtain plasmids pHR_A3_AtKCS, pHR_F1-3_CraKCS, pHR_AXP_CgKCS-opt. The "UAS4B+TEF-MaELO3" fragment was cut through restriction sites NheI and BssHII to obtain plasmid pHR_F1_MaELO3.

(4) Transform the plasmid pHR_F1_MaELO3 and sgRNA plasmid pCRISPRyl_F1 obtained in step (3) into Yarrowia lipolytica Po1f-ΔPEX10 at the same time, and recover the screening marker to obtain the strain GQ06. After verification, the MaELO3 gene was knocked into the strain GQ06 . Wherein, the transformation uses the kit Frozen EZ Yeast Transformation II ^TM (purchased from Zymo Research), and operates according to the method described in the kit instruction manual.

(5) Transform the plasmid pHR_A3_AtKCS and sgRNA plasmid pCRISPRyl_A3 obtained in step (3) into Yarrowia lipolytica Po1f-ΔPEX10-F1MaELO3 at the same time, and recover the screening marker to obtain strain NA01. After verification, the knock-in AtKCS gene. Wherein, the transformation uses the kit Frozen EZ Yeast Transformation II ^TM (purchased from Zymo Research), and operates according to the method described in the kit instruction manual.

(6) Transform the plasmid pHR_F1-3_CraKCS and sgRNA plasmid pCRISPRyl_F1-3 obtained in step (3) into Yarrowia lipolytica Po1f-ΔPEX10-F1MaELO3-A3AtKCS at the same time, and obtain the strain NA02 after recovering the selection marker (that is, the patent The bacterial strain GQ07 in the application CN 111979135 A) was verified, and the CraKCS gene was knocked into the bacterial strain NA02. Wherein, the transformation uses the kit Frozen EZ Yeast Transformation II ^TM (purchased from Zymo Research), and operates according to the method described in the kit instruction manual.

(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, and the selection marker was recovered to obtain strain NA03, It was verified that the CgKCS-opt gene was knocked into the strain NA03. Wherein, the transformation uses the kit FrozenEZ Yeast Transformation II ^TM (purchased from Zymo Research), and operates according to the method described in the kit instruction manual.

Example 2 Construction of Yarrowia lipolytica genetically engineered bacterium producing neuric acid

(1) The fusion expression form of elongase gene and desaturase gene CgKCS-opt-L-MaD15D, the fusion expression form of endogenous Δ9 desaturase OLE1 and DGA1 OLE1-L-DGA1 and diacylglycerol transferase gene DGA1 Construction of expression plasmids p32UTCgKCS-opt-L-MaD15D, p32UTOLE1-L-DGA1, p32DGA1, p69DGA1. Use primers 32UTCgKCS-opt-L-MaD15D-f and 32UTCgKCS-opt-L-MaD15D-r (nucleotide sequences respectively as shown in sequence table SEQID NO:21～22), 32UTOLE1-L-DGA1-f and 32UTOLE1 in sequence -L-DGA1-r (the nucleotide sequences are respectively shown in the sequence listing SEQ ID NO: 23～24), 69-DGA1-f and 69-DGA1-r (the nucleotide sequences are respectively shown in the sequence listing SEQ ID NO: 25-26), 32DGA1-f and 32DGA1-r (the nucleotide sequences are respectively shown in the sequence table SEQ ID NO: 27-28), the elongase gene CgKCS derived from Cardamine graeca -opt and the fusion gene CgKCS-opt-L-MaD15D (its nucleotide sequence is shown in SEQ ID NO:39) derived from the optimized sequence of the desaturase gene MaD15D of Mortierella alpine (Mortierella alpine), lipolysis Endogenous Δ9 desaturase OLE1 and the fusion gene OLE1-L-DGA1 of the optimized sequence of diacylglycerol transferase gene DGA1 (its nucleotide sequence is shown in SEQ ID NO: 40), and diacylglycerol transferase gene The optimized sequence of DGA1 (its nucleotide sequence is shown in SEQ ID NO: 38), was constructed into the plasmid p3204 whose promoter is UAS4B+TEF through restriction sites Pmll and BamHI, and the plasmid p32UTCgKCS-opt-L- MaD15DCgKCS-opt-L-MaD15D, p32UTOLE1-L-DGA1, p32DGA1 and pINA69. Wherein, the codon-optimized CgKCS-opt and MaD15D genes are obtained by performing the nucleotide sequences of CgKCS-opt and MaD15D derived from Cardamine graeca and Mortierella alpine respectively. obtained after optimization.

(2) Based on the existing CRISPR/Cas9 operating system, the 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 respectively constructed. They can be combined with sgRNA plasmids pCRISPRyl_A1-2 and pCRISPRyl_E1-3 in turn to form a pair of knock-in plasmids.

The knock-in plasmid pair in this example is a conventional recombinant vector in the field, wherein the sgRNA plasmid contains a leucine selection marker, and the donor plasmid contains a uracil selection marker, which can transform uracil and leucine auxotrophic solutions lipolytica, the knock-in plasmid pair can sequentially knock-in the above-mentioned optimized CgKCS-opt-L-MaD15D gene.

Specific steps are as follows:

(1) Firstly, the target gene needs to be obtained, and the primers 32UTCgKCS-opt-L-MaD15D-f and 32UTCgKCS-opt-L-MaD15D-r are sequentially used (the nucleotide sequences are respectively shown in SEQ ID NO: 1-2 in the sequence table) 1. Obtain the "CgKCS-opt-L-MaD15D" fragment of the p32UTCgKCS-opt-L-MaD15D plasmid. The plasmids pHR_A1-2_hrGFP and pHR_E1-3_hrGFP were respectively constructed through restriction sites NheI and BssHII to obtain plasmids pHR_A1-2_CgKCS-opt-L-MaD15D and pHR_E1-3_CgKCS-opt-L-MaD15D.

(2) The plasmid pHR_A1-2_CgKCS-opt-L-MaD15D obtained in step (1) and the sgRNA plasmid pCRISPRyl_A1-2 were simultaneously transformed into Yarrowia lipolytica Po1f-ΔPEX10-F1MaELO3-A3AtKCS-F1-3CraKCS-AXPCgKCS-opt In NA04, the strain NA04 was obtained after recovering the screening marker, and it was verified that the CgKCS-opt-L-MaD15D gene was knocked into the strain NA04. Wherein, the transformation uses the kit Frozen EZ Yeast Transformation II ^TM (purchased from Zymo Research), and operates according to the method described in the kit instruction manual.

(3) The plasmid pHR_E1-3_CgKCS-opt-L-MaD15D obtained in step (1) and the sgRNA plasmid pCRISPRyl_E1-3 were simultaneously transformed into Yarrowia lipolytica Po1f-ΔPEX10-F1MaELO3-A3AtKCS-F1-3CraKCS-AXPCgKCS-opt - In A1-2CgKCS-opt-L-MaD15D, strain NA05 was obtained after recovering the screening marker, and it was verified that the CgKCS-opt-L-MaD15D gene was knocked into strain NA05. Wherein, the kit Frozen EZ YeastTransformation II ^TM (purchased from Zymo Research) was used for the transformation, and the operation was performed according to the method described in the kit instruction manual.

(4) The plasmids p32UTOLE1-L-DGA1 and p69DGA1 obtained in step (1) were transformed into Yarrowia lipolytica NA05 to obtain a Yarrowia lipolytica strain NA06 producing neural acid.

(5) The plasmid p32DGA1 obtained in step (1) was transformed into Yarrowia lipolytica NA03 to obtain a Yarrowia lipolytica strain NA07 producing neuramic acid.

The construction steps of the plasmid p32DGA1 are as follows: the DGA1 gene derived from Yarrowia lipolytica is constructed into the plasmid p3204 through the restriction sites PmlI and BamHI to obtain the plasmid p32DGA1. The nucleotide sequence of the DGA1 gene is as follows: Shown in SEQID NO:38.

According to the present invention, the construction step of the plasmid p32UTOLE1-L-DGA1 in step (4) is as follows: the OLE1 gene and DGA1 gene derived from Yarrowia lipolytica are constructed into the plasmid through NEB ligase and restriction sites PmlI and BamHI In p3204, the plasmid p32UTOLE1-L-DGA1 was obtained, and the nucleotide sequence of the OLE1-L-DGA1 gene was shown in SEQ ID NO:40.

The steps for constructing the plasmid p69DGA1 are as follows: constructing the DGA1 gene derived from Yarrowia lipolytica into the plasmid pINA1269 to obtain the plasmid p69DGA1.

Example 3 Determination of strains producing neuramic acid

The bacterial strain GQ06 that embodiment 1 and embodiment 2 prepare, NA01, NA02, NA03, NA04, NA05, NA06 and NA07 are added with 3mL YPD (this YPD medium is made of 2% glucose, 2% peptone and 1% yeast extract 100 μL of bacterial solution was inoculated into a test tube with the balance being water, and the stated percentages were mass percentages), and cultured at 37° C. and 220 rpm for 24 hours. Then the bacterial strain was inoculated in a conical flask with 50 mL of YPD, and the volume of the inoculation was determined according to the OD600=0.01 of the bacterial liquid in the shake flask after inoculation. Fermentation was carried out at 37°C and 220rpm for 3 days, and all shake flask experiments were set up in parallel.

Extraction of fatty acids: fully mix the bacterial liquid in the shake flask after fermentation, and take 20mL of culture liquid into a 50mL screw-top centrifuge tube. Centrifuge at 6400rpm for 4min. Pour off the supernatant and add 20 mL of deionized water, mix well with a vortex shaker, centrifuge under the same conditions, pour off the supernatant, and repeat the steps once. Add 5 mL of 4 mol/L hydrochloric acid into the centrifuge tube, mix well, shake at 30°C and 220 rpm for 30 min. Take out the centrifuge tube from the shaker, put it in a boiling water bath and keep it for 5 minutes, insert it on ice quickly after the time is up, keep it for 5 minutes, and repeat the step once. Add 20 mL of a well-mixed solution, wherein V methanol: V chloroform = 1:2, shake at 220 rpm for 30 min at 30° C., and then centrifuge at 6400 rpm for 4 min. When significant stratification occurs, the clear underlying liquid is aspirated and transferred to a stoppered glass test tube. The glass test tubes were dried at 105°C in advance to constant weight and then weighed.

Derivation of fatty acids: In this experiment, GC (gas chromatography) was used to detect the products. Since GC cannot detect fatty acids, fatty acids can only be derivatized into corresponding fatty acid methyl esters for detection. The specific process is as follows: put the Add 3mL of the now prepared 0.5mol/L methanolic potassium hydroxide solution (2.8g potassium hydroxide dissolved in 100mL methanol), cover with a glass stopper, and dissolve the grease in ultrasonication. After the fat is dissolved, keep it warm in a constant temperature water bath at 75°C for 20 minutes. After 20 minutes, take out the glass tube, add 3mL of 14% boron trichloride solution to the test tube, and keep it warm at 75°C for 20 minutes. Take out the test tube and add 1 mL of saturated NaCl and 500 μL of n-hexane, and keep it on a vortex shaker at 150 rpm for 1.5 min. After shaking, the liquid in the test tube will be separated. Take the upper layer liquid into a 1.5mL EP tube and centrifuge at 12000rpm for 1min. After centrifugation, take 50 μL of the upper layer liquid into 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 well, that is, V internal standard: V sample = 1:4. Filter through a 0.22μm organic phase filter membrane to a gas phase bottle for GC detection.

Determination of fatty acid content: GC detection method is adopted, and the chromatographic column is DB-5HT (30m) T, which is detected by gas chromatography (0.1 μm). The inlet temperature was 280°C, the injection volume was 1 μL, the detector temperature was 200°C, and the split ratio was 20:1. The specific procedure is: the initial column temperature is 150°C, keep it for 2min, then raise it to 180°C at a rate of 20°C/min, then raise it to 215°C at a rate of 4°C/min, keep it for 1.5min, and finally The heating rate was increased to 300°C at a rate of 20°C/min.

Inoculated in 2mL YPD medium (the YPD medium is composed of 2% glucose, 2% peptone and 1% yeast extract, the balance is water, and the percentage is mass percentage), cultured for 24 hours, and then the initial _OD600 The inoculum of 0.01 was inoculated into new 50mL YPD medium for culture. After 3 days of fermentation and cultivation, extract nervonic acid and use GC (gas chromatography) to detect the content of each nervonic acid component.

Determination of nervonic acid content: after the fermentation, extract the total oil in the Yarrowia lipolytica engineered bacteria from the fermentation 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, filtered through a 0.22 μm organic phase filter, and then detected by GC.

The chromatographic column is DB-5HT. The initial column temperature was 150°C, kept for 2min, then raised to 180°C at a rate of 20°C/min, then raised to 200°C at a rate of 8°C/min, and then increased to 1°C/min to 218°C, and finally raised to 350°C at a rate of 3.5°C/min and maintained for 10 minutes. The injection volume was 1 μL, the detector temperature was 200 °C, the injection port temperature was 280 °C, and the split ratio was 20:1.

The results of the determination of nervonic acid are shown in Fig. 2 . Among them, the yield of NA06 engineered bacteria is 111.6mg/L, which is the strain with the highest yield of nervonic acid among the strains of Yarrowia lipolytica.

Fermentation culture of embodiment 4 nervonic acid production strain

In this experiment, the Yarrowia lipolytica strain NA07, which produces neural acid, was used as the fermentation strain. By adding different oils/oleic acid to 50 mL of YPD fermentation medium, the optimal medium composition of Yarrowia lipolytica for the synthesis of neural acid was explored. Streak the strains preserved in the glycerol seed tube on the YPD solid plate, pick the monoclonal strains, and inoculate them into 15 mL short test tubes with 3 mL of YPD medium (add ampicillin resistance and kanamycin resistance to the test tubes). In order to prevent infection), the test tube was placed in a constant temperature shaker at 30°C at 220rpm for overnight culture, and the cells in the exponential growth phase were inoculated into a 250mL shake flask. Set 0.25mL of different oils/oleic acid, including rapeseed oil (CO, coleseed oil), soybean oil (SO, soybean oil), sunflower seed (SSO, sunflower seed oil), oleic acid (OA, ω-9octadecanoic acid), Kitchen waste oil (WCO, wastecooking oil). The results are shown in Figure 3A, in which the control (control) group indicated that no additional oil/oleic acid was added, and the best auxiliary carbon source measured in the experiment was rapeseed oil, and 0.25 mL of rapeseed oil was added to the YPD medium. The acid content reached 95.2mg/L.

In this experiment, Yarrowia lipolytica strain NA07, which produces neural acid, was used as the fermentation strain. By adding different volumes of rapeseed oil to 50 mL of YPD fermentation medium, the optimal medium composition of Yarrowia lipolytica for synthesizing neural acid was explored. . Streak the strains preserved in the glycerol seed tube on the YPD solid plate, pick the monoclonal strains, and inoculate them into 15 mL short test tubes with 3 mL of YPD medium (add ampicillin resistance and kanamycin resistance to the test tubes). In order to prevent infection), the test tube was placed in a constant temperature shaker at 30°C at 220rpm for overnight culture, and the cells in the exponential growth phase were inoculated into a 250mL shake flask. Set different rapeseed oil addition gradients 0, 0.25mL, 0.5mL, 0.75mL, 1mL, 1.25mL (the corresponding volume percentages are 0%, 0.5%, 1%, 1.5%, 2%, 2.5%). The results are shown in Figure 3B. Under the condition of 0.5mL of rapeseed oil added in 50mL of YPD culture medium, the results of fermentation and culture of strain NA06 are shown in Figure 3C. Highest yield of neuramic acid in yeast.

The results in Figure 3B show that in the experiment, adding 0, 0.25mL, 0.5mL, 0.75mL, 1mL, 1.25mL of different gradients of rapeseed oil, among which 0.5mL or 1% rapeseed oil is more conducive to the effect of nervonic acid on lipolysis Synthesis in Yarrowia. The results in Fig. 3C show: in 50mL YPD with 0.5mL rapeseed oil added, the fermentation results of the strain NA06 showed that the production of nervonic acid in Yarrowia lipolytica reached the highest level of 185.1mg/L.

Example 5 The impact of codon optimization on gene CgKCS

(1) Construction of elongase gene AtKCS, CraKCS, CgKCS and CgKCS-opt expression plasmids p32UTAtKCS, p32UTCraKCS, p32UTCgKCS and p32UTCgKCS-opt. The optimized sequence of the elongase gene AtKCS derived from Arabidopsis thaliana (Arabidopsisthaliana) (its nucleotide sequence is shown in SEQ ID NO: 34), the optimized sequence of the elongase gene CraKCS of Crabapple (Crambe abyssinica) (its nucleus Nucleotide sequence is shown in SEQ ID NO:35), the unoptimized sequence (its nucleotide sequence is shown in SEQ ID NO:33) and the optimization of the elongase gene CgKCS of the cardamine graeca (Cardamine graeca) elongase gene CgKCS The sequence CgKCS-opt (its nucleotide sequence is shown in SEQ ID NO: 36), was constructed into the plasmid p3204 whose promoter is UAS4B+TEF through the enzyme cutting sites PmlI and BamHI, and the plasmids p32UTAtKCS, p32UTCraKCS, p32UTCgKCS and p32UTCgKCS-opt. Among them, the CgKCS without codon optimization is derived from Cardamine graeca; the codon-optimized AtKCS, CraKCS, and CgKCS-opt genes are derived from Arabidopsis thaliana, Crabapple (Crambe abyssinica) and Cardamine graeca (Cardamine graeca) AtKCS, CraKCS and CgKCS were obtained after optimization.

(2) Based on the existing CRISPR/Cas9 operating system, construct the donor plasmid pHR_A3_AtKCS of the optimized AtKCS gene containing the promoter UAS4B+TEF, the donor plasmid pHR_F1-3_CraKCS of the optimized CraKCS gene, and the donor plasmid pHR_AXP_CgKCS of the optimized CgKCS gene And the donor plasmid pHR_AXP_CgKCS-opt of the optimized CgKCS-opt gene. A pair of knock-in plasmids can be formed with sgRNA plasmids pCRISPRyl_A3, pCRISPRyl_F1-3, pCRISPRyl_AXP and pCRISPRyl_AXP respectively.

The knock-in plasmid pair in this example is a conventional recombinant vector in the field, wherein the sgRNA plasmid contains a leucine selection marker, and the donor plasmid contains a uracil selection marker, which can transform uracil and leucine auxotrophic solutions lipolytica, the knock-in plasmid pair can sequentially knock-in the above-mentioned optimized AtKCS gene, CraKCS gene, CgKCS gene and CgKCS-opt gene.

Specific steps are as follows:

(1) First, the expression cassette of the target gene needs to be obtained, and the primers are sequentially used to use primers 32UTAtKCS-f and 32UTAtKCS-r (the nucleotide sequences are respectively shown in the sequence table SEQ ID NO: 1-2), 32UTCraKCS-f and 32UTCraKCS -r (the nucleotide sequences are respectively shown in the sequence table SEQ ID NO: 3 ~ 4), 32UTCgKCS-f and 32UTCgKCS-r (the nucleotide sequences are respectively shown in the sequence table SEQ ID NO: 29 ~ 30) 32UTCgKCS- opt-f and 32UTCgKCS-opt-r (the nucleotide sequences are respectively shown in the sequence table SEQ ID NO: 5 ~ 6) to obtain the corresponding "AtKCS", "CraKCS", "CraKCS", "CgKCS" and "CgKCS-opt" were sequentially constructed into plasmids pHR_A3_hrGFP, pHR_F1-3_hrGFP, pHR_AXP_hrGFP, and pHR_AXP_hrGFP through restriction sites NheI and pteI, respectively, to obtain plasmids pHR_A3_AtKCS, pHR_F1-3_CraKCS, pHR_AXP_CgKCS, pHR_AXP_Cg KCS-opt.

(2) Transform the plasmid pHR_AXP_CgKCS and the sgRNA plasmid pCRISPRyl_AXP obtained in step (1) into Yarrowia lipolytica Po1f-ΔPEX10-F1MaELO3 at the same time, and recover the screening marker to obtain the strain JL00. After verification, the knock-in CgKCS gene. Wherein, the kit Frozen EZ Yeast Transformation II ^TM (purchased from Zymo Research) was used for transformation, and the operation was performed according to the method described in the kit instruction manual.

(3) Transform the plasmid pHR_AXP_CgKCS_opt and the sgRNA plasmid pCRISPRyl_AXP obtained in step (1) into Yarrowia lipolytica Po1f-ΔPEX10-F1MaELO3 at the same time, and recover the screening marker to obtain the strain JL01. After verification, the knock-in CgKCS_opt gene. Wherein, the kit Frozen EZ YeastTransformation II ^TM (purchased from Zymo Research) was used for the transformation, and the operation was performed according to the method described in the kit instruction manual.

(4) Transform the plasmid pHR_A3_AtKCS and the sgRNA plasmid pCRISPRyl_A3 obtained in step (1) into Yarrowia lipolytica Po1f-ΔPEX10-F1MaELO3-AXPCgKCS or Po1f-ΔPEX10-F1MaELO3-AXPCgKCS_opt at the same time, and recover the screening markers. , the strain knocked in the AtKCS gene. Wherein, the transformation uses the kit FrozenEZ Yeast Transformation II ^TM (purchased from Zymo Research), and operates according to the method described in the kit instruction manual.

(5) The plasmid pHR_F1-3_CraKCS obtained in step (1) and the sgRNA plasmid pCRISPRyl_F1-3 were simultaneously transformed into Yarrowia lipolytica Po1f-ΔPEX10-F1MaELO3-A3AtKCS-AXPCgKCS or Po1f-ΔPEX10-F1MaELO3-A3AtKCS-AXPCgKCS_opt, The strains JL02 and JL03 were obtained after recovering the screening markers. It was verified that the CgKCS gene was knocked in in the strain JL02, and the CgKCS_opt gene was knocked in in the strain JL03. Wherein, the transformation uses the kit Frozen EZ Yeast Transformation II ^TM (purchased from Zymo Research), and operates according to the method described in the kit instruction manual.

The constructed Yarrowia lipolytica genetically engineered bacteria were drawn from the preservation glycerol tube to inoculate 100-200 μL of the bacterial liquid into 5 mL of YPD medium and cultured for 16-24 hours. Then inoculate them into 50 mL of new YPD medium with an initial OD600 of 0.01. 30°C, 220rpm fermentation culture for 3 days and harvested bacteria. After oil extraction and methyl esterification, use GC (gas chromatography) to detect the fatty acid content of various chain lengths.

The results shown in Fig. 4: It can be seen that the C20 and C22 fatty acid contents of the control strain GQ07 are significantly higher than those of JL02 and JL03. It shows that after overexpression of CgKCS, it is more beneficial for cells to synthesize ultra-long-chain fatty acids, and C24 fatty acids change from a very small amount to a significant increase, especially in the bacterial strains that overexpress the codon-optimized CgKCS_opt gene, the cells can synthesize 4.6mg/L Nervous acid was 4.75 times higher than that of the control strain, which proved that the optimized CgKCS_opt gene combined with the other three fatty acid elongases could enable cells to synthesize nervonic acid more efficiently.

The above descriptions are only preferred implementations of the present invention. It should be pointed out that those skilled in the art can make some improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (10)

1. A genetically engineered strain of yarrowia lipolytica (Yarrowia lipolytica) which has gene PEX10 knocked out in yarrowia lipolytica Po1f and expresses C16/18 elongase gene MaELO3 derived from mortierella alpina (Mortierella alpine), elongase gene atccs derived from arabidopsis thaliana (Arabidopsis thaliana) and elongase gene CraKCS derived from crassel cabbage (Crambe abyssinica), characterized in that the genetically engineered strain further expresses elongase gene CgKCS derived from Cardamine graeca.

2. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium comprises 1 copy of the elongase gene CgKCS; or the genetically engineered bacterium contains 1 copy of the elongase gene CgKCS and 2 copies of the fusion gene CgKCS of the elongase gene CgKCS and the desaturase gene MaD D from Mortierella alpina, and CgKCS-L-MaD D.

3. The genetically engineered bacterium of claim 1, further overexpressing its own diacylglycerol transferase gene DGA1;

preferably, the genetically engineered bacterium contains 2 copies of the diacylglycerol transferase gene DGA1 in total.

4. The genetically engineered bacterium of claim 2, wherein the genetically engineered bacterium comprises 2 copies of its own diacylglycerol transferase gene DGA1, and further comprises 1 copy of a fusion gene OLE1-L-DGA1 of the diacylglycerol transferase gene DGA1 and its own Δ9 desaturase gene OLE 1.

5. The genetically engineered bacterium of any one of claims 1 to 4, wherein 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 as SEQ ID NO. 34; and/or the nucleotide sequence of the elongase gene CraKCS is shown as SEQ ID NO. 35; and/or the nucleotide sequence of the elongase gene CgKCS is shown as SEQ ID NO. 33 or SEQ ID NO. 36; and/or the nucleotide sequence of the diacylglycerol transferase gene DGA1 is shown as SEQ ID NO. 38; and/or the nucleotide sequence of the fusion gene CgKCS-L-MaD D is shown as SEQ ID NO. 39; and/or the nucleotide sequence of the fusion gene OLE1-L-DGA1 is shown as SEQ ID NO. 40.

6. The method of constructing genetically engineered bacteria of any one of claims 1 to 5, comprising integrating a gene to be expressed into the genome of yarrowia lipolytica by transferring an integrated expression plasmid comprising the gene to be expressed in yarrowia lipolytica; and/or, the construction method is based on a CRISPR/Cas9 operating system.

7. The construction method of claim 6, comprising the steps of, when the construction method is based on a CRISPR/Cas9 operating system:

(1) Constructing a plasmid pair containing a donor plasmid and an sgRNA plasmid, wherein the donor plasmid contains a gene to be expressed, and the sgRNA plasmid contains a sgRNA sequence of a target locus to be integrated on a genome;

(2) The plasmid pairs are simultaneously transformed into yarrowia lipolytica, and the target strain is obtained by screening.

8. The construction method according to claim 6 or 7, wherein the promoter of the gene to be expressed in the donor plasmid or the integrated expression plasmid is uas4b+tef; and/or, the skeleton of the donor plasmid is pHR_hrGFP; and/or, the skeleton of the sgRNA plasmid is pCRISPRyl; and/or the backbone of the integrated expression plasmid is pINA1269 or p3204;

Preferably, the donor plasmid is a pHR_hrGFP plasmid comprising the elongase gene CgKCS or the fusion gene CgKCS-L-MaD D, and the integrated expression plasmid is a pINA1269 plasmid comprising the diacylglycerol transferase gene DGA1 or a p3204 plasmid comprising the fusion gene OLE1-L-DGA 1.

9. A method for producing a nervonic acid, comprising fermenting the genetically engineered bacterium of any one of claims 1 to 5 in a medium and isolating the nervonic acid therefrom; the culture medium is preferably YPD culture medium;

preferably, the culture medium is additionally added with grease or oleic acid; the grease is preferably rapeseed oil;

more preferably, the rapeseed oil is added in a volume percentage of 1%.

10. The use of the genetically engineered bacterium of any one of claims 1 to 5 for producing nervonic acid.

Images