Use of fatty acid elongase and esterase genes in yeast synthesis of nervonic acid and fats.

Abstract

The present invention provides an engineered fungus for producing nervonic acid and / or fats and oils, wherein the engineered fungus has incorporated into its genome an expression cassette that expresses a protein encoded by a 3-ketoacyl-CoA synthase (KCS) gene and / or an esterase gene.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, and more particularly to the use of fatty acid elongase genes and esterase genes in the synthesis of nervonic acid by yeast. [Background technology]

[0002] Nervonic acid (cis-15-tetracosenoic acid, 24:1Δ15) is a very long-chain monounsaturated fatty acid (VLCMFAs) found primarily in the form of glycosphingolipids and sphingophospholipids in the cerebral white matter and myelin sheath of animal nerve fibers, where it is an important component of biological membranes. Nervonic acid has important medical and health benefits, including promoting cerebral development, improving memory, regulating blood lipids, and enhancing immunity, making it useful for treating neurological disorders such as multiple sclerosis. Research has also shown that nervonic acid promotes nervous system development, particularly in the growth and development of brain neurons and optic nerve cells in infants.

[0003] The synthesis of very long-chain fatty acids, such as lignoceric acid (C24:0) and nervonic acid, is now relatively well-known in plants. Plants use oleic acid as a substrate and elongate the carbon chain of the very long-chain fatty acid through a chain elongation cycle, in which the carbon chain of the original fatty acid is elongated by two carbon atoms per cycle. This elongation process is catalyzed and regulated by a combination of four enzyme complexes (KCS, KCR, HCD, and ECR), of which KCS (3-ketoacyl-CoA synthase) is currently considered the key enzyme that determines the length and synthesis rate of very long-chain fatty acid chains in plant tissues.

[0004] Nervonic acid has a wide range of uses and is highly beneficial to human health. However, it is difficult for the human body to synthesize it internally and must be ingested. Currently, nervonic acid is synthesized using chemical methods, but it produces many by-products, has a long synthesis process, low yields, and costs over $1,000 per kilogram, making it uncompetitive in the market. While there is a huge market demand for nervonic acid, its production is limited by its characteristics of being mainly derived from plants, its slow growth, and its high environmental requirements. Microbial production of nervonic acid offers great potential. However, many of the known nervonic acid-producing microorganisms are pathogenic or lack potential production capacity, preventing their application to industrialized production.

[0005] Biological oils are useful as raw materials for renewable energy, functional foods, health foods, and special dietary supplements. Currently, they are mainly obtained from plants, requiring the use of large amounts of arable land. Oil-producing microorganisms can synthesize oils from renewable raw materials, providing an important complement to the methods for obtaining oils. Furthermore, synthetic biology methods can be used to control the composition of microbial oils, showing great potential in the fields of functional foods and medicine.

[0006] The choice of microorganism and the efficiency of raw material conversion significantly affect the cost of fat synthesis. In recent decades, the synthesis of fats and oils by photosynthetic microorganisms such as algae has been gaining attention. However, even with low-cost carbon dioxide as a carbon source, oil production by microalgae has been difficult to overcome due to challenges such as low biomass, high water consumption, and a tendency toward contamination, making it costly and difficult to commercialize. Unlike microalgae, oleaginous yeast is a heterotrophic microorganism that can synthesize large amounts of fats and oils using sugars, with fat content accounting for 70% of the cell dry weight and fat production exceeding 90 g / L. Furthermore, oleaginous yeast has relatively abundant genetic tools and is easily modified, making it an excellent chassis cell for developing specialized functional fatty acids. Nervonic acid synthesized by yeast is primarily present in the form of triglyceride within the cell. The precursor for nervonic acid synthesis is oleic acid, and when oleic acid is esterified into triglyceride, further elongation to nervonic acid is prevented, thereby affecting nervonic acid production. Therefore, screening for very long chain fatty acid esterases would favor the rapid conversion of nervonic acid to triglycerides, promote the conversion of upstream precursors, and further improve the production of nervonic acid.

[0007] Therefore, there is a strong demand in this field for the development of fatty acid elongation enzymes and esterases having specific substrate preferences so as to improve the production of nervonic acid and fats and oils. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a new method for producing nervonic acid and fats and oils in high yields. [Means for solving the problem]

[0009] In a first aspect, the present invention provides an engineered fungus for producing nervonic acid and / or fats and oils, wherein a gene expression cassette is incorporated into the genome of the engineered fungus, and the gene expression cassette expresses a protein encoded by a 3-ketoacyl-CoA synthase (KCS) gene and / or an esterase gene.

[0010] In another preferred embodiment, the engineered fungus comprises a yeast fungus. In another preferred embodiment, the engineered fungus is selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Methylobacterium sp., Pichia pastoris, Hansenula yeast, Kluyveromyces yeast, Candida yeast, Cryptococcus yeast, Isachenkia orientalis, Rhodotorula yeast, or a combination thereof.

[0011] In another preferred embodiment, the 3-ketoacyl-CoA synthase gene is derived from Malania oleifera, Cardamine graeca, Acer truncatum, Lunaria annua, Ximenia caffra, Tropaeolum speciosum, Xanthoceras sorbifolia, Brassica, or Capsella.

[0012] In another preferred embodiment, the engineered fungus is Yarrowia lipolytica, preferably Yarrowia lipolytica Polg-G3-MaoleKCS or Yarrowia lipolytica YL-88-B1.

[0013] In another preferred embodiment, the esterase gene is derived from Malania oleifera, Cardamine graeca, Acer truncatum, Lunaria annua, Ximenia caffra, Tropaeolum speciosum, Xanthoceras sorbifolia, Brassica, or Capsella.

[0014] In another preferred embodiment, the esterase gene is selected from the group consisting of diacylglycerol acyltransferase (DGAT2), glycerol-3-phosphate acyltransferase (GPAT) genes, or a combination thereof.

[0015] In another preferred embodiment, 1 to 20 (preferably 1 to 10, more preferably 1 to 5) of the gene expression cassettes are integrated into the genome. In another preferred embodiment, the gene expression cassette is integrated into one or more promoters selected from the group consisting of TEF, TEFintro, EXP, GPD, GPAT, YAT, hp4d, hp8d, and XPR.

[0016] In another preferred embodiment, the 3-ketoacyl-CoA synthase (KCS) gene is a codon-optimized 3-ketoacyl-CoA synthase (KCS) gene. In another preferred embodiment, the esterase gene is a codon-optimized esterase gene.

[0017] In another preferred embodiment, the sequence of the 3-ketoacyl-CoA synthase (KCS) gene is set forth in any of SEQ ID NOs: 1-11. In another preferred embodiment, the sequence of the esterase gene is set forth in any one of SEQ ID NOs: 48, 49, 50, 51, and 52.

[0018] In another preferred embodiment, the 3-ketoacyl-CoA synthase (KCS) gene or esterase gene is driven by a constitutive or inducible promoter, such as TEF, TEFintro, EXP, GPD, GPAT, YAT, hp4d, hp8d, or XPR.

[0019] In another preferred embodiment, the promoter is selected from the group consisting of TEF, TEFintro, EXP, GPD, GPAT, YAT, hp4d, hp8d, XPR, or a combination thereof. In another preferred embodiment, the engineered fungus has a production of nervonic acid of ≧20 g / L, preferably 20-30 g / L.

[0020] In another preferred embodiment, the production of nervonic acid by the engineered fungus is increased by ≧25%, preferably 26-30%. In another preferred embodiment, the engineered fungus has an oil production of ≧16 g / L, preferably 16-18 g / L, during shake fermentation.

[0021] In another preferred embodiment, the engineered fungus has a nervonic acid / total oil ratio (mass percentage) of ≧18%, preferably 18-20%. In another preferred embodiment, the fatty acid composition of the oil or fat mainly comprises C16:1, C18:1, and C24:1, and the mass percentage of each component is 15-40%, preferably 18-40%.

[0022] In another preferred embodiment, the engineered fungus has a fermentation biomass (cell dry weight) of ≧120 g / L, preferably 130-200 g / L. In another preferred embodiment, the engineered fungus has a nervonic acid / total fatty acid ratio of ≧20%, preferably 20-65%, more preferably 25-65%.

[0023] In a second aspect of the present invention, there is provided a method for producing nervonic acid and / or fats and oils, the method comprising the steps of: (i) obtaining a fermentation product comprising nervonic acid and / or an oil by culturing an engineered fungus according to the first aspect of the present invention; and (ii) separating nervonic acid and / or oil from the fermentation product;

[0024] In a third aspect of the present invention, there is provided a method of constructing an engineered fungus according to the first aspect of the present invention, the method comprising the steps of: (a) constructing a vector containing a gene expression cassette having a screening marker gene, a resistance gene element, an rDNA homologous recombination gene fragment, and a target gene element, which is a 3-ketoacyl-CoA synthase (KCS) gene and / or an esterase gene; and (b) The vector containing the gene expression cassette obtained in step (a) is introduced into a receptor strain to obtain a strain in which the gene expression cassette has been integrated into the receptor genome.

[0025] In another preferred embodiment, the method further comprises the following step: step (c): verifying the genotype of the strain into which the gene expression cassette obtained in step (b) has been integrated by PCR and DNA sequencing; and / or Step (d): The amount of nervonic acid and / or fat produced by fermenting the strain into which the gene expression cassette has been inserted is detected.

[0026] In another preferred embodiment, the vector in step (a) is a plasmid, a cosmid or a nucleic acid fragment. In another preferred embodiment, the expression cassette in step (a) further comprises a strong promoter element.

[0027] In another preferred embodiment, the strong promoter element comprises TEFintro, GPAT, EXP, GPD, GPAT, YAT, hp4d, hp8d, or XPR. In another preferred embodiment, the strong promoter element is TEFintro.

[0028] In another preferred embodiment, the number of the strong promoter elements is 1-3, preferably 1-2, more preferably 2. In another preferred embodiment, the screening marker gene is selected from the group consisting of URA, leu, HGR, or a combination thereof. In another preferred embodiment, the resistance gene element comprises an ampicillin resistance gene.

[0029] In another preferred embodiment, the expression cassette in step (a) comprises TEFintro as a strong promoter element, a 3-ketoacyl-CoA synthase (KCS) gene and / or an esterase gene as a target gene, a yeast screening marker gene including one or more of URA, leu, or HGR, and an ampicillin resistance gene AMP element. In another preferred embodiment, the number of gene expression cassettes integrated into the genome of the receptor in step (b) is 1-5, preferably 1-3.

[0030] In a fourth aspect of the present invention, there is provided the use of an engineered strain according to the first aspect of the present invention as a strain for producing nervonic acid and / or oils by fermentation.

[0031] Of course, it is understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be described here one by one due to space limitations. [Brief explanation of the drawings]

[0032] [Figure 1] FIG. 1 is a phylogenetic tree of the protein sequences of different 3-ketoacyl-CoA synthases. [Figure 2]Figure 2 shows an alignment of the protein sequences of different 3-ketoacyl-CoA synthases. Boxes depict the three functionally conserved domains of the KCS family: "FGNTSSSS," "GSGFKCNSAVW," and "GMGCSA."

[0033] [Figure 3] Figure 3 shows diagnostic PCR validation of recombinant strains. A: Downstream PCR results. CK is the blank control Polg-G3, and numbers 1-8 correspond to different transformants of recombinant strain Polg-G3-MaoleKCS398, Polg-G3-MaoleKCS461, or Polg-G3-MaoleKCS467. B: Downstream PCR results of different transformants of recombinant strain Polg-G3-MaoleKCS817. C: Upstream PCR results. CK is the blank control Polg-G3, and numbers 1-8 correspond to different transformants of recombinant strain Polg-G3-MaoleKCS398, Polg-G3-MaoleKCS461, Polg-G3-MaoleKCS467, or Polg-G3-MaoleKCS817.

[0034] [Figure 4] Figure 4 shows the results of shake fermentation of the recombinant strains. The dry weight, total lipid production, and total lipid content were compared among the different strains. Strain CK: control strain Polg-G3; strain 398: Polg-G3-MaoleKCS398; strain 461: Polg-G3-MaoleKCS461; strain 467: Polg-G3-MaoleKCS467; strain 817: Polg-G3-MaoleKCS81. [Figure 5] Figure 5 shows the results of gas-phase analysis of fatty acids of the recombinant strains. CK: control strain Polg-G3; MaoleKCS: Polg-G3-MaoleKCS398. Numbers 1-11 represent different fatty acid components, which are C16:0, C16:1, C18:0, C18:1, C18:2, C20:0, C20:1, C22:0, C22:1, C24:0, and C24:1, respectively.

[0035] [Figure 6] Figure 6 shows the results of shake fermentation of the overexpressed MaoleKCS recombinant strains. A: Comparison of dry weight, total lipid production, and total lipid content among different strains. B: Analysis of fatty acid composition of different strains. Strain CK: control strain Polg-G3-MaoleKCS; strain#1: Polg-G3-3MaoleKCS398; strain#2: Polg-G3-3MaoleKCS461; strain#3: Polg-G3-3MaoleKCS467; strain#4: Polg-G3-3MaoleKCS817.

[0036] [Figure 7] Figure 7 shows the gas-phase analysis spectra of fatty acid components of the overexpressing MaoleKCS recombinant strains. a: Control strain Polg-G3-MaoleKCS; b: Polg-G3-3MaoleKCS398; c: Polg-G3-3MaoleKCS461; d: Polg-G3-3MaoleKCS467; e: Polg-G3-3MaoleKCS817. Numbers 1-11 represent different fatty acid components, respectively: C16:0, C16:1, C18:0, C18:1, C18:2, C20:0, C20:1, C22:0, C22:1, C24:0, and C24:1.

[0037] [Figure 8] FIG. 8 shows the results of fermentation tank fermentation of the recombinant strain. [Figure 9] Figure 9 shows the gas-phase analysis spectrum of fatty acid components after 168 hours of fermentation in a fermentation tank of the recombinant strain. Numbers 1-11 represent the different fatty acid components, which are C16:0, C16:1, C18:0, C18:1, C18:2, C20:0, C20:1, C22:0, C22:1, C24:0, and C24:1, respectively.

[0038] [Figure 10] FIG. 10 shows the analysis of oil production of the esterase recombinant strains. [Figure 11] FIG. 11 shows the analysis of nervonic acid production of the esterase recombinant strains. [Figure 12] FIG. 12 shows the changes in fatty acid composition of the esterase engineered strains. [Figure 13] FIG. 13 shows the results of identifying positive clones of the esterase recombinant strains.

[0039] Here, in Figures 10-12, CK is Polg-G3-CgKCS. DETAILED DESCRIPTION OF THE INVENTION

[0040] After extensive and in-depth research, the present inventors have unexpectedly constructed, for the first time, a type of engineered yeast strain, which has a gene expression cassette integrated into its genome, and which expresses proteins encoded by the 3-ketoacyl-CoA synthase (KCS) gene and / or esterase gene. The engineered strain of the present invention can significantly improve the production of nervonic acid and / or fats and oils, and can also change the fatty acid composition of fats and oils. Based on this, the present invention has been completed.

[0041] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0042] As used herein, when used with a specific exemplified numerical value, the term "about" means that the value may vary from the exemplified numerical value by up to 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0043] As used herein, the terms "comprise" or "include" may refer to open, semi-closed, and closed systems. In other words, the terms also include "consisting essentially of" or "consisting of." As used herein, the terms "greater than or equal to" and "less than or equal to" are inclusive, for example, "20% or greater" means ≧20% and "8:1 or greater" means ≧8:1.

[0044] starting strain As used herein, the terms "starting strain of the present invention" and "starting microorganism of the present invention" can be used interchangeably to refer to Yarrowia lipolytica strains Y. lipolytica Polg (MATa, leu2-270, ura3-302::URA3, xpr2-3) and Y. lipolytica Polg-KCS (MATa, leu2-270, ura3-302::URA3, xpr2-3,KCS), where Y. lipolytica Polg was purchased from Yeastern Biotech Co., Ltd. and Y. lipolytica Polg-KCS was obtained from Qingdao Institute of Bioenergy and Bioprocessing, Chinese Academy of Sciences. Of course, the starting strain includes not only the Yarrowia lipolytica Polg of the present invention, but also its derivative strains.

[0045] Nervonic acid Nervonic acid (cis-15-tetracosenoic acid, 24:1Δ15) is a very long-chain monounsaturated fatty acid (VLCMFAs) found primarily in the form of glycosphingolipids and sphingophospholipids in the cerebral white matter and myelin sheath of animal nerve fibers, where it is an important component of biological membranes. Nervonic acid has important medical and health benefits, including promoting cerebral development, improving memory, regulating blood lipids, and enhancing immunity, making it useful for treating neurological disorders such as multiple sclerosis. Research has also shown that nervonic acid promotes nervous system development, particularly in the growth and development of brain neurons and optic nerve cells in infants.

[0046] fats and oils Microbial oils have important application value in fields such as bioenergy, food, and medicine. Yarrowia lipolytica is an obligately aerobic, oil-producing yeast capable of accumulating large amounts of neutral lipids, the most important of which are palmitoleic acid and oleic acid. Through enhanced oil production, the combined content of these two acids can reach over 70% of total fatty acids. Palmitoleic acid has therapeutic effects on certain chronic diseases, such as metabolic syndrome, diabetes, and inflammation. It can also inhibit melanin production in human cells and improve skin pigmentation. Oleic acid, with its cis structure, has some effect on vascular softening and plays an important role in the metabolic processes of humans and animals. Therefore, consuming edible oils with a high oleic acid content is beneficial to health. Oleic acid is also used as a paint drier, a lubricant thickener, and an important raw material in the wool spinning and printing and dyeing industries. The mixed oils synthesized by oleaginous yeasts are useful as feedstocks for biodiesel refineries.

[0047] 3-Ketoacyl-CoA synthase (KCS) gene In the present invention, the protein product encoded by the "3-ketoacyl-CoA synthase (KCS) gene" described herein is 3-ketoacyl-CoA synthase, which is involved in catalyzing the synthesis of nervonic acid.

[0048] The protein product encoded by the 3-ketoacyl-CoA synthase (KCS) gene is 3-ketoacyl-CoA synthase, which catalyzes the final synthesis of nervonic acid. 3-Ketoacyl-CoA synthase (KCS) contains a single reading frame and encodes a protein of 512-653 amino acids.

[0049] In the present invention, through extensive creative experiments, it was found that by incorporating an expression cassette expressing the 3-ketoacyl-CoA synthase (KCS) gene into engineered bacteria, the production of nervonic acid can be improved and the content of unsaturated fatty acids can be reduced. Therefore, the modified engineered bacteria provided by this method have broad practical value and future prospects.

[0050] Those skilled in the art can obtain the sequence of the 3-ketoacyl-CoA synthase (KCS) gene by conventional methods, for example, from NCBI.

[0051] The 3-ketoacyl-CoA synthase (KCS) genes of the present invention further include 3-ketoacyl-CoA synthase (KCS) genes of different origins, different homologous sequences of the same origin, and codon-optimized 3-ketoacyl-CoA synthase (KCS) genes.

[0052] Esterase In the present invention, the "esterase genes" described herein are diacylglycerol acyltransferase (DGAT2) and glycerol-3-phosphate acyltransferase (GPAT) genes, and the encoded protein products are diacylglycerol acyltransferase (DGAT2) and glycerol-3-phosphate acyltransferase (GPAT), with diacylglycerol acyltransferase involved in catalyzing the synthesis of triacylglycerol and glycerol-3-phosphate acyltransferase involved in catalyzing the synthesis of lysophosphatidic acid.

[0053] Diacylglycerol acyltransferase (DGAT2) includes MoDGAT2 (Maole_015949) and MoDGAT2 (Maole_010035), and encodes proteins of 403-522 amino acids.

[0054] Glycerol-3-phosphate acyltransferase (GPAT) genes include MoGPAT (Maole_006088), MoGPAT (Maole_006089), and MoGPAT (Maole_006090), and encode proteins of 231–370 amino acids.

[0055] In the present invention, through extensive creative experiments, it has been found that by incorporating an expression cassette for expressing a 3-ketoacyl-CoA synthase (KCS) gene and / or an esterase into an engineered fungus, it is possible to improve the production of nervonic acid or fats and oils, reduce the content of unsaturated fatty acids, change the fatty acid composition of the fats and oils, increase the proportion of oleic acid in the total fats and oils, increase the proportion of nervonic acid in the total fats and oils, and improve the fermentation concentration of nervonic acid. Therefore, the modified engineered fungus provided by this method has broad practical value and future prospects.

[0056] Those skilled in the art can obtain the sequence of the esterase gene by conventional methods, for example, from NCBI. The esterase genes of the present invention further include esterase genes of different origins and different homologous sequences of the same origin.

[0057] Primer As used herein, the term "primer" refers to an oligonucleotide that matches a template and initiates synthesis of a DNA strand complementary to the template under the action of a DNA polymerase. A primer may be natural RNA or DNA, or any form of natural nucleotide. A primer may also be unnatural nucleotide, such as LNA or ZNA.

[0058] A primer is "nearly" (or "essentially") complementary to one specific sequence on one strand of the template. A primer will not be extended unless it is sufficiently complementary to one strand of the template, but the primer sequence does not have to be perfectly complementary to the template sequence. For example, if a primer whose 3' end is complementary to the template has a sequence at its 5' end that is not complementary to the template, such a primer will still be nearly complementary to the template. As long as the primer is long enough to bind to the template, even a primer that is not perfectly complementary can form a primer-template complex with the template and cause amplification.

[0059] Construction of engineered bacteria The present invention provides a method for constructing a genetically engineered strain of nervonic acid-rich bacteria, the method including, but not limited to, the following steps:

[0060] 1. Construction of a Vector Containing a Gene Expression Cassette After codon optimization, the gene was synthesized, PCR primers for the 3-ketoacyl-CoA synthase (KCS) gene were designed, and the target gene was obtained by cloning. A plasmid was then constructed using the seamless cloning method of Gibson assembly. 2. The resulting vector containing the gene expression cassette was then introduced into a receptor strain, resulting in a strain with the gene expression cassette integrated into the receptor genome. In one preferred example, the vector containing the constructed expression cassette was introduced into a starting strain by intergeneric conjugation, and positive zygotes were selected under specific resistance screening conditions.

[0061] In one preferred embodiment, further steps include step 3: verifying the genotype of the strain into which the gene expression cassette obtained in step 2 has been integrated by PCR; and / or step 4: detecting the amount of nervonic acid produced by fermenting the strain into which the gene expression cassette has been integrated. In one preferred embodiment, positive zygotes having a predetermined resistance are cultured in a liquid medium, and the total DNA of the cells is extracted and the genotype is verified by PCR. By designing multiple pairs of primers for the target gene, it may be possible to verify that the corresponding gene has been integrated into the target genome. The base fragments of the correct size amplified by PCR are recovered, and the accuracy of the genetically engineered strain is finally confirmed by DNA sequencing.

[0062] The strain obtained in the present invention, Yarrowia lipolytica strain Polg-G3--MaoleKCS, has significantly improved nervonic acid production capacity through fermentation.

[0063] In one preferred embodiment, the technical solution used in the present invention is as follows: (1) Screening and analysis of 3-ketoacyl-CoA synthase from Malania oleifera was performed by bioinformatics analysis. (2) Using Yarrowia lipolytica as a starting strain, the 3-ketoacyl-CoA synthase gene of Malania oleifera (see SEQ ID NOs: 1-11) is introduced into Yarrowia lipolytica to give or enhance the ability to synthesize nervonic acid.

[0064] Furthermore, Yarrowia lipolytica Pol1g-G3 was used as the starting strain, and the MaoleKCS gene was introduced by means of homologous recombination to obtain the strain Pol1g-G3-MaoleKCS.

[0065] Furthermore, first, a homologous recombination plasmid pYLEX-rDNA-MaoleKCS-URA was constructed, and the resulting plasmid was then enzymatically digested and used to transform the Yarrowia lipolytica strain Pol1g-G3-ΔURA. Transformants were then screened on uracil auxotrophic plates to obtain the recombinant strain Pol1g-G3-MaoleKCS, into which the MaoleKCS gene had been introduced.

[0066] Furthermore, the homologous recombination plasmid pYLEX-rDNA-MaoleKCS-URA uses the plasmid pYLEX-URA as a backbone template, and two fragments of Yarrowia lipolytica 26S rDNA are located upstream and downstream of the MaoleKCS expression cassette (see SEQ ID NO: 12 and SEQ ID NO: 13), respectively.

[0067] (3) Using Yarrowia lipolytica as a starting strain, the 3-ketoacyl-CoA synthase gene of Malania oleifera (see SEQ ID NOs: 1-11) is overexpressed to give or enhance the ability of Yarrowia lipolytica to synthesize nervonic acid.

[0068] Furthermore, Yarrowia lipolytica Pol1g-G3-MaoleKCS was used as the starting strain, and the MaoleKCS gene was introduced by means of homologous recombination to obtain the strain Pol1g-G3-3MaoleKCS.

[0069] Furthermore, first, a homologous recombination plasmid pYLEX-FAD2-MaoleKCS-URA was constructed, and the resulting plasmid was then enzymatically digested and used to transform the Yarrowia lipolytica strain Pol1g-G3-MaoleKCS-ΔURA. Transformants were then screened on uracil-auxotrophic plates to obtain the recombinant strain Pol1g-G3-3MaoleKCS, into which the MaoleKCS gene had been introduced.

[0070] Furthermore, the homologous recombination plasmid pYLEX-FAD2-MaoleKCS-URA uses the plasmid pYLEX-URA as a backbone template, and two fragments of the Yarrowia lipolytica FAD2 gene are located upstream and downstream of the MaoleKCS expression cassette (see SEQ ID NO: 14 and SEQ ID NO: 15).

[0071] (4) The recombinant strain was cultured in a fermentation tank, and the ratio of nervonic acid to total fatty acids reached 36.9%, with a nervonic acid production rate of 25.7 g / L.

[0072] The present invention provides a method for constructing a nervonic acid genetically engineered strain, and also provides a method for constructing a high fat and oil producing strain, including but not limited to the following steps:

[0073] 1. Construction of a Vector Containing a Gene Expression Cassette After codon optimization, the gene was synthesized, PCR primers for diacylglycerol acyltransferase (DGAT2) and glycerol-3-phosphate acyltransferase (GPAT) enzyme genes were designed, and the target gene was obtained by cloning. The plasmid was then constructed using the seamless cloning method of Gibson assembly. 2. The vector containing the gene expression cassette thus obtained is introduced into a receptor strain to obtain a strain in which the gene expression cassette has been integrated into the receptor genome. In one preferred example, the vector containing the constructed expression cassette is introduced into a starting strain by intergeneric conjugation, and positive zygotes are selected under predetermined resistance screening conditions.

[0074] In one preferred embodiment, further steps include step 3: verifying the genotype of the strain into which the gene expression cassette obtained in step 2 has been integrated by PCR; and / or step 4: detecting the amount of nervonic acid produced by fermenting the strain into which the gene expression cassette has been integrated. In one preferred embodiment, positive zygotes having a predetermined resistance are cultured in a liquid medium, and the total DNA of the cells is extracted and the genotype is verified by PCR. By designing multiple pairs of primers for the target gene, it may be possible to verify that the corresponding gene has been integrated into the target genome. The base fragments of the correct size amplified by PCR are recovered, and the accuracy of the genetically engineered strain is finally confirmed by DNA sequencing. The Yarrowia lipolytica strain YL-88-B1 obtained in the present invention has significantly improved production capacity of oils and fats and nervonic acid through fermentation.

[0075] In another preferred embodiment, the technical solution used in the present invention is as follows: (1) Through bioinformatics analysis, screening and analysis will be carried out for diacylglycerol acyltransferase (DGAT2) and glycerol-3-phosphate acyltransferase (GPAT) enzymes. (2) Using the Yarrowia lipolytica strain Polg-G3-CgKCS, which already expresses the fatty acid elongase CgKCS, as the starting strain, the Malania oleifera diacylglycerol acyltransferase (DGAT2) and glycerol-3-phosphate acyltransferase (GPAT) enzymes (see SEQ ID NOs: 48, 49, 50, 51, and 52) are introduced to impart or enhance the ability of Yarrowia lipolytica to synthesize oils and nervonic acid.

[0076] Furthermore, using Yarrowia lipolytica Polg-G3-CgKCS as the starting strain, the diacylglycerol acyltransferase (DGAT2) and glycerol-3-phosphate acyltransferase (GPAT) genes were introduced by homologous recombination to obtain strains YL-35-F5, YL-49-C6, YL-88-B1, and YL-90-B6. When the elongase CgKCS and esterase were expressed using the method of the present invention, the oil production of strain YL-88-B1 at the shake fermentation level increased by 22.1%, and the proportion of nervonic acid in total fatty acids reached 18-20%.

[0077] Fermentative production of nervonic acid The genetically modified strains of the present invention are useful for producing nervonic acid by fermentation, and in one preferred embodiment, the medium includes, but is not limited to, the following, calculated in weight-to-volume ratios relative to the total volume of the medium: YPD liquid medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract. If solid medium is required, add 1.5% agar. Used for general cultivation.

[0078] Uracil auxotrophic medium (YNB-Δura): YNB (yeast nitrogen base without amino acids and ammonium sulfate) 1.7 g / L, glucose 20.0 g / L, CSM-Ura (MP Biomedicals) 0.67 g / L, ammonium sulfate 5 g / L. If solid medium is required, add 1.5% agar. Used for screening and activating recombinant strains. Shaking fermentation medium: glucose 150 g / L, yeast extract 6 g / L, ammonium sulfate 12 g / L.

[0079] The incubation temperature used for E. coli is 37°C, the shaker rotation speed is 200 rpm for liquid incubation, and the media used are as follows: LB medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L. If solid medium is required, add 1.5% agar. Used for routine cultivation. Amp-resistant medium: LB medium to which a high-concentration Amp solution has been added to make the final Amp concentration 100 μg / ml. If solid medium is required, 1.5% agar is added. This medium is used for screening and amplifying recombinant bacterial strains. The fermentation products of the strains of the present invention can be used to produce nervonic acid.

[0080] Drug Compositions and Methods of Application Nervonic acid, a fermentation product of the strain of the present invention, is useful for the production of drugs. The nervonic acid and oil of the present invention can be administered to mammals (e.g., humans) by oral, rectal, extragastrointestinal (intravenous, intramuscular, or subcutaneous), or topical means. The nervonic acid and oil may be administered alone or in combination with other pharmaceutically acceptable compounds. The nervonic acid and oil of the present invention can also be administered as a mixture.

[0081] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is usually mixed with at least one inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or may contain (a) a filler or compatibilizer, such as starch, lactose, sucrose, glucose, mannitol, or silicic acid; (b) a binder, such as hydromethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, or gum arabic; (c) a humectant, such as glycerin; or (d) a disintegrant, such as agar, calcium carbonate, or the like. The pharmaceutical composition may be mixed with ingredients such as calcium, potato starch, tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retardants such as paraffin, (f) absorption promoters such as ammonium compounds, (g) wetting agents such as cetanol and glycerin monostearate, (h) adsorbents such as kaolin, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0082] Solid dosage forms, such as tablets, pills, capsules, pills, and granules, can be prepared with coatings or shells, such as enteric coatings and other materials known in the art. Opacifying agents may be included, and in such compositions, the release of the active substance or compound may be delayed in a certain part of the digestive tract. Examples of encapsulating materials that can be used include polymeric and wax-based materials. If necessary, the active compound may also be formed into a microcapsule with one or more of the above-mentioned excipients.

[0083] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, compatibilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0084] Besides these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifying agents, suspending agents, sweetening agents, and flavoring agents.

[0085] In addition to the active compound, suspensions may contain a suspending agent such as, for example, ethoxylated isooctadecanol, polyoxyethylene sorbitol or sorbitan esters, microcrystalline cellulose, aluminum methoxy or agar, or mixtures of these substances.

[0086] Compositions for parenteral injection include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions and emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0087] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants, as required.

[0088] When using the pharmaceutical composition, a safe and effective amount of the nervonic acid and oil of the present invention is administered to a mammal (e.g., a human) in need of treatment, at a dosage that is considered pharmacologically effective, typically 1-1000 mg, preferably 20-500 mg daily for an individual weighing 60 kg. Of course, the specific dosage should be determined taking into account factors such as the mode of administration and the individual's health condition, all of which are within the skill of a skilled physician.

[0089] The main advantages of the present invention are as follows: (1) In the present invention, the production amount of nervonic acid is significantly improved by expressing the 3-ketoacyl-CoA synthase (KCS) gene. (2) In the present invention, efficient expression of a single enzyme alone can endow or enhance the ability of yeast engineered fungi, such as Yarrowia lipolytica, to synthesize nervonic acid.

[0090] (3) In the recombinant strain of the present invention, the ratio of nervonic acid to total fatty acids is 36.9%, and the nervonic acid production reaches 25.7 g / L. The Malania oleifera 3-ketoacyl-CoA synthase provided by the present invention is useful for constructing yeast strains that produce high levels of nervonic acid, and has wide application potential in fields such as chemical industry, medicine, and health care.

[0091] (4) In the present invention, the expression of esterase alone or the co-expression of esterase and ketoacyl-CoA synthase (KCS) genes significantly improves the production of nervonic acid and fats and oils, changes the fatty acid composition of fats and oils, improves the ratio of oleic acid in the total fats and oils, improves the ratio of nervonic acid in the total fats and oils, and improves the fermentation concentration of nervonic acid.

[0092] The present invention will be further described below with reference to specific examples. It should be understood that these examples are used only to illustrate the present invention and do not limit the scope of the present invention. Experimental methods for which specific conditions are not specified in the following examples generally follow conventional conditions, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989), or the manufacturer's recommended conditions. Unless otherwise specified, percentages and parts are by weight. Unless otherwise stated, all materials and reagents used in the examples of the present invention are commercially available.

[0093] Example 1: Synthesis of nervonic acid by expression of MaoleKCS in yeast 1. Materials and Methods 1.1 Strains and culture conditions The starting strain of Y. lipolytica used in the examples was Y. lipolytica Po1g (MATa, leu2-270, ura3-302::URA3, xpr2-3), which was purchased from Yeastern Biotech Co., Ltd. Strain Po1g-G3-ΔURA was obtained as follows. Specifically, strain Po1g-G3 was obtained by overexpressing the endogenous genes DGAT1, ACC1, and SCD of Y. lipolytica (Qiao and Stephanopoulos, Metabolic Engineering, 2015, 29:56-65). Further reverse screening using the 5-FOA method yielded the uracil-auxotrophic strain Po1g-G3-ΔURA.

[0094] The incubation temperature used for Yarrowia lipolytica was 28°C, and the shaker rotation speed for liquid culture was 220 rpm. The following media were used: YPD liquid medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract. If solid medium is required, add 1.5% agar. Used for general cultivation.

[0095] Uracil auxotrophic medium (YNB-Δura): YNB (yeast nitrogen base without amino acids and ammonium sulfate) 1.7 g / L, glucose 20.0 g / L, CSM-Ura (MP Biomedicals) 0.67 g / L, ammonium sulfate 5 g / L. If solid medium is required, add 1.5% agar. Used for screening and activating recombinant strains. Shaking fermentation medium: glucose 150 g / L, yeast extract 6 g / L, ammonium sulfate 12 g / L.

[0096] The incubation temperature used for E. coli is 37°C, the shaker rotation speed is 200 rpm for liquid incubation, and the media used are as follows: LB medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L. If solid medium is required, add 1.5% agar. Used for routine cultivation. Amp-resistant medium: LB medium to which a high-concentration Amp solution has been added to make the final Amp concentration 100 μg / ml. If solid medium is required, 1.5% agar is added. This medium is used for screening and amplifying recombinant bacterial strains.

[0097] 1.2 Screening and expression of Malania oleifera 3-ketoacyl-CoA synthase gene 1.2.1 Bioinformatics analysis of Malania oleifera 3-ketoacyl-CoA synthase genes Twelve potential Malania oleifera 3-ketoacyl-CoA synthase genes were obtained from genome data in the literature (Xu et al., GigaScience, 2019, 8:1-14), and they are Maole_002411, Maole_003085, Maole_004215, Maole_005654, Maole_008020, Maole_009817, Maole_016461, Maole_016463, Maole_016466, Maole_016467, Maole_017397, and Maole_017398, respectively. Furthermore, six of these genes were selected and named KCS397 (Maole_017397), KCS398 (Maole_017398), KCS461 (Maole_016461), KCS463 (Maole_016463), KCS467 (Maole_016467), and KCS817 (Maole_009817), respectively, and the corresponding amino acid sequences were obtained, which are Maole_017397.T1, Maole_017398.T1, Maole_016461.T1, Maole_016463.T1, Maole_016467.T1, and Maole_009817.T1, respectively. See Table 1 for details. The NCBI database identified MoKCS (GenBank: QDA34238.1, from M. oleifera), CgKCS (GenBank: ACJ61778.1, from C. graeca), LaKCS (GenBank: EU871787.1, from Lunaria annua), BrKCS (GenBank: GU325723, from Brassica rapa), BtKCS (GenBank: KF664165, from Brassica tournefortii), and BeKCS (GenBank: KF664168, from Brassica elongata) as 3-ketoacyl-CoA synthases, and their genes and amino acid sequences were found.Using ClusterW (included in MEGA5.1), we performed multiple protein alignments against the amino acid sequences of KCS397, KCS398, KCS461, KCS463, KCS467, KCS817, MoKCS, CgKCS, LaKCS, BrKCS, BtKCS, and BeKCS. Then, using the neighbor-joining algorithm (1000 bootstraps), we constructed a phylogenetic tree, as shown in Figure 1. Four KCS genes (KCS398, KCS461, KCS467, and KCS817) were selected and aligned against the protein sequences of CgKCS, MoKCS, and the four selected KCSs using the NCBI online domain analysis software (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) and DNAMAN software. The results are shown in Figure 2.

[0098] 1.2.2 Vector construction The KCS398, KCS461, KCS467, and KCS817 genes were synthesized by Wuxi Qinglan Biotechnology Co., Ltd. with codon optimization (the optimized gene sequences are SEQ ID NOs: 2, 3, 5, and 6, respectively). A shuttle plasmid carrying the MaoleKCS gene and ampicillin resistance gene was obtained and named pMv-MaoleKCS#-AMP (Note: # represents the number of the different MaoleKCS genes, i.e., 398, 461, 467, or 817; the same applies below).

[0099] Plasmid vectors were constructed using the Gibson assembly method, and all assembled fragments were obtained by PCR amplification. The plasmid backbone was derived from the pYLEX plasmid (Yeastern Biotech Co., Ltd.). All PCR amplifications were performed using KAPA HiFi high-fidelity DNA polymerase in a 50 μl volume (25 μl of 2× KAPA Mix, 1.5 μl of each 10 μM primer, 1 μl of template, and water added to 50 μl). The amplification conditions were 95°C for 3 minutes of pre-denaturation, 98°C for 20 seconds, 60–72°C for 15 seconds of annealing, and 72°C for extension. The extension time was calculated as 30 seconds per kb, with 29–35 cycles and 10 minutes of extension at 72°C.

[0100] [Table 1]

[0101] The pYLEX-URA plasmid was constructed by substituting the URA3 gene for the LEU2 gene in the pYLEX plasmid using the Gibson assembly method. Furthermore, the pYLEX-URA backbone was used to construct the homologous recombination plasmid pYLEX-rDNA-CgKCS-URA (CgKCS is the Cardamine KCS gene in 1.2). The upstream and downstream sequences of the CgKCS expression cassette were part of the Yarrowia lipolytica 26S rDNA sequence (see SEQ ID NO: 12 and SEQ ID NO: 13), respectively. Therefore, a homologous recombination plasmid with a site-specific integration site in the 26S rDNA region was obtained. The pYLEX-rDNA-CgKCS-URA backbone was used to construct the homologous recombination plasmid pYLEX-rDNA-MaoleKCS-URA by substituting the MaoleKCS gene for the CgKCS gene using the Gibson assembly method.

[0102] The specific construction process is as follows: The pYLEX-rDNA-CgKCS-URA plasmid was obtained directly in the laboratory by the patent applicant. First, a backbone DNA fragment containing the AMP and URA screening markers was amplified using the pYLEX-rDNA-CgKCS-URA plasmid as a template with primers P#-F and P#-R. The MaoleKCS gene fragment was then amplified using the pMv-MaoleKCS#-AMP plasmid containing the MaoleKCS gene with primers pMv#-F and pMv#-R, respectively. After Gibson assembly, the backbone and MaoleKCS gene fragments were transformed into susceptible E. coli Trans-T1 (Beijing Zenshi Jin Biotechnology Co., Ltd.). Colony PCR and sequencing verification was performed using primers rDNA-SF and rDNA-SR, resulting in the homologous recombination plasmid pYLEX-rDNA-MaoleKCS#-URA (see Table 2 for the primers).

[0103] [Table 2]

[0104] 1.2.3 Yeast transformation The recombinant plasmid pYLEX-rDNA-MaoleKCS-URA was digested with NotI restriction enzyme, and the resulting fragment was confirmed by agarose gel electrophoresis. The fragment was then transformed into Yarrowia lipolytica Polg-G3-ΔURA by LiAc transformation. The transformation system is shown in Table 3, and the steps are as follows: [Table 3]

[0105] (1) Each component was added according to the transformation system in Table 3 and mixed uniformly. (2) After treatment in a 30°C water bath for 1 hour, the cells were vortexed and then heat-stimulated at 39°C for 10 minutes. (3) The transformation mixture was directly applied to a screening plate of YNB-Δura medium and cultured at 28°C for 2-4 days to obtain a recombinant strain into which the MaoleKCS gene had been introduced, which was named Po1g-G3-MaoleKCS#.

[0106] 1.2.4 Diagnostic PCR verification of recombinant strains The different transformants of each recombinant strain obtained above were verified by diagnostic PCR. A single colony of the recombinant strain was taken from the screening plate and inoculated onto a YPD plate. After growth for approximately 24 hours, the fungal cells were collected and the genomic DNA of the recombinant strain was extracted using a fungal genome rapid extraction kit (Sengong Bioengineering Co., Ltd.). Using the genomic DNA as a template, the upstream portion where the MaoleKCS gene was introduced was verified by PCR using primers ylr-duf and ylr-dur, and the downstream portion was verified using primers ylr-ddf and ylr-ddr (see Table 1 for the primers).

[0107] PCR amplification was performed using EasyTaq DNA polymerase. The amplification system was 20 μl (10 μl of 2× EasyTaq Mix, 0.6 μl of each 10 μM primer, 1 μl of template, and water added to 20 μl). The amplified fragments were confirmed by agarose gel electrophoresis. The amplification conditions were pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C. The extension time was calculated as 60 seconds / kb, and the number of cycles was 36, with extension at 72°C for 10 minutes.

[0108] 1.2.5 Shaking fermentation of recombinant strains Both the upstream and downstream recombinant strains were verified to be successful (see Figure 3). The strains were inoculated into 20 ml YPD medium (in a 250 ml Erlenmeyer flask) and grown for approximately 24 hours. After that, they were transferred to a YPD plate and grown for approximately 24 hours. Then, they were inoculated into 5 ml YNB-Δura medium (in a 50 ml Erlenmeyer flask) and grown for 36 hours. After that, 100 μl was aspirated and transferred to fresh 5 ml YNB-Δura medium for 36 hours. Finally, they were transferred to 30 ml shaker fermentation medium and standardized to an initial OD of 0.08. Each group was cultured in three parallel batches for 168 hours. The dry weight of the strains was obtained by weighing.

[0109] 1.2.6 Extraction of total fats and oils by acid-heat method (1) The Yarrowia lipolytica fermentation broth was collected in a 50 ml centrifuge tube and centrifuged at 6000 rpm for 5 min to collect the yeast cells. (2) Approximately 10 ml of 4 M hydrochloric acid was added per 1 g of wet cells, shaken uniformly, and then shaken in a shaker at 28°C for approximately 1-2 hours. (3) The mixture was treated in a boiling water bath for 6-8 minutes and immediately cooled to -20°C for 30 minutes. (4) 20 ml of chloroform:methanol = 1:1 (V / V) was added and mixed thoroughly. (5) The lower layer of chloroform was separated and its volume was measured, and an equal volume of 0.15% sodium chloride was added, followed by centrifugation at 4000 rpm for 10 minutes. (6) The lower layer was removed and transferred to a new weighed glass tube, the solvent phase was dried using a nitrogen blower, and the weight was then measured to calculate the amount of oil produced. The total lipid content was the ratio of the total lipid production to the dry weight.

[0110] 1.2.7 Methyl esterification of fatty acids (1) 2.6 ml of a methanol:sulfuric acid=98:2 (V / V) solution was added to a glass tube and mixed uniformly, followed by reaction at 85°C for 3 hours and then cooling in a refrigerator. (2) 1 ml of saturated NaCl solution and 1 ml of n-hexane were added, and after shaking, the mixture was centrifuged at 5000 rpm for 5 minutes. The supernatant was then aspirated into an EP tube and filtered through an organic phase filter membrane before a gas chromatography experiment.

[0111] 1.2.8 Analysis of fatty acid components by gas chromatography Gas chromatography (Agilent 7890B-GC) was used to analyze fatty acid components. Chromatographic detection conditions: The chromatography column was HP-INOWAX (30 m × 0.32 mm × 0.5 μm). The loading temperature was 250°C, the detector temperature was 250°C, and the loading volume was 1 μL. The column initial temperature was 140°C, maintained for 1 min, then increased to 180°C at 10°C / min and maintained for 2 min. The temperature was then increased to 210°C at 5°C / min and maintained for 4 min. The temperature was then further increased to 250°C at 5°C / min and maintained for 4 min. The relative content of each fatty acid component was obtained by the area percentage method.

[0112] 1.3 Overexpression of MaoleKCS in Yarrowia lipolytica 1.3.1 Construction of overexpression recombinant plasmids The plasmid vector was constructed using the Gibson assembly method, with all assembled fragments obtained by PCR amplification, as described in 1.2.2. The homologous recombination plasmid pYLEX-FAD2-CgKCS-URA, constructed using pYLEX-URA as the backbone, was obtained directly in the patent applicant's laboratory. The upstream and downstream sequences of the CgKCS expression cassette are portions of the Yarrowia lipolytica FAD2 gene (see SEQ ID NO: 14 and SEQ ID NO: 15), respectively, resulting in a homologous recombination plasmid with a site-specific integration site at the FAD2 locus. Using the plasmid pYLEX-FAD2-CgKCS-URA as the backbone, the Gibson assembly method was used to construct the homologous recombination plasmid pYLEX-FAD2-MaoleKCS-URA, substituting two consecutive MaoleKCS expression cassettes for the CgKCS expression cassette.

[0113] 1.3.2 Yeast transformation and verification of recombinant strains by diagnostic PCR Reverse screening using the 5-FOA method yielded a uracil-auxotrophic strain of Polg-G3-MaoleKCS, Polg-G3-MaoleKCS-ΔURA. The recombinant plasmid pYLEX-FAD2-MaoleKCS-URA was digested with the NotI restriction enzyme, confirmed by agarose gel electrophoresis, and the transformation fragment was recovered and transformed into Yarrowia lipolytica Polg-G3-MaoleKCS-ΔURA by the LiAc transformation method. The resulting recombinant strain, designated Polg-G3-3MaoleKCS, contained the MaoleKCS gene. Different transformants were selected and verified by diagnostic PCR. The method was the same as in 1.2.4. The upstream part was verified by PCR with primers FAD-df (5'-CTACGGCACGATAAAGATGG-3') and ylr-dur, and the downstream part was verified by PCR with primers ylr-ddf and FAD-dr (5'-TTACAATGGGACCGTGCTTG-3'). Different transformants that were verified as successful both upstream and downstream were cultured under shaking fermentation. The culture method and the analytical method for each indicator after culture were the same as in 1.2.

[0114] 2. Results and Analysis 2.1 Screening and expression of Malania oleifera 3-ketoacyl-CoA synthase genes 2.1.1 Bioinformatics analysis of Malania oleifera 3-ketoacyl-CoA synthase genes Twelve 3-ketoacyl-CoA synthases were identified in the Malania oleifera genome, but their functions and applications have not been reported. Of these, six 3-ketoacyl-CoA synthases have been annotated by literature authors as simultaneously performing KCS, KCR, HCD, and ECR functions in the fatty acid elongation process. After multiple alignment of the amino acid sequences of the six KCSs with the MoKCS (GenBank: QDA34238.1) and CgKCS (GenBank: ACJ61778.1) in the NCBI database, a phylogenetic tree (see Figure 1) was constructed using the neighbor-joining algorithm. MaoleKCS463 shared a perfect amino acid sequence identity with MoKCS, and MaoleKCS397 shared a high degree of similarity with MaoleKCS398. As shown in Figure 2, KCS398, KCS461, KCS467, and KCS817, as well as the previously reported CgKCS and MoKCS, all contain the three functionally conserved domains of the KCS family, "FGNTSSSS," "GSGFKCNSAVW," and "GMGCSA" (boxed in red), indicating that these proteins belong to the KCS family. KCS398, KCS461, KCS467, and KCS817 were selected for further study. These four KCS genes were codon-optimized and synthesized by Wuxi Qinglan Biotechnology Co., Ltd., to obtain the shuttle plasmid pMv-MaoleKCS#-AMP, which contains the MaoleKCS gene and ampicillin resistance gene (# represents the numbers 398, 463, 467, and 817, respectively).

[0115] 2.1.2 Diagnostic PCR verification of recombinant yeast strains The recombinant transformants were selected from the screening plates and inoculated onto YPD plates. Seven transformants were selected from strain Po1g-G3-MaoleKCS398, and eight each from strains Po1g-G3-MaoleKCS461, Po1g-G3-MaoleKCS467, and Po1g-G3-MaoleKCS817. At the same time, the control strain Po1g-G3 was inoculated onto YPD plates and grown for approximately 24 hours. Genomic DNA was then extracted from the recombinant transformants and the control strain. Using the genomic DNA as a template, PCR amplification of the upstream and downstream regions of the introduced gene, MaoleKCS, was performed to verify gene integration and expression. Genomic DNA from Po1g-G3 was used as a control. The agarose gel electrophoresis pattern of the PCR product is shown in Figure 3. First, in the control group Po1g-G3, there was no upstream or downstream PCR product (see the leftmost lane CK in Figure 3A and Figure 3C), and the transformant that showed a positive PCR result for the downstream region (the target band was 1632 bp) was Po1g-G3-MaoleKCS398 nos. 1, 3, 4, 5 and 6; Po1g-G3-MaoleKCS461 nos. 1, 2, 3 and 4; Po1g-G3-MaoleKCS467 nos. 2, 3, 4, 6 and 7; 2, 4, 6, and 8 of Polg-G3-MaoleKCS817 (Figure 3B).

[0116] Furthermore, we verified the integration of the upstream region in the transformants of the recombinant strains that showed a positive downstream region. Po1g-G3-MaoleKCS398 nos. 1, 3, 4, 5 and 6; Po1g-G3-MaoleKCS461 nos. 1, 2 and 4; Po1g-G3-MaoleKCS467 nos. 2, 3, 4, 6 and 7; Polg-G3-MaoleKCS817 numbers 2, 4, 6 and 8.

[0117] The brightness of the upstream and downstream PCR products was adjusted to obtain the final product, Po1g-G3-MaoleKCS. No. 1 of 398, No. 2 of Polg-G3-MaoleKCS461, No. 3 of Polg-G3-MaoleKCS467, and No. 8 of Polg-G3-MaoleKCS817 were selected and subjected to subsequent shaking fermentation culture.

[0118] 2.1.3 Results of shake fermentation of recombinant strains As shown in Figure 4 , compared with the control strain Po1g-G3, the dry weight, total lipid production, and total lipid content of strain Po1g-G3-MaoleKCS817 were all significantly reduced, the dry weight, total lipid production, and total lipid content of strains Po1g-G3-MaoleKCS398 and Po1g-G3-MaoleKCS467 were slightly improved, and the dry weight, total lipid production, and total lipid content of the strains were not significantly changed.

[0119] Fatty acid composition analysis showed that all strains significantly produced nervonic acid and lignoceric acid compared with the control strain Polg-G3, and the production levels were similar, reaching 8.9% and 6.2% of total fatty acids, respectively (Figure 5). At the same time, the C18:1 and C18:0 fatty acid ratios of the strains were significantly lower than those of the control, and small amounts of C20 and C22 fatty acids were also detected (see Figure 5). This suggests that MaoleKCS can gradually elongate the C18:1 and C18:0 carbon chains to produce nervonic acid and lignoceric acid. Therefore, through screening for fatty acid elongases and expression analysis in yeast, the present invention found that the fatty acid elongase MaoleKCS from Malania oleifera catalyzes the synthesis of nervonic acid and that the corresponding gene is useful for constructing yeast with high nervonic acid production.

[0120] 2.2 Overexpression of MaoleKCS in Yarrowia lipolytica As shown in Figure 6A, the dry weights of the different recombinant transformants were reduced to different degrees, respectively, at 26.7 g / L, 27.4 g / L, 25.8 g / L, and 28.7 g / L, compared to 29.2 g / L for the control strain. Total lipid production was 14.16 g / L for the control strain, while transformants 1, 2, and 3 showed significant decreases, with transformant 3 showing the most significant decrease (32%). Transformant 4 showed no significant change, with a total lipid production of 14.18 g / L. Similar to total lipid production, transformants 1, 2, and 3 showed significant decreases, with transformant 3 showing the most significant decrease (32%). Transformant 4 showed no significant change in lipid content.

[0121] The ratios of different fatty acids in each transformant of the recombinant strain are shown in Figures 6B and 7. In the control strain, the ratio of nervonic acid to total fatty acids was 8.96% (Figure 7a). In transformants 1, 2, and 4, the ratios of nervonic acid were all significantly improved, reaching 13.27%, 13.26%, and 12.60%, respectively (Figures 7b, c, and e). In transformant 3, the ratio was slightly reduced to 8.61% (Figure 7d). At the same time, the ratio of lignoceric acid to total fatty acids in all four transformants was significantly improved, with transformant 3 showing the greatest improvement, nearly two-fold higher than the control. Correspondingly, the ratios of C18:0 and C18:1 in the control strain were reduced to different degrees, indicating that overexpression of MaoleKCS further catalyzed the elongation of C18:0 and C18:1 carbon chains, among which the synthesis effect of nervonic acid in transformant No. 4 was most significant. In transformant No. 4, the total lipid production was not significantly changed compared with the control, and the proportion of nervonic acid was increased by 40%, thereby enhancing the yeast's ability to synthesize nervonic acid.

[0122] Example 2: Fermentation tank fermentation of recombinant strains 1. Materials and Methods 1.1 Fermentation medium Seed medium (YNB medium): YNB 3 g / L, glucose 60 g / L, ammonium sulfate 35 g / L. YNB and ammonium sulfate were sterilized and filtered before addition. Fermentation medium (FM medium): glucose 50 g / L, ammonium sulfate 35 g / L, yeast powder 38 g / L, potassium dihydrogen phosphate 8 g / L, disodium hydrogen phosphate dodecahydrate 6 g / L, magnesium sulfate heptahydrate 5 g / L, antifoam agent 1 mL / L. Control by addition: Control was performed according to the residual sugar concentration in the fermentation tank, and sterilized glucose was added at 600 g / L.

[0123] 1.2 Fermentation process in 50 L fermentation tanks The deposited strain was removed from -80°C and thawed on ice. 100 μl of the broth was inoculated into 250 mL of YNB medium and cultured for 24 hours in a shaker at 220 rpm and 28°C. After 24 hours, the broth was transferred to a 5 L seed fermentation tank containing FM fermentation medium with a liquid volume of 2.5 L. The culture conditions for the seed tank were a temperature of 28°C, circulating cooling with a chiller, pH controlled to 5.5 with 5 mol / L sodium hydroxide, and DO of 20%. After 24 hours of culture, the broth was transferred to a 50 L fermentation tank (Zhenjiang Huinengda Co., Ltd., HND-BJ-5L-50L) to begin fermentation. The FM fermentation medium was prepared according to the experimental plan, thoroughly mixed, and then poured into a 50 L fermentation tank. The liquid filling volume of the fermentation tank was 25 L. When the inoculum volume was 10% and the temperature had dropped to 28°C, 150 ml of seed liquid was inoculated through the inoculation port.

[0124] Fermentation conditions: Fermentation temperature was 28°C, circulating and cooled by a chiller. pH was controlled at 5.5 with 5 mol / L sodium hydroxide. The initial aeration rate was 1 vvm, gradually increased to the maximum of 2 vvm over the first 24 hours. Dissolved oxygen was controlled using cascade control with the rotation speed. DO was set at 20% and adjusted to 10% after 48 hours. The rotation speed ranged from 200 rpm to 600 rpm. The added glucose concentration was 600 g / L, and was added according to changes in the residual sugar concentration in the tank, until the concentration dropped below 30 g / L. For the analytical method of the indicators during the fermentation process, see Example 1. Crude nervonic acid production (g / L) = total lipid production (g / L) x proportion of nervonic acid in total fatty acids (%).

[0125] 2. Results and Analysis The recombinant strain Po1g-G3-3Maole398 was fermented in a 50 L fermentation tank using shaker fermentation, and the results are shown in Figure 8. The lipid, dry weight, and fatty acid composition of the recombinant strain were analyzed, leading to the following conclusions: After 168 hours of fermentation, the dry weight (DCW) reached 149.4 g / L, the total lipid production was 69.6 g / L, and the total lipid content was 46.6%. The proportion of nervonic acid in total fatty acids was 36.9%. Gas chromatography spectra of the fatty acid components are shown in Figure 9, showing that nervonic acid production reached 25.7 g / L.

[0126] Example 3. Excavation and analysis of esterases in Malania oleifera Based on bioinformatics analysis, two diacylglycerol acyltransferase (DGAT2) and three glycerol-3-phosphate acyltransferase (GPAT) enzyme genes were identified from the genome data of Malania oleifera (Xu et al., 2019, 8:1-14). The two DGAT2 genes were named MoDGAT2 (Maole_010035) and MoDGAT2 (Maole_015949), respectively, and the three GPAT genes were named MoGPAT (Maole_006088), MoGPAT (Maole_006089), and MoGPAT (Maole_006090), respectively.

[0127] >SEQ ID NO:38 MoDGAT2(Maole_010035) gene sequence

[0128] (a) Sequence characteristics: Length: 1566 ●Type: DNA sequence Chain type: Single chain ● Phase structure: Linear (b) Type of molecule: DNA (c) Assumption: No (d) Antisense: No (e) Original origin: Malania oleifera Sequence characteristics: The product encoded by the gene is diacylglycerol acyltransferase (DGAT).

[0129] >SEQ ID NO: 39 MoDGAT2(Maole_010035) amino acid sequence MDGSSQIKELSCLVKFVEETVRIEHASNPNKPVYLVGDSFGGCLALAVAARNPTFDLVLILVNPATSFGRSQLQPLLPVLEALPDGLHFTVPYLLSFIMGTCSAALKPYRDPLKLAMVNIETRFPPALVL EQLSGNLTALLPRLSGLANIIPKETLLWKLKLLKSAAAYANSRLHAVKAEVLVLASGSDNMLPSKDEAERLSRSLQNCIVRLFKDNGHTLLLEDGYSLLTIIKCTSKYRCSRKHDFITDFLPPSMSEFKQ ELNQRFGLFRVATSPILFSTLPDGKIVKGLAGIPNEGPVLLVGYHMLLGCELGPLVEAFLKEKNIMIRGVAHPEMFSKKHEGPSNEFGPFDLVKLFGGLPVTPRNLFKLFSTKSHILLYPGGAREALHRK GEKYKLLWPNQPEFVRMAARFGATIVPFGTVGEDDIAEAESTMLAKKITLRSSDGETFEVDKIVALESQMIKHMIEDDCADNGIPLPNVTSWILVKVIEYCKKHVKAPKIEKRGDVNKELKSWDAEFVKID

[0130] (a) Sequence characteristics: Length: 521 ●Type: Amino acid sequence Chain type: Single chain ● Phase structure: Linear (b) Type of molecule: Amino acid (c) Assumption: No (d) Antisense: No (e) Original origin: Malania oleifera Characteristics of the sequence: The amino acid sequence is that of diacylglycerol acyltransferase (DGAT).

[0131] >SEQ ID NO: 40 MoDGAT2(Maole_015949) gene sequence

[0132] (a) Sequence characteristics: Length: 1209 ●Type: DNA sequence Chain type: Single chain ● Phase structure: Linear (b) Type of molecule: DNA (c) Assumption: No (d) Antisense: No (e) Original origin: Malania oleifera Sequence characteristics: The product encoded by the gene is diacylglycerol acyltransferase (DGAT).

[0133] >SEQ ID NO: 41 MoDGAT2(Maole_015949) amino acid sequence MAETEVRESPLTAAPAPEVTVIESPRVPLLHSVLATALWGCIHLFFAVMFTATFLLPLSKSIAVFALLLVLIVVPVEADSKFGMKVRRGNNHHDNEGDDEEFSNIHGWVVKKMVVVYCHSVTMPELEVTMLPSNGSVLSFTVFALEPHSVLPVGVISLMHLSNAVPLPKTRVLASSAVFRTPFLRHIWTWMGLAAVTRKN FISLLAAGYSCAIIPGGTRETLLMVQDHEVYLLKLSVALLRIAARSTALEWNLCPFQKIAFLKTRKGFVRTAIETGVPLIPVFSFGQVFFFFWKDQVYTDVLLGSFGMLHLITWTGLLSTHHTRMPSFFIMRRAATVPLTPMPRRLPLHVVVGRPIEVKQNSQPTAEEVNEVHSQFVGALQDLFERHKARVGHADRELKII

[0134] (a) Sequence characteristics: Length: 402 ●Type: Amino acid sequence Chain type: Single chain ● Phase structure: Linear (b) Type of molecule: Amino acid (c) Assumption: No (d) Antisense: No (e) Original origin: Malania oleifera Characteristics of the sequence: The amino acid sequence is that of diacylglycerol acyltransferase (DGAT).

[0135] >SEQ ID NO: 42 MoGPAT(Maole_006088) gene sequence ATGGACTTTGTACCCGACATCTCCGCCATCAAGACCGACAAGATTAAAATTGACAAGGAAACCACCGACATGCTTACCGCTCGGCATGACGGATCTTCTGAAGTTAGGGAGGACGAGTTGGTGGCTGCGCCAGTGCCAACTTCTCCGTTCGGTTGTTGGGCCGGCGGACCCGGTAGGAGCATCAACCGTCGCACAAATCAAATACCTCTCGAACCTTGCAGAGACATCTCAGTTCATAAACTAATCGAACCGATGTGCTCAAAAAGACTAGGGAGTGATTTCTCCGACCAGTTAGCGTCACCTTGTGTGTCTCATTCGTGGTTGCCGGCGACGACGAAGTGCGATCATGTGCACTGCCTGTGCATGGAACGTGCCTGCTGCTGGAATTGGTCCTCTGATGATGTGCTTTCAGCTGCTGGCGGATCAAGTCGTGTTGAATCTAATAGTGTTCATGGCGTATTAAGAAAACTCCAGCAATGGTCTGCAATGAACAGTGCTGCCGATGGGGGGACAAAGGGGCTGGAATTTGACAGAAAAGACACGAAGAACAAGAATGTGATGAAAGAGAGTACAGTGACCACTCCAAAGACCTTGAGGGTTGCATTGAAATGCTGCACAAGGGAAGATACTGCACCGGAGGCAAAAGAGAATATGAATTATTTCCGTGGTATAGAATGTTGTGAGGTTAAGGAGGCATATTCGAAGGCACTGCATGATTCTGTTACTGAACAATACAATGTGCTCAAATCTGCCATACATGGCAAACAAGGACTGGAGGCATCCATTCCAGATGTCTCATTATCACAACCATGGCAGTAG

[0136] (a) Characteristics of the sequence: ● Length: 819 ● Type: DNA sequence ● Strand type: Single-stranded ● Topology: Linear (b) Type of molecule: DNA (c) Hypothesis: No (d) Antisense: No (e) Original origin: Malania oleifera Sequence characteristics: The product encoded by the gene is glycerol-3-phosphate acyltransferase (GPAT).

[0137] >SEQ ID NO: 43 MoGPAT(Maole_006088) amino acid sequence MAETEVRESPLTAAPAPEVTVIESPRVPLLHSVLATALWGCIHLFFAVMFTATFLLPLSKSIAVFALLLVLIVVPVEADSKFGMKVRRGNNHHDNEGDDEEFSNIHGWVVKKMVVVYCHSVTMPELEVTMLPSNGSVLSFTVFALEPHSVLPVGVISLMHLSNAVPLPKTRVLASSAVFRTPFLRHIWTWMGLAAVTRKN FISLLAAGYSCAIIPGGTRETLLMVQDHEVYLLKLSVALLRIAARSTALEWNLCPFQKIAFLKTRKGFVRTAIETGVPLIPVFSFGQVFFFFWKDQVYTDVLLGSFGMLHLITWTGLLSTHHTRMPSFFIMRRAATVPLTPMPRRLPLHVVVGRPIEVKQNSQPTAEEVNEVHSQFVGALQDLFERHKARVGHADRELKII

[0138] (a) Sequence characteristics: Length: 402 ●Type: Amino acid sequence Chain type: Single chain ● Phase structure: Linear (b) Type of molecule: Amino acid (c) Assumption: No (d) Antisense: No (e) Original origin: Malania oleifera Sequence characteristics: The product encoded by the gene is glycerol-3-phosphate acyltransferase (GPAT).

[0139] >SEQ ID NO:44 MoGPAT(Maole_006089) gene sequence

[0140] (a) Sequence characteristics: Length: 1110 ●Type: DNA sequence Chain type: Single chain ● Phase structure: Linear (b) Type of molecule: DNA (c) Assumption: No (d) Antisense: No (e) Original origin: Malania oleifera Sequence characteristics: The product encoded by the gene is glycerol-3-phosphate acyltransferase (GPAT).

[0141] >SEQ ID NO: 45 MoGPAT(Maole_006089) amino acid sequence MVLWQCGGSGLRGSDEGLRWHGAAMLATRTVPAAAAGLDSGRHSHNVQGSQPQEICAKESLGNIDGVRGDSINDAEWAGKAVTYVAGDRVLTDILCKPFSMGRNLMCVYSKKHMLDDPKLAEMKKKANIRSLKEMAMLLRGGSQIVWIAPSGGRDRPDPQTGEWHPAPFDASSVDNNMRRLAEN AGVPAHIYPLALICYNIMPPPPKVEKEIGEKRVVSFHGVGLSVIPDISYAEIAAACENSEETGDADTNQSHYQCHRRIPQAEKFGILAALPDDESRAKATSPRSTPSKRLRYKIAGFSAHLMKRIQKGPFRGISLKLQEEEHERRMDFVPDISAIKTDKIKVDKETRSTPSRRLRNKIATSPPTS

[0142] (a) Sequence characteristics: Length: 272 ●Type: Amino acid sequence Chain type: Single chain ● Phase structure: Linear (b) Type of molecule: Amino acid (c) Assumption: No (d) Antisense: No (e) Original origin: Malania oleifera Sequence characteristics: The product encoded by the gene is glycerol-3-phosphate acyltransferase (GPAT).

[0143] >SEQ ID NO: 46 MoGPAT(Maole_006090) gene sequence ATGCAGCTAAGAATACTTTTAGATGTTTTGACTCTTCTGTTTTGCAGCATTTCTTTATTTGGAATCTAATTTGGAGGTATTGTTTTCTTGTCTTGCTGGGTTTCCTGCAGAGCTGCTTTCTGGAATAAAGAAGGAAACAGAAGCTGGCAGATTGCCTTCAAATTTTGCTTCAGT TCTGGTTTTCCAAAGTGGAATTCCAGATGCTGATGAAATTATATTGTCAAACACAACTGCTTTGTTTGATCGTGTTTTACTGGATGCAGAGGACTCTTTTGTTTTCCCCCACATCATAAAGCAATGCGAGAGCCTTTTGATTACTACATGTTTGGTCAAAATTATATCCGTC CTTTGATAGATTTTGGGAATTCATATGTTGGCAATATCAACATTTTTCATGAAATGGAAGAGAAGCTGCAGCAGGTCAAGAATCTCAATGGGATTGAGTGGGTCAGCCATGGAGGAGTCTCCACTCTCTGCAAATAGAGAGTAAATCCTTAGATCTGCGACTGGAAAAGGTT GGGAAGGGTTTCTTTTTGGCTGGATGCTGTTTTCGGTTGCAAAGGGAAATTGTCAAGTTAAATTCTTGGATTTGGGTTATACTCATTTGGAAAGGCTTATTCTATTTTTGTTCTATGGGGTTTTGATGGGGAGTGATGGCAACGTTTGTTGAGATGTTGCAGCCGACGGTAA

[0144] (a) Sequence characteristics: Length: 693 ●Type: DNA sequence Chain type: Single chain ● Phase structure: Linear (b) Type of molecule: DNA (c) Assumption: No (d) Antisense: No (e) Original origin: Malania oleifera Sequence characteristics: The product encoded by the gene is glycerol-3-phosphate acyltransferase (GPAT).

[0145] >SEQ ID NO: 47 MoGPAT(Maole_006090) amino acid sequence MQLRILLDVLTLLFAAFLYLESNLEVLFSCLAGFPAELLSGIKKETEAGRLPSNFASVLVFQSGIPDADEIILSNTTALFDRVLLDAEDSFVFPPHHKAMREPFDYYMFGQNYIR PLIDFGNSYVGNINIFHEMEEKLQQVKNLNGIEWGQPWRSLHSLQIESKSLDLRLEKVGKGFFLAGCCFRLQREIVKLNSWIWGYTHLERLILFYGVLMGSDGNGLLRCCSRR

[0146] (a) Sequence characteristics: Length: 230 ●Type: Amino acid sequence Chain type: Single chain ● Phase structure: Linear (b) Type of molecule: Amino acid (c) Assumption: No (d) Antisense: No (e) Original origin: Malania oleifera Sequence characteristics: The product encoded by the gene is glycerol-3-phosphate acyltransferase (GPAT). The codon-optimized sequences of the five esterases mentioned above in Yarrowia lipolytica are as follows:

[0147] >SEQ ID NO: 48 MoDGAT2(Maole_010035) gene sequence

[0148] (a) Sequence characteristics: Length: 1566 ●Type: DNA sequence Chain type: Single chain ● Phase structure: Linear (b) Type of molecule: DNA (c) Assumption: No (d) Antisense: No (e) Original origin: Malania oleifera Sequence characteristics: The product encoded by the gene is diacylglycerol acyltransferase (DGAT).

[0149] >SEQ ID NO: 49 MoDGAT2(Maole_015949) gene sequence

[0150] (a) Sequence characteristics: Length: 1209 ●Type: DNA sequence Chain type: Single chain ● Phase structure: Linear (b) Type of molecule: DNA (c) Assumption: No (d) Antisense: No (e) Original origin: Malania oleifera Sequence characteristics: The product encoded by the gene is diacylglycerol acyltransferase (DGAT).

[0151] >Sequence number 50 MoGPAT(Maole_006088) gene sequence ATGGACTTCGTGCCCGACATCTCTGCCATCAAGACCGACAAGATCAAGATCGACAAGGAGACTACCGACATGCTGACCGCCAGACATGATGGCTCTTCTGAGGTGAGAGAAGACGAGCTGGTGGCCGCCCCTGTGCCTACTTCTCCTTTTGGATGTTGGGCTGGCGGACCCGGCAGATCTATCAACAGAAGAACTAATCAGATCCCCCTGGAGCCCTGCCGAGACATTTCTGTGCATAAACTGATCGAGCCCATGTGCTCTAAGCGACTGGGCTCTGACTTCTCTGACCAGCTGGCCTCTCCCTGCGTGTCTCACTCTTGGCTGCCTGCTACTACCAAATGCGACCATGTGCACTGCCTGTGCATGGAGCGAGCCTGTTGTTGGAATTGGTCTTCTGACGACGTGCTGTCTGCCGCCGGAGGATCTTCTAGAGTGGAATCTAACTCTGTGCACGGCGTGCTGCGAAAGCTGCAGCAATGGTCTGCCATGAACTCTGCCGCCGATGGAGGCACTAAAGGACTGGAGTTTGATCGAAAGGACACCAAGAACAAGAACGTGATGAAGGAGTCTACCGTGACCACCCCCAAGACCCTGAGAGTGGCTCTGAAATGCTGTACCCGAGAGGATACCGCCCCCGAAGCTAAAGAGAACATGAACTACTTCCGAGGCATCGAGTGCTGCGAGGTGAAGGAGGCTTATTCTAAGGCCCTGCACGACTCTGTGACCGAGCAGTATAATGTGCTGAAGTCTGCCATCCACGGCAAGCAGGGCCTGGAGGCTTCTATTCCCGATGTGTCTCTGTCTCAGCCCTGGCAGTAA

[0152] [[ID=]](a) Characteristics of the sequence: ● Length: 819 ● Type: DNA sequence ● Strand type: Single-stranded ● Topology: Linear (b) Type of molecule: DNA (c) Hypothesis: No (d) Antisense: No (e) Original origin: Malania oleifera Sequence characteristics: The product encoded by the gene is glycerol-3-phosphate acyltransferase (GPAT).

[0153] >SEQ ID NO:51 MoGPAT(Maole_006089) gene sequence

[0154] (a) Sequence characteristics: Length: 1110 ●Type: DNA sequence Chain type: Single chain ● Phase structure: Linear (b) Type of molecule: DNA (c) Assumption: No (d) Antisense: No (e) Original origin: Malania oleifera Sequence characteristics: The product encoded by the gene is glycerol-3-phosphate acyltransferase (GPAT).

[0155] >SEQ ID NO:52 MoGPAT(Maole_006089) gene sequence ATGCAGCTGCGAATCCTGCTGGACGTGCTGACCTTACTGTTTGCCGCTTTTCTGTACCTGGAGTCTAACCTGGAGGTGCTGTTCTCTTGCCTGGCCGGCTTTCCTGCTGAACTGCTGTCTGGAATTAAGAAGGAGACTGAGGCCGGCCGACTGCCCTCTAATTTTGCATCTGT GCTGGTGTTCCAGTCTGGCATCCCCGACGCTGATGAAATCATTCTGTCTAACACCACCGCCCTGTTCGACCGAGTGCTGCTTGATGCTGAAGACTCTTTCGTGTTCCCCCCCCACAAGGCCATGAGAGAACCTTTTGATTACTACATGTTCGGCCAGAACTACATCCGAC CCCTGATCGACTTCGGCAACTCTTACGTGGGCAACATCAACATCTTCCACGAGATGGAGGAGAAGCTGCAGCAGGTGAAGAACCTGAACGGCATCGAGTGGGGCCAGCCCTGGAGATCTCTGCATTCTCTGCAGATCGAGTCTAAGTCTCTGGACCTGCGACTGGAGAAGGTG GGCAAAGGATTTTTCCTGGCCGGATGCTGTTTTCGACTGCAGCGAGAGATCGTGAAGCTGAACTCTTGGATCTGGGGCTACACCCACCTGGAGCGACTGATCCTTTTTCTGTTCTACGGCGTGCTGATGGGCTCTGACGGCAATGGACTGCTGAGATGTTGCTCTCGACGATAA

[0156] (A) Sequence characteristics: Length: 693 ●Type: DNA sequence Chain type: Single chain ● Phase structure: Linear (B) Type of molecule: DNA (C) Assumption: No (D) Antisense: No (E) Original origin: Malania oleifera Sequence characteristics: The product encoded by the gene is glycerol-3-phosphate acyltransferase (GPAT).

[0157] Example 3. Synthesis of esterase in Malania oleifera, plasmid construction and yeast transformation The genes MoDGAT2(Maole_010035), MoDGAT2(Maole_015949), GPAT, MoGPAT(Maole_006088), MoGPAT(Maole_006089), and MoGPAT(Maole_006090) were synthesized by Wuxi Qinglan Biotechnology Co., Ltd. with optimized codons (the optimized gene sequences are SEQ ID NOs: 48, 49, 50, 51, and 52, respectively). Shuttle plasmids carrying the esterase gene and ampicillin resistance gene were obtained: pMV-MoDGAT2(Maole_010035)-AMP, pMV-MoDGAT2(Maole_015949)-AMP, pMV-MoGPAT(Maole_006088)-AMP, pMV-MoGPAT(Maole_006089)-AMP, and pMV- It was named MoGPAT(Maole_006090)-AMP.

[0158] Plasmid vectors were constructed using the Gibson assembly method, and all assembled fragments were obtained by PCR amplification. The plasmid backbone was derived from the pYL-PEX10-CgKCS plasmid (obtained from the Qingdao Institute of Bioenergy and Bioprocessing, Chinese Academy of Sciences). The esterase promoter used was TEFintro. All PCR amplifications were performed using KAPA HiFi high-fidelity DNA polymerase in a 50 μl volume (25 μl of 2× KAPA Mix, 1.5 μl of each 10 μM primer, 1 μl of template, and water added to 50 μl). The amplification conditions were: pre-denaturation at 95°C for 3 minutes, denaturation at 98°C for 20 seconds, annealing at 60–72°C for 15 seconds, and extension at 72°C. The extension time was calculated as 30 seconds per kb, with cycles of 29–35, and extension at 72°C for 10 minutes.

[0159] After Gibson assembly of the backbone and esterase gene fragments, they were transformed into susceptible E. coli Trans-T1 (Beijing Quanshi Jin Biotechnology Co., Ltd.). Colony PCR and sequencing verification was performed using primers TEFin-PF and XPR2-PR (Qingdao Jinke Biotechnology Co., Ltd.). Homologous recombination plasmids pYLEX-PEX10-MoDGAT2(Maole_010035)-URA, pYLEX-PEX10-MoGPAT(Maole_006088)-URA, pYLEX-PEX10-MoGPAT(Maole_006089)-URA, and pYLEX-PEX10-MoGPAT(Maole_006090)-URA were obtained. The primers are listed in Table 1.

[0160] [Table 4]

[0161] PCR amplification of the recombinant plasmids pYLEX-PEX10- MoDGAT2(Maole_010035)-URA, pYLEX-PEX10- MoGPAT(Maole_006088)-URA, pYLEX-PEX10- MoGPAT(Maole_006089)-URA, and pYLEX-PEX10- MoGPAT(Maole_006090)-URA was performed using 10up and 10dn PCR products to obtain transforming fragments. All PCR amplifications were performed using KAPA HiFi high-fidelity DNA polymerase. Each amplification system was 50 μl (25 μl of 2× KAPA Mix, 1.5 μl of each 10 μM primer, 1 μl of template, and water was added to 50 μl). The amplification conditions were pre-denaturation at 95°C for 3 minutes, denaturation at 98°C for 20 seconds, annealing at 60–72°C for 15 seconds, and extension at 72°C. The extension time was calculated as 30 seconds / kb, and the number of cycles was 29–35, with extension at 72°C for 10 minutes.

[0162] The PCR results were confirmed by agarose gel electrophoresis, and the transformation fragment was excised from the gel and then transformed into Yarrowia lipolytica Polg-G3-CgKCS, which already expressed the elongase CgKCS, by LiAc transformation. The resulting transformant was plated on a YNB-Δura medium screening plate and cultured at 28°C for 2–4 days to obtain a recombinant strain carrying the relevant esterase gene. The strains transformed with pYLEX-PEX10-MoDGAT2(Maole_010035)-URA, pYLEX-PEX10-MoDGAT2(Maole_015949)-URA, pYLEX-PEX10-MoGPAT(Maole_006088)-URA, pYLEX-PEX10-MoGPAT(Maole_006089)-URA, and pYLEX-PEX10-MoGPAT(Maole_006090)-URA are abbreviated as YL-35, YL-49, YL-88, YL-89, and YL-90, respectively, from left to right. Verification of the recombinant strains was performed using diagnostic PCR. Single colonies were picked from the screening plates, streaked onto YPD plates, and grown for approximately 24 hours. Fresh cells were harvested and genomic DNA was extracted from the recombinant strains using a fungal genome rapid extraction kit (Sangong Bioengineering Co., Ltd.). Genomic DNA was used as a template and positive clones were verified using the primers listed in Table 2. PCR amplification was performed using EasyTaq DNA polymerase in a 20 μl amplification system (10 μl of 2× EasyTaq Mix, 0.6 μl each of 10 μM primers, 1 μl of template, and water added to 20 μl). Amplified fragments were confirmed by agarose gel electrophoresis. The amplification conditions were 94°C for 3 minutes of pre-denaturation, 94°C for 30 seconds, 55°C for 30 seconds of annealing, and 72°C for extension. The extension time was calculated as 60 seconds per kb, with 36 cycles and 10 minutes of extension at 72°C.

[0163] [Table 5]

[0164] The recombinant strains were then inoculated onto YPD plates, with 48 sites selected for each of YL-35, YL-49, YL-88, YL-89, and YL-90. A control strain was also inoculated onto a YPD plate. After growth for approximately 24 hours, genomic DNA was extracted from the recombinant and control strains. PCR amplification of the transgenes was performed using the genomic DNA as a template to verify gene integration and expression. Five positive clones were selected from the group and named YL-35-F5 (co-expressing CgKCS and MoDGAT2 (Maole_010035)), YL-49-C6 (co-expressing CgKCS and MoDGAT2 (Maole_015949)), YL-88-B1 (co-expressing CgKCS and MoGPAT (Maole_006088)), and YL-90-B6 (co-expressing CgKCS and MoGPAT (Maole_006090)), as shown in Figure 13. The CK strain was Po1g-G3-CgKCS, which expressed the elongase CgKCS, followed by shaking fermentation. The starting strain served as a control.

[0165] Example 4 Effect of esterase on oil and nervonic acid in Malania oleifera The strain and culture conditions were the same as in Example 1, 1.1. The specific method and steps for the shaking fermentation culture were the same as those in section 1.2.5 of Example 1.

[0166] After shaking fermentation, oils were extracted from YL-35-F5, YL-49-C6, YL-88-B1, and YL-90-B6. The production of nervonic acid in YL-35-F5, YL-49-C6, YL-88-B1, and YL-90-B6 was significantly improved compared to the control, Po1g-G3-CgKCS(CK), by 15.6%, 17.2%, 22.1%, and 18.7%, respectively. The results are shown in Table 10. The combined ratio of nervonic acid to total oils indicates that the production of nervonic acid in YL-35-F5, YL-49-C6, YL-88-B1, and YL-90-B6 was significantly improved compared to the control. Among them, YL-88-B1 showed the greatest improvement, with nervonic acid reaching 3.36 g / L, a 28.16% increase compared to the control. The results are shown in Figure 11. Compared to the starting strain Po1g-G3-CgKCS, the ratio of oleic acid to total oil in YL-88-B1 increased from 24.2% to 28.0%, the ratio of nervonic acid to total oil increased from 17.3% to 18.1%, and the nervonic acid fermentation concentration increased by 28.1%. The results are shown in Figure 12.

[0167] All documents related to the present invention are incorporated herein by reference as if each document were individually incorporated by reference. After reading the above content of the present invention, it should be understood that those skilled in the art can make various changes and modifications to the present invention, and that equivalents thereof are within the scope of the claims of the present invention.

Claims

1. An engineered yeast for producing nervonic acid and / or fats and oils, wherein a gene expression cassette is integrated into the genome of the engineered yeast, and the gene expression cassette expresses a protein encoded by a 3-ketoacyl-CoA synthase (KCS) gene and / or an esterase gene; wherein the sequence of the 3-ketoacyl-CoA synthase (KCS) gene is set forth in any of SEQ ID NOs: 2 or 3, and / or the sequence of the esterase gene is set forth in any of SEQ ID NOs: 48, 49, 50, or 52; wherein the oil or fat is an oil or fat containing nervonic acid.

2. 2. The engineered yeast of claim 1, wherein the sequence of the esterase is as set forth in SEQ ID NO:

50.

3. 2. The engineered yeast of claim 1, wherein the engineered yeast is selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Pichia pastoris, Isachenkia orientalis, or a combination thereof.

4. 2. The engineered yeast of claim 1, wherein the sequence of the 3-ketoacyl-CoA synthase gene is as set forth in SEQ ID NO:

2.

5. 2. The engineered yeast of claim 1, wherein the engineered yeast is Yarrowia lipolytica.

6. 2. The engineered yeast of claim 1, wherein the 3-ketoacyl-CoA synthase (KCS) gene or esterase gene is a codon-optimized 3-ketoacyl-CoA synthase (KCS) gene or a codon-optimized esterase gene.

7. 2. The engineered yeast of claim 1, wherein the sequence of the 3-ketoacyl-CoA synthase (KCS) gene is as set forth in SEQ ID NO:

3.

8. 2. The engineered yeast of claim 1, wherein the esterase gene is selected from the group consisting of diacylglycerol acyltransferase (DGAT2), glycerol-3-phosphate acyltransferase (GPAT) enzyme genes, or a combination thereof.

9. 1. A method for producing nervonic acid and / or fats and oils, comprising the steps of: (i) obtaining a fermentation product containing nervonic acid and / or oil by culturing the engineered yeast of claim 1; and (ii) separating nervonic acid and / or oil from the fermentation product; The method comprising:

10. 10. A method for constructing the engineered yeast of claim 1, comprising the steps of: (a) constructing a vector containing a gene expression cassette having the following elements: a screening marker gene, a resistance gene element, a homologous recombination gene fragment, and a target gene, wherein the target gene is a 3-ketoacyl-CoA synthase (KCS) gene and / or an esterase gene; and (b) introducing the vector containing the gene expression cassette obtained in step (a) into a recipient strain to obtain a strain in which the gene expression cassette has been integrated into the genome of the recipient. The method comprising:

11. 10. The use of the engineered yeast of claim 1, wherein said engineered yeast is used as a strain for producing nervonic acid and / or oils by fermentation.

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