Opo type structure ester producing genetically engineered bacteria, construction method and application thereof

Abstract

This invention relates to a genetically engineered bacterium producing OPO-type structural esters, which is a recombinant yeast cell containing the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase. Specifically, this genetically engineered bacterium is constructed by replacing the OLE1 gene encoding Δ9 desaturase with the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase in Saccharomyces cerevisiae strain YS58, and by overexpressing the GAT1 and / or SLC and / or PAP and / or DGAT genes. This effectively increases the proportion of OPO-type structural esters in the lipids of Saccharomyces cerevisiae, making the content of OPO-type structural esters in triglycerides in the genetically engineered Saccharomyces cerevisiae reach 17.2%, thus overcoming the limitations of OPO-type structural ester sources.

Description

Technical Field

[0001] This invention belongs to the field of biosynthesis and genetic engineering technology, and relates to genetically engineered bacteria that produce OPO-type structural esters, their construction methods, and applications. Background Technology

[0002] OPO-type structural esters, chemically named 1,3-dioleoyl-2-palmitoylglycerol, are an important component of breast milk fat. Studies have shown that adding OPO to infant formula can increase the content of palmitic acid at the Sn-2 position. After milk fat enters the infant's body, it is first initially digested by gastric lipases in the stomach, with 10%–30% of the triglycerides being digested in this process. The fat globules then pass through the stomach into the small intestine, where they are hydrolyzed by pancreatic lipases into sn-2 monoglycerides and free fatty acids. Sn-2 monoglycerides can be directly absorbed by the small intestine, providing energy for the infant. This reduces constipation and bowel movement difficulties in infants, improves the absorption and utilization of energy and minerals, reduces the loss of calcium, magnesium, and other bone minerals and nutrients, alleviates symptoms of insufficient calcium and energy intake in infants due to infant formula, activates immune cells, and enhances the infant's natural resistance.

[0003] OPO structured esters can be synthesized through chemical and enzymatic methods. Chemical synthesis suffers from problems such as high catalyst toxicity, high reaction temperatures, strong reaction randomness, and poor selectivity, and is now largely obsolete. In recent years, with the development of enzyme engineering technology, enzyme-catalyzed synthesis has improved the yield of structured esters to some extent. However, enzymes have drawbacks such as high catalyst cost, poor stability, and limited batch usage. Furthermore, enzyme catalysis is heavily constrained by raw materials, and factors such as conversion rate and cost also limit the development of structured esters. Therefore, developing new production technologies and processes for structured esters and reducing their production costs is of significant value and importance for meeting the growing nutritional and health needs of the people and promoting the "Healthy China Strategy." Summary of the Invention

[0004] One of the objectives of this invention is to provide a genetically engineered bacterium that produces OPO-type structural esters, which produces high levels of OPO-type structural esters and is easy to industrialize.

[0005] The second objective of this invention is to provide a method for constructing the above-mentioned genetically engineered bacteria that produce OPO-type structural esters. This method can overcome the drawbacks of enzymatic synthesis of OPO-type structural esters, such as large constraints on raw materials, low conversion rate, high cost, poor stability, and limited batch use. It has important value and significance for meeting the growing nutritional and health needs of the people and promoting the development of the "Healthy China Strategy".

[0006] The third objective of this invention is to provide the application of the above-mentioned genetically engineered bacteria in the production of OPO-type structural esters. Fermentation culture of the genetically engineered bacteria to produce OPO-type structural esters has high conversion rate, low cost, good stability, and is easy to industrialize.

[0007] Therefore, the present invention first provides a genetically engineered bacterium that produces OPO-type structural esters.

[0008] According to some embodiments of the first aspect of the present invention, the genetically engineered bacteria that produce OPO-type structural esters are recombinant yeast cells containing the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase.

[0009] In some embodiments of the present invention, the genetically engineered bacteria are recombinant yeast cells modified with a chassis; preferably, the chassis modification includes the inhibition of the C16:1 anabolic pathway; more preferably, the inhibition of the C16:1 anabolic pathway is the knockout of the OLE1 gene encoding Δ9 desaturase.

[0010] In some preferred embodiments of the present invention, the OLE1 gene encoding Δ9 desaturase in the genetically engineered bacteria is replaced by the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase.

[0011] According to some embodiments of the second aspect of the present invention, the genetically engineered bacteria are recombinant yeast cells optimized through lipid or lipid precursor synthesis pathways.

[0012] Preferably, the optimization of the lipid or lipid precursor synthesis pathway includes overexpression of one or more of the following genes in recombinant yeast cells: the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, and the DGAT gene encoding diacylglycerol acyltransferase; more preferably, the lipid or lipid precursor is a triglyceride or a fatty acid; even more preferably, the fatty acid includes one or more of C16:0, C16:1, C18:0, and C18:1.

[0013] In some specific preferred embodiments of the present invention, the lipid or lipid precursor synthesis pathway is optimized by overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, or the DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cells.

[0014] In some specific preferred embodiments of the present invention, the lipid or lipid precursor synthesis pathway is optimized by overexpressing the SLC gene encoding phosphatidyl acyltransferase and the PAP gene encoding phosphatidyl phosphatase in recombinant yeast cells.

[0015] In some specific preferred embodiments of the present invention, the lipid or lipid precursor synthesis pathway is optimized by overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, and the PAP gene encoding phosphatidylphosphatase in recombinant yeast cells.

[0016] In some specific preferred embodiments of the present invention, the lipid or lipid precursor synthesis pathway is optimized by overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, and the DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cells.

[0017] In this invention, the yeast cells are selected from the group consisting of the following yeast cells: Yeastra lipolytica, Rhodotorula rubrum, lipophilic yeast, lipophilic yeast, Rhodotorula glutinis, Cryptococcus curvifolius, Candida curvifolius, Myxospora fermentans, Candida lakoffii, Candida ferruginosa, Candida rubrum, Candida tropicalis, Candida utilis, Myxospora dermoidosa, and Saccharomyces cerevisiae, preferably Saccharomyces cerevisiae.

[0018] The present invention also provides a method for constructing genetically engineered bacteria as described in the embodiments of the first to third aspects of the present invention, comprising:

[0019] Step (A): Construct recombinant yeast cell A containing the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase;

[0020] Step (Z) optimizes lipid or lipid precursor synthesis pathways.

[0021] According to some embodiments of the present invention, step (A) includes:

[0022] Step M: Express the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase in yeast cells;

[0023] Step N: Knock out the OLE1 gene encoding Δ9 desaturase from yeast cells;

[0024] In some preferred embodiments of the present invention, step (A) includes replacing the OLE1 gene encoding Δ9 desaturase with the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase to construct a recombinant yeast cell A containing the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase.

[0025] According to other embodiments of the present invention, step (Z) includes:

[0026] Recombinant yeast cells B were obtained by overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, or the DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cells.

[0027] Alternatively, recombinant yeast cells C can be obtained by overexpressing the SLC gene encoding phosphatidyl transferase and the PAP gene encoding phosphatidyl phosphatase in recombinant yeast cells.

[0028] Alternatively, recombinant yeast cells D can be obtained by overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, and the PAP gene encoding phosphatidylphosphatase in recombinant yeast cells.

[0029] Alternatively, recombinant yeast cells E can be obtained by overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, and the DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cells.

[0030] The present invention also provides the application of genetically engineered bacteria as described in the embodiments of the first to third aspects of the present invention, or genetically engineered bacteria constructed by the above-described methods of the present invention, in the production of OPO-type structural esters.

[0031] According to the present invention, the application includes inoculating the genetically engineered bacteria into a fermentation medium, performing fermentation culture, and then separating and purifying the obtained fermentation culture broth to obtain an OPO-type structural ester; more preferably, the fermentation medium includes one or more of YPD fermentation medium, YNB-Trp fermentation medium, and YNB-Trp / Ura fermentation medium; even more preferably, the fermentation culture temperature is 30°C; and / or, the fermentation culture time is 72 h.

[0032] The beneficial effects of this invention are mainly reflected in the following aspects: This invention increases the content of OPO-type structural esters in *Saccharomyces cerevisiae*, overcoming the limitations of OPO-type structural ester sources and providing a new method for achieving microbial sources of structural esters. In *Saccharomyces cerevisiae* strain YS58, this invention expresses the ChDes9-2 gene and overexpresses GAT1 and / or SLC and / or PAP and / or DGAT genes to construct a genetically engineered *Saccharomyces cerevisiae* strain, effectively increasing the proportion of OPO-type structural esters in the lipids of *Saccharomyces cerevisiae*, achieving a content of 17.2% of OPO-type structural esters in triglycerides in the genetically engineered *Saccharomyces cerevisiae* strain. Attached Figure Description

[0033] The present invention will now be described in further detail with reference to the accompanying drawings:

[0034] Figure 1 The metabolic pathway of triglyceride synthesis in Saccharomyces cerevisiae is shown.

[0035] Figure 2 The effect of ChDes9-2 gene expression on fatty acid production in Saccharomyces cerevisiae is shown.

[0036] Figure 3 This study demonstrates the effect of the ChDes9-2 gene on the production of OPO-type structural esters by Saccharomyces cerevisiae.

[0037] Figure 4 The effects of overexpression of the GAT1 gene, SLC gene, PAP gene, and DGAT gene on fatty acid production by engineered bacteria are shown.

[0038] Figure 5 The effects of overexpression of GAT1, SLC, PAP, and DGAT genes on the production of OPO-type esters by engineered bacteria are shown.

[0039] Figure 6 The effects of co-overexpression of SLC and PAP genes, co-overexpression of GAT1, SLC and PAP genes, and co-overexpression of GAT1, SLC, PAP and DGAT genes on the production of OPO-type esters by engineered bacteria are shown. Detailed Implementation

[0040] To facilitate understanding of the present invention, it will be described in detail below with reference to the accompanying drawings and embodiments. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0041] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods and materials are now described.

[0042] I. Terminology

[0043] The term "chassis microorganism" used in this invention, also known as "chassis microbial cell," refers to the use of microbial cells as a platform to place functionalized biological systems, enabling the cells to possess the functions required by humans for biosynthesis. Chassis microbial cells need to have simplified functions themselves, but must possess the most basic self-replication and metabolic capabilities, thus becoming a blank platform on which functions can be continuously added.

[0044] In this invention, the term "genetically engineered bacteria" refers to bacteria into a host organism (i.e., host cells, chassis microorganisms, or bacterial cells) to express a target gene and produce the desired protein, such as *Saccharomyces cerevisiae*. The core technology of genetic engineering is DNA recombination technology; therefore, in this invention, genetically engineered bacteria are also referred to as recombinant microorganisms, such as recombinant yeast cells.

[0045] The term "recombination" as used in this invention refers to the process of using the genetic material of a donor organism or artificially synthesized genes, cutting them in vitro or in vitro with restriction enzymes, and then linking them with a suitable vector to form recombinant DNA molecules. These recombinant DNA molecules are then introduced into recipient cells or recipient organisms to construct transgenic organisms, which can then exhibit certain traits of another organism according to a pre-designed blueprint.

[0046] In this invention, the term "endogenous gene" refers to a gene within the genome of a host cell or a cell of the same species as the host cell. For example, in this invention, the host cell used to construct the genetically engineered bacteria is the yeast cell Saccharomyces cerevisiae YS58, and its endogenous genes can come from the yeast cell Saccharomyces cerevisiae YS58.

[0047] In this invention, the term "heterologous gene" refers to a gene from another species or cell introduced into the host bacterium through genetic engineering during the construction of genetically engineered bacteria. It can also be a gene that has been artificially optimized, modified, or synthesized.

[0048] In this invention, the term "expression" refers to the expression of genes in metabolic pathways, specifically the expression of their basal promoters.

[0049] In this invention, the term "overexpression" refers to the expression of a gene in a host cell or genetically engineered bacteria using a strong or super-strong promoter.

[0050] In this invention, the term "screening gene" refers to a marker gene, which is a biosynthetic gene with a known function or sequence that serves as a specific marker. In the context of genetic engineering, it is an important marker for recombinant DNA vectors, typically used to verify the success of transformation. In the context of gene localization, it is a tool for marking target genes, usually used to detect the location of target genes in cells. Examples include the recombinant tryptophan biosynthesis gene (TRP) and the recombinant uracil biosynthesis gene (URA3).

[0051] In this invention, the term "water" refers to deionized water, distilled water, or ultrapure water unless otherwise specified or limited.

[0052] II. Implementation Plan

[0053] As mentioned earlier, existing enzymatic synthesis methods for OPO structured esters suffer from problems such as high catalyst costs, poor stability, and limited batch usage. Furthermore, enzymatic catalysis is heavily constrained by raw materials, and factors such as conversion rate and cost also limit the development of structured esters. Therefore, the inventors have conducted extensive research on biological methods for synthesizing OPO structured esters.

[0054] The inventors have noted that, with the continuous development of synthetic biology technology, the pathways and key gene functions related to lipid synthesis within cells have been gradually elucidated in recent years, laying a theoretical foundation for the construction of engineered bacteria that produce structured lipids. For example... Figure 1 As shown, yeast fatty acid synthesis begins with acetyl-CoA carboxylase encoded by ACC1. Through the catalysis of fatty acid synthase FAS and the consumption of NADPH, long-chain acyl-CoA is elongated, leading to the synthesis of triglycerides (TAG), phospholipids, and other fatty acids. Acyl-CoA is then used by fatty acid elongase Elo and fatty acid desaturase Ole1 on the endoplasmic reticulum membrane to synthesize long-chain fatty acids and unsaturated fatty acids, respectively. In the de novo synthesis of triglycerides, acetyl-CoA is catalyzed by acetyl-CoA carboxylase Acc1 to generate malonyl-CoA. Acetyl-CoA and malonyl-CoA then react under the cyclic catalysis of fatty acid synthase FAS to synthesize C16 and C18 acyl-CoA. Acyl-CoA and glycerol-3-phosphate undergo a reaction catalyzed by glycerol-3-phosphoacyltransferase (GPAT) to attach fatty acids to the sn-1 position of glycerol-3-phosphate, generating lysophosphatidic acid. Then, lysophosphatidic acid and one molecule of acyl-CoA are catalyzed by phosphatidyl acyltransferase (SLC) to attach a fatty acid to the sn-2 position, generating phosphatidic acid. Phosphatidic acid is then catalyzed by phosphatidyl phosphatase (PAP) to remove a phosphate group, generating diglyceride. Finally, diglyceride and acyl-CoA are catalyzed by diacylglycerol acyltransferase (DGAT) to generate TAG.

[0055] Based on the above, the inventors have researched, designed, and discovered that by using yeast cells as the expression host, expressing the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase and overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase and / or the SLC gene encoding phosphatidyltransferase and / or the PAP gene encoding phosphatidylphosphatase and / or the DGAT gene encoding diacylglycerol acyltransferase, a genetically engineered strain of *Saccharomyces cerevisiae* can be constructed. This effectively increases the proportion of OPO-type structural esters in the lipids of *Saccharomyces cerevisiae*, thereby enabling the construction of a highly efficient OPO-type structural ester-producing genetically engineered strain, thus obtaining the present invention.

[0056] Therefore, the genetically engineered bacteria producing OPO-type structural esters provided by this invention are constructed using *Saccharomyces cerevisiae* as the expression host; the yeast cells are selected from the group consisting of the following yeast cells: *Yacinthia lipolytica*, *Rhodotorula rubrum*, *Oleopterinarian*, *Oleopterinarian*, *Rhodotorula glutinis*, *Cryptococcus curvifolius*, *Candida curvifolius*, *Myriophyllum flavum*, *Candida lakowiczianum*, *Candida ferruginosa*, *Candida tropicalis*, *Candida utilis*, *Myriophyllum demersum*, and *Saccharomyces cerevisiae*, preferably *Saccharomyces cerevisiae*; more preferably, *Saccharomyces cerevisiae* YS58 (strain preservation number ST12300, purchased from Beijing Keruisibo Biotechnology Co., Ltd.) is used as the expression host in this invention.

[0057] In embodiments of the present invention, the genetically engineered bacteria that produce OPO-type structural esters are recombinant yeast cells containing the ChDes9-2 gene (referred to as the ChDes9-2 gene) encoding stearoyl-CoAΔ9 desaturase. In the present invention, they are also referred to as the initial genetically engineered bacteria that produce OPO-type structural esters or the initial recombinant yeast cells that produce OPO-type structural esters.

[0058] In this invention, the ChDes9-2 gene, which encodes stearoyl-CoAΔ9 desaturase, is a key gene for maintaining the C18:1 anabolic pathway. In yeast cells, the expression of the ChDes9-2 gene, which encodes stearoyl-CoAΔ9 desaturase using C18:0 as a substrate, can maintain the C18:1 anabolic pathway.

[0059] Those skilled in the art should understand that the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase is a gene not found in the chassis strain (i.e., the host cell, Saccharomyces cerevisiae) used; in this invention, the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase is derived from Daphnia davidii, and its nucleotide sequence is shown in positions 1045-2085 of SEQ ID No. 1, and it is expressed in genetically engineered bacteria.

[0060] In some embodiments of the present invention, the genetically engineered bacteria are recombinant yeast cells with modified chassis.

[0061] Specifically, the chassis modification includes inhibiting the expression of the endogenous OLE1 gene (referred to as the OLE1 gene) encoding Δ9 desaturase, which uses C16:0 and C18:0 as substrates, thereby inhibiting the C16:1 anabolic pathway of Saccharomyces cerevisiae itself; preferably, the inhibition of the C16:1 anabolic pathway is the knockout of the OLE1 gene encoding Δ9 desaturase.

[0062] In some preferred embodiments of the present invention, the OLE1 gene encoding Δ9 desaturase in the genetically engineered bacteria is replaced by the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase, thereby inhibiting the expression of the endogenous OLE1 gene encoding Δ9 desaturase with C16:0 and C18:0 as substrates, while expressing the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase with C18:0 as substrate, increasing the fatty acid ratio of C18:1 and C16:0 in the recombinant bacteria; in the present invention, the corresponding recombinant Saccharomyces cerevisiae cell is referred to as recombinant yeast cell A producing OPO-type structural esters.

[0063] In some preferred embodiments of the present invention, the replacement of the OLE1 gene encoding Δ9 desaturase with the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase is achieved through homologous recombination technology. By introducing the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase from the Arctic water flea in the form of a recombinant DNA fragment into the oil-producing Saccharomyces cerevisiae, the OLE1 gene encoding Δ9 desaturase in the Saccharomyces cerevisiae is replaced with the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase, inhibiting the C16:1 anabolic pathway while maintaining the C18:1 anabolic pathway, which is beneficial for the accumulation of C16:0 and C18:1. In this invention, the nucleotide sequence of the recombinant DNA fragment containing the ChDes9-2 gene is shown in SEQ ID No. 1.

[0064] Positions 1-60 of SEQ ID No. 1 are the upstream homologous arm of OLE1, positions 61-1044 are the PGK1 promoter, positions 1045-2085 are the ChDes9-2 gene, positions 2086-2250 are the ADH1 terminator, positions 2251-3206 are the TRP selection gene, and positions 3207-3266 are the downstream homologous arm of OLE1.

[0065] In order to improve the OPO-type structural ester production capacity of the initial recombinant yeast, in some preferred embodiments of the present invention, the lipid or lipid precursor synthesis pathway of the obtained initial recombinant yeast cells producing OPO-type structural esters is optimized to obtain recombinant yeast cells with optimized lipid or lipid precursor synthesis pathways, which are also referred to in the present invention as recombinant yeast cells producing OPO-type structural esters with optimized lipid or lipid precursor synthesis pathways.

[0066] Preferably, in this invention, lipid or lipid precursor synthesis pathways are optimized by overexpressing one or more of the following genes in recombinant yeast cells: the GAT1 gene (GAT1 gene) encoding glycerol-3-phosphoacyltransferase, the SLC gene (SLC gene) encoding phosphatidyltransferase, the PAP gene (PAP gene) encoding phosphatidylphosphatase, and the DGAT gene (DGAT gene) encoding diacylglycerol acyltransferase; more preferably, the lipid or lipid precursor is a triglyceride or a fatty acid; even more preferably, the fatty acid includes one or more of C16:0, C16:1, C18:0, and C18:1.

[0067] Those skilled in the art should understand that, therefore, the fatty acid composition of *Saccharomyces cerevisiae* must first be regulated in order to regulate triglycerides. Thus, the lipids or lipid precursors described herein comprise both triglycerides and fatty acids. The triglycerides referred to here are mixed triglycerides found in *Saccharomyces cerevisiae*, including OPO-type structured lipids.

[0068] In this invention, the nucleotide sequence of the GAT1 gene encoding glycerol-3-phosphoacyltransferase is shown in SEQ ID No. 2; the nucleotide sequence of the SLC gene encoding phosphatidyltransferase is shown in SEQ ID No. 3; the nucleotide sequence of the PAP gene encoding phosphatidylphosphatase is shown in SEQ ID No. 4; and the nucleotide sequence of the DGAT gene encoding diacylglycerol acyltransferase is shown in SEQ ID No. 5.

[0069] In some specific preferred embodiments of the present invention, the optimization of the lipid or lipid precursor synthesis pathway is to overexpress the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, or the DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cells; in the present invention, the corresponding recombinant Saccharomyces cerevisiae cells are referred to as recombinant yeast cells B that produce OPO-type structural esters.

[0070] In some specific preferred embodiments of the present invention, the optimization of the lipid or lipid precursor synthesis pathway is to overexpress the SLC gene encoding phosphatidyl acyltransferase and the PAP gene encoding phosphatidyl phosphatase in recombinant yeast cells; in the present invention, the corresponding recombinant Saccharomyces cerevisiae cells are referred to as recombinant yeast cells C that produce OPO-type structural esters.

[0071] In some specific preferred embodiments of the present invention, the optimization of the lipid or lipid precursor synthesis pathway is to overexpress the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, and the PAP gene encoding phosphatidylphosphatase in recombinant yeast cells; in the present invention, the corresponding recombinant Saccharomyces cerevisiae cells are referred to as recombinant yeast cells D that produce OPO-type structural esters.

[0072] In some specific preferred embodiments of the present invention, the optimization of the lipid or lipid precursor synthesis pathway is to overexpress the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, and the DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cells; in the present invention, the corresponding recombinant Saccharomyces cerevisiae cells are referred to as recombinant yeast cells E that produce OPO-type structural esters.

[0073] Based on the above, it is easy to understand that the recombinant yeast cells B to E that produce OPO-type structural esters are obtained by optimizing the lipid or lipid precursor synthesis pathway of the recombinant yeast cell A that produces OPO-type structural esters.

[0074] The results show that the genetically engineered bacteria that produce OPO-type structural esters provided by this invention, after fermentation culture, contain more than 10% lipids in the dry weight of yeast cells.

[0075] To achieve the above implementation scheme, the present invention also provides a method for constructing a genetically engineered bacterium producing OPO-type structural esters as described in the embodiments of the present invention, comprising:

[0076] Step (A): Construct recombinant yeast cell A containing the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase;

[0077] Step (Z) optimizes lipid or lipid precursor synthesis pathways.

[0078] According to some embodiments of the present invention, step (A) includes:

[0079] Step M: Express the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase in yeast cells;

[0080] Step N: Knock out the OLE1 gene encoding Δ9 desaturase from yeast cells;

[0081] In some preferred embodiments of the present invention, step (A) includes replacing the OLE1 gene encoding Δ9 desaturase with the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase to construct a recombinant yeast cell A containing the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase (also referred to in the present invention as an OPO-type structural ester-producing genetically engineered bacterium A).

[0082] Specifically, in step (A), the expression of the endogenous OLE1 gene encoding Δ9 desaturase with C16:0 and C18:0 substrates is suppressed, while the expression of stearoyl-CoA Δ9 desaturase ChDes9-2 with C18:0 substrate can be achieved through gene substitution via homologous recombination.

[0083] The nucleotide sequence of the gene encoding the stearoyl-CoAΔ9 desaturase ChDes9-2 with C18:0 as substrate is shown in positions 1045-2085 of SEQ ID No. 1.

[0084] In a specific embodiment of the present invention, this is achieved through homologous recombination technology. By introducing the Δ9 desaturase ChDes9-2 gene from the Arctic water flea into the oil-producing Saccharomyces cerevisiae in the form of a recombinant DNA fragment, the OLE1 gene of Saccharomyces cerevisiae is replaced by the ChDes9-2 gene, inhibiting the C16:1 anabolic pathway while maintaining the C18:1 anabolic pathway, which is beneficial for the accumulation of C16:0 and C18:1. The nucleotide sequence of the recombinant DNA fragment is shown in SEQ ID No. 1.

[0085] Positions 1-60 of SEQ ID No. 1 are the upstream homologous arm of OLE1, positions 61-1044 are the PGK1 promoter, positions 1045-2085 are the ChDes9-2 gene, positions 2086-2250 are the ADH1 terminator, positions 2251-3206 are the TRP selection gene, and positions 3207-3266 are the downstream homologous arm of OLE1.

[0086] According to other embodiments of the present invention, step (Z) includes:

[0087] Step (B): Overexpress the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, or the DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cells to obtain recombinant yeast cells B.

[0088] Alternatively, in step (C), the SLC gene encoding phosphatidyl transferase and the PAP gene encoding phosphatidyl phosphatase are overexpressed in recombinant yeast cells to obtain recombinant yeast cells C.

[0089] Alternatively, in step (D), recombinant yeast cells D are obtained by overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, and the PAP gene encoding phosphatidylphosphatase in recombinant yeast cells.

[0090] Alternatively, in step (E), recombinant yeast cells E are obtained by overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, and the DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cells.

[0091] Further, in step (B), overexpression of the GAT1 gene encoding glycerol-3-phosphoacyltransferase in *Saccharomyces cerevisiae* can be achieved by introducing the encoding gene of the GAT1 gene into *Saccharomyces cerevisiae*; overexpression of the SLC gene encoding phosphatidyltransferase in *Saccharomyces cerevisiae* can be achieved by introducing the encoding gene of the SLC gene into *Saccharomyces cerevisiae*; overexpression of the PAP gene encoding phosphatidylphosphatase in *Saccharomyces cerevisiae* can be achieved by introducing the encoding gene of the PAP gene into *Saccharomyces cerevisiae*; and overexpression of the DGAT gene encoding diacylglycerol acyltransferase in *Saccharomyces cerevisiae* can be achieved by introducing the encoding gene of the DGAT gene into *Saccharomyces cerevisiae*.

[0092] The nucleotide sequence of the GAT1 gene encoding glycerol-3-phosphoacyltransferase in *Saccharomyces cerevisiae* is shown in SEQ ID No. 2; the nucleotide sequence of the SLC gene encoding phosphatidyltransferase in *Saccharomyces cerevisiae* is shown in SEQ ID No. 3; the nucleotide sequence of the PAP gene encoding phosphatidylphosphatase in *Saccharomyces cerevisiae* is shown in SEQ ID No. 4; and the nucleotide sequence of the DGAT gene encoding diacylglycerol acyltransferase in *Saccharomyces cerevisiae* is shown in SEQ ID No. 5.

[0093] In a specific embodiment of the present invention, overexpression of the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, or the DGAT gene encoding diacylglycerol acyltransferase is achieved by constructing a plasmid with a strong promoter to drive the expression of the target gene. Using the YS58 genome of *Saccharomyces cerevisiae* as a template, the GAT1 gene fragment was amplified by PCR. The GAT1 gene fragment was ligated into plasmid pSP-GM2 to obtain plasmid pSP-GM2-GAT1. Plasmid pSP-GM2-GAT1 was then transformed into the strain obtained in step (A) using lithium acetate conversion. The nucleotide sequence of GAT1 is shown in SEQ ID No. 3. Using the YS58 genome of *Saccharomyces cerevisiae* as a template, the SLC gene fragment was amplified by PCR. The SLC gene fragment was ligated into plasmid pSP-GM2 to obtain plasmid pSP-GM2-SLC. Plasmid pSP-GM2-SLC was then transformed into the strain obtained in step (A) using lithium acetate conversion. The nucleotide sequence of SLC is shown in SEQ ID No. 3. As shown in No. 4; using the genome of Saccharomyces cerevisiae YS58 as a template, PCR amplification was performed to obtain the PAP gene fragment. The PAP gene fragment was ligated to plasmid pSP-GM2 to obtain plasmid pSP-GM2-PAP. Plasmid pSP-GM2-PAP was then transferred into the strain obtained in step (A) using lithium acetate conversion. The nucleotide sequence of the PAP is shown in SEQ ID No. 5.

[0094] In step (C), the simultaneous overexpression of the SLC gene encoding phosphatidyl transferase and the PAP gene encoding phosphatidyl phosphatase in the Saccharomyces cerevisiae can be achieved by simultaneously introducing the encoding genes of the SLC gene encoding phosphatidyl transferase and the PAP gene encoding phosphatidyl phosphatase into the Saccharomyces cerevisiae.

[0095] In a specific embodiment of the present invention, overexpression of the SLC gene encoding phosphatidyl transferase and the PAP gene encoding phosphatidyl phosphatase is achieved by constructing a plasmid with a strong promoter driving the expression of the target gene. Using the genome of *Saccharomyces cerevisiae* YS58 as a template, the PAP gene fragment is amplified by PCR and ligated into the plasmid pSP-GM2-SLC obtained in step (B), resulting in plasmid pSP-GM2-SLC-PAP. Plasmid pSP-GM2-SLC-PAP is then transformed into the bacterial strain obtained in step (A) using lithium acetate conversion. The nucleotide sequence of the SLC gene is shown in SEQ ID No. 3; the nucleotide sequence of the gene encoding the PAP gene is shown in SEQ ID No. 4.

[0096] In step (D), the simultaneous overexpression of the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, and the PAP gene encoding phosphatidylphosphatase in the Saccharomyces cerevisiae can be achieved by simultaneously introducing the encoding genes of the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, and the PAP gene encoding phosphatidylphosphatase into the Saccharomyces cerevisiae.

[0097] In a specific embodiment of the present invention, overexpression of the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, and the PAP gene encoding phosphatidylphosphatase is achieved by constructing plasmids with strong promoters driving the expression of the target genes. Using the genome of *Saccharomyces cerevisiae* YS58 as a template, the GAT1 gene fragment was amplified by PCR and ligated to the pSP-GM2-SLC-PAP plasmid obtained in step (C), resulting in the plasmid pSP-GM2-GAT1-SLC-PAP. The obtained plasmid pSP-GM2-GAT1-SLC-PAP was then transformed into the bacterial strain obtained in step (A) using lithium acetate conversion. The nucleotide sequence of GAT1 is shown in SEQ ID No. 2; the nucleotide sequence of SLC is shown in SEQ ID No. 3; and the nucleotide sequence of PAP is shown in SEQ ID No. 4.

[0098] In step (E), the simultaneous overexpression of the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, and the DGAT gene encoding diacylglycerol acyltransferase in the *Saccharomyces cerevisiae* can be achieved by simultaneously introducing the encoding genes of the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, and the DGAT gene encoding diacylglycerol acyltransferase into the *Saccharomyces cerevisiae*.

[0099] In a specific embodiment of the present invention, overexpression of the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, and the DGAT gene encoding diacylglycerol acyltransferase is achieved by constructing plasmids with strong promoters driving the expression of the target genes. Using the genome of *Saccharomyces cerevisiae* YS58 as a template, the DGAT gene fragment was amplified by PCR and ligated into the pSP-GM2-GAT1-SLC-PAP plasmid obtained in step (D) to obtain the plasmid pSP-GM2-GAT1-SLC-PAP-DGAT. The obtained plasmid pSP-GM2-GAT1-SLC-PAP-DGAT was then transformed into the strain obtained in step (A) using lithium acetate conversion. The nucleotide sequence of GAT1 is shown in SEQ ID No. 2; the nucleotide sequence of SLC is shown in SEQ ID No. 3; the nucleotide sequence of PAP is shown in SEQ ID No. 4; and the nucleotide sequence of DGAT is shown in SEQ ID No. 5.

[0100] In some specific preferred embodiments of the present invention, the method for constructing the OPO-type structural ester-producing genetically engineered bacteria comprising step (A) can be understood as a method for increasing the proportion of C16:0 and C18:1 fatty acids in Saccharomyces cerevisiae. This method involves replacing the OLE1 gene encoding Δ9 desaturase with the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase according to the method in step (A), thereby constructing a recombinant yeast cell A (also referred to in this invention as the OPO-type structural ester-producing genetically engineered bacteria A) containing the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase. This increases the proportion of C16:0 and C18:1 fatty acids in Saccharomyces cerevisiae.

[0101] In some other preferred embodiments of the present invention, the method for constructing the OPO-type structural ester-producing genetically engineered bacteria comprising steps (A) and S2 can be understood as a method for increasing the proportion of fatty acids C16:0 and C18:1 in Saccharomyces cerevisiae. First, the method in step (A) is followed to replace the OLE1 gene encoding Δ9 desaturase with the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase, thereby modifying the oil-producing Saccharomyces cerevisiae to increase the proportion of fatty acids C16:0 and C18:1 in Saccharomyces cerevisiae. Then, based on this, the method is followed to modify the bacteria according to steps (B), (C), (D), and (E) to construct the OPO-type structural ester-producing genetically engineered bacteria and increase the proportion of fatty acids C16:0 and C18:1 in Saccharomyces cerevisiae.

[0102] As can be seen from the above, the genetically engineered strain producing OPO-type structural esters provided by the present invention is obtained by first modifying the initial genetically engineered strain A in Saccharomyces cerevisiae using step (A), and then further modifying it using steps (B) and / or (C) and / or (D) and / or (E): wherein, step (A) inhibits the expression of the endogenous OLE1 gene encoding Δ9 desaturase with C16:0 and C18:0 as substrates, while expressing the stearoyl-CoA Δ9 desaturase ChDes9-2 with C18:0 as substrate; step (B) while expressing the stearoyl-CoA Δ9 desaturase ChDes9-2 with C18:0 as substrate, overexpresses the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, or the DG gene encoding diacylglycerol acyltransferase in Saccharomyces cerevisiae. AT gene; Step (C) While expressing stearoyl-CoAΔ9 desaturase ChDes9-2 with C18:0 substrate, overexpress the SLC gene encoding phosphatidyl transferase and the PAP gene encoding phosphatidyl phosphatase from *Saccharomyces cerevisiae*; Step (D) While expressing stearoyl-CoAΔ9 desaturase ChDes9-2 with C18:0 substrate, overexpress the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyl transferase, and the PAP gene encoding phosphatidyl phosphatase from *Saccharomyces cerevisiae*; Step (E) While expressing stearoyl-CoAΔ9 desaturase ChDes9-2 with C18:0 substrate, overexpress the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyl transferase, the PAP gene encoding phosphatidyl phosphatase, and the DGAT gene encoding diacylglycerol acyltransferase from *Saccharomyces cerevisiae*. This invention increases the content of OPO-type structural esters in brewer's yeast, overcoming the limitations of OPO-type structural ester sources.

[0103] Those skilled in the art should understand that the steps (A) to (B), (C), (D) or (E) of constructing the genetically engineered bacteria that produce OPO-type structural esters can also be understood as genetic modification of the host organism. Therefore, in this invention, the genetically engineered bacteria that produce OPO-type structural esters are also referred to as genetically modified yeast cells that produce OPO-type structural esters.

[0104] The gene modification is an engineered gene modification that includes the regulation of protein expression, specifically, the gene modification increases or decreases the expression of the protein.

[0105] For example, the genetically modified yeast cell A obtained after step (A), which is otherwise identical to a yeast cell lacking the genetic modification, has the following genetic modification: the genetic modification increases the activity level of stearoyl-CoA Δ9 desaturase (ChDes9-2) protein and decreases the activity level of Δ9 desaturase (OLE1) protein with C16:0 and C18:0 substrates.

[0106] The genetically modified yeast cell B obtained after step (B), which is otherwise identical to the yeast cell lacking the genetic modification, has increased the protein activity levels of glycerol-3-phosphoacyltransferase (GAT1), phosphatidyltransferase (SLC), phosphatidylphosphatase (PAP), or diacylglycerol acyltransferase (DGAT).

[0107] The genetically modified yeast cell C obtained after step (C), which is otherwise identical to the yeast cell lacking the genetic modification, has increased the activity levels of phosphatidyl acyltransferase (SLC) and phosphatidyl phosphatase (PAP) proteins.

[0108] The genetically modified yeast cell D obtained after step (D), which is otherwise identical to the yeast cell lacking the genetic modification, has increased the activity levels of glycerol-3-phosphoacyltransferase (GAT1), phosphatidyltransferase (SLC), and phosphatidylphosphatase (PAP) proteins.

[0109] The genetically modified yeast cell E obtained after step (E), which is otherwise identical to the yeast cell lacking the genetic modification, has increased the protein activity levels of glycerol-3-phosphoacyltransferase (GAT1), phosphatidyltransferase (SLC), phosphatidylphosphatase (PAP), and diacylglycerol acyltransferase (DGAT).

[0110] Those skilled in the art should also understand that the above-mentioned OPO-type structural ester-producing gene-modified yeast cells B to E are all obtained by optimizing the lipid or lipid precursor synthesis pathway of gene-modified yeast cell A. Therefore, compared with yeast cells that are otherwise identical but lack the gene modification, the gene modification of the OPO-type structural ester-producing gene-modified yeast cells B to E increases the activity level of stearoyl-CoA Δ9 desaturase (ChDes9-2) protein and decreases the activity level of Δ9 desaturase (OLE1) protein with C16:0 and C18:0 as substrates.

[0111] The results show that the genetically engineered bacteria that produce OPO-type structural esters provided by this invention, after fermentation culture, have a dry weight of more than 10% lipids in the yeast cells.

[0112] The application of the genetically engineered bacteria or the genetically engineered bacteria constructed by the above-mentioned method in the production of OPO-type structural esters can be understood as a method for producing OPO-type structural esters using the genetically engineered bacteria or the genetically engineered bacteria constructed by the above-mentioned method. It can also be further understood as a method for preparing or producing C16:0 and C18:1 using the genetically engineered bacteria or the genetically engineered bacteria constructed by the above-mentioned method.

[0113] Based on the foregoing, it is not difficult to understand that the method for constructing OPO-type structural ester-producing genetically engineered bacteria provided by the present invention can also be understood as the application of the OPO-type structural ester-producing genetically engineered bacteria described in the above embodiments of the present invention in increasing the proportion of C16:0 and C18:1 in the fatty acids of Saccharomyces cerevisiae.

[0114] Alternatively, the application of the genetically engineered bacteria producing OPO-type structural esters described in the embodiments of the present invention in increasing the proportion of fatty acids at the sn-2 position of C16:0 in triglycerides of Saccharomyces cerevisiae.

[0115] Alternatively, the application of the genetically engineered bacteria that produce OPO-type structural esters as described in the embodiments of the present invention in increasing the proportion of fatty acids at the sn-1, 3 position of C18:1 in triglycerides of Saccharomyces cerevisiae.

[0116] Alternatively, the application of the genetically engineered bacteria that produce OPO-type structural esters as described in the embodiments of the present invention in the preparation or production of OPO-type structural esters.

[0117] In some embodiments of the present invention, the genetically engineered bacteria are inoculated into a fermentation medium for fermentation culture, and then the obtained fermentation culture is separated and purified to obtain an OPO-type structural ester.

[0118] Specifically, the process of inoculating the genetically engineered bacteria into the fermentation medium and carrying out fermentation culture includes inoculating the engineered bacteria provided by the present invention into the YNB auxotrophic medium, activating it by shaking in a flask at 30°C and 200 rpm to obtain a seed culture; then inoculating the seed culture into the YNB auxotrophic medium and culturing it in a flask at 30°C and 200 rpm for 72 h, and collecting the fermentation product.

[0119] In some specific embodiments of the present invention, the method for preparing or producing C16:0 and C18:1 using the above-described genetically engineered bacteria or the genetically engineered bacteria constructed by the above-described method includes the following steps:

[0120] (1) The recombinant bacteria described in the first aspect above were fermented in YNB auxotrophic medium, and the fermentation products were collected.

[0121] In a specific embodiment of the present invention, this step specifically involves: inoculating the engineered bacteria into the YNB auxotrophic medium, culturing and activating it in a shake flask at 30°C and 200 rpm to obtain a seed culture; and then inoculating the seed culture into the YNB auxotrophic medium and culturing it in a shake flask at 30°C and 200 rpm for 72 hours.

[0122] (2) The fatty acids in the fermentation product are extracted by saponification to obtain C16:0 and C18:1.

[0123] In a specific embodiment of the present invention, this step is as follows: the fermentation product is placed in a methanol solution containing 10% (volume percentage) KOH; then refluxed at 80°C for 2 hours, the refluxed liquid is collected to obtain fatty acid salts, and fatty acids are obtained after acidification with hydrochloric acid (4 mL of 6M hydrochloric acid is added).

[0124] In other specific embodiments of the present invention, the method for preparing or producing OPO-type structural esters using the above-described genetically engineered bacteria or the genetically engineered bacteria constructed by the above-described method includes the following steps:

[0125] (1) Ferment the engineered bacteria described in the first aspect above in YNB auxotrophic medium and collect the fermentation products.

[0126] In a specific embodiment of the present invention, this step is as follows: the engineered bacteria are inoculated into the YNB auxotrophic medium and activated by shaking in a flask at 30°C and 200 rpm to obtain a seed culture; then the seed culture is inoculated into the YNB auxotrophic medium and cultured in a shaking flask at 30°C and 200 rpm for 72 h, and the fermentation product is collected.

[0127] (2) Extraction method is used to extract the oil from the fermentation product to obtain OPO type structure ester.

[0128] In a specific embodiment of the present invention, this step is as follows: the collected bacterial cells are repeatedly frozen and thawed four times, and then ultrasonically disrupted. The disrupted bacterial cell suspension is placed in a chloroform-methanol (v:v = 2:1) solution, shaken to mix, and the lower layer solution is collected and rotary evaporated to obtain the oil. The obtained oil is dissolved in diethyl ether, and triglycerides are separated by thin-layer chromatography using hexane:diethyl ether:glacial acetic acid (50:50:1, v / v / v) as the developing solvent.

[0129] III. Correlation Analysis and Detection Methods

[0130] (1) Detection of triglyceride fatty acid composition.

[0131] The fatty acid composition was determined in accordance with GB / T 17376—2008 "Preparation of Fatty Acid Methyl Esters from Animal and Vegetable Oils" and GB / T17377—2008 "Gas Chromatographic Analysis of Fatty Acid Methyl Esters from Animal and Vegetable Oils".

[0132] (2) Triglyceride structure analysis.

[0133] The determination of the Sn-2 fatty acid composition was carried out in accordance with GB / T 24894—2010 "Determination of the 2-position fatty acid composition of triglycerides in animal and vegetable oils". Based on the Sn-1,3-random-2-random distribution theory, the content of Sn-1 and 3 fatty acids in Saccharomyces cerevisiae and the composition and content of triglycerides in Saccharomyces cerevisiae were calculated and analyzed.

[0134] In this invention, in *Saccharomyces cerevisiae* YS58, the endogenous OLE1 gene is first replaced with the ChDes9-2 gene. This maintains the C18:1 anabolic pathway in *Saccharomyces cerevisiae* while inhibiting the C16:1 anabolic pathway, thereby increasing the proportion of C16:0 and C18:1 fatty acids in *Saccharomyces cerevisiae*. Based on this, the endogenous GAT1 and / or SLC and / or PAP and / or DGAT genes in *Saccharomyces cerevisiae* are overexpressed to construct engineered *Saccharomyces cerevisiae* strains, increasing the proportion of OPO-type esters in the yeast's lipid content.

[0135] IV. Examples

[0136] The present invention will be specifically described below through specific embodiments. Unless otherwise specified, the experimental methods described below are conventional laboratory methods using conventional experimental equipment. Unless otherwise specified, the experimental materials described below are all commercially available.

[0137] The culture media used in the following examples are as follows, where all percentages are by mass:

[0138] The final concentration of LB medium is: 0.5% yeast extract, 1% peptone, 0.5% sodium chloride, and 1.7% agar (for solid media), in deionized water, with a natural pH. All percentages are in g / 100ml.

[0139] The final concentration of YPD medium is: 1% yeast extract, 2% peptone, 2% glucose, and 1.7% agar (for solid media), in deionized water, with a natural pH. All percentages are expressed in g / 100ml.

[0140] The final composition of the YNB-Trp fermentation medium was: 0.17% amino-free yeast nitrogen source (Shanghai Yuanye Biotechnology Co., Ltd., S24483), 0.5% ammonium sulfate, 0.129% Dropout Supplement-TRP (Beijing Coollab Technology Co., Ltd., PM2270), 2% glucose, in deionized water, pH 6.0. The addition of DS amino acids was determined by the selection markers of the expression vector. For solid culture media, 1.7% agar was added to the above components. All percentages represent g / 100ml.

[0141] The final composition of the YNB-Ura / Trp fermentation medium was: 0.17% amino-free yeast nitrogen source (Shanghai Yuanye Biotechnology Co., Ltd., S24483), 0.5% ammonium sulfate, 0.12% Dropout Supplement-Ura / Trp (Beijing Coollab Technology Co., Ltd., PM2270), 2% glucose, in deionized water, pH 6.0. The addition of DS amino acids was determined by the selection markers of the expression vector. For solid culture media, 1.7% agar was added to the above components. All percentages represent g / 100ml.

[0142] In the following examples: the nucleotide sequence of ChDes9-2 is positions 1045-2085 of SEQ ID No. 1; the nucleotide sequence of the GAT1 gene encoding glycerol-3-phosphoacyltransferase is SEQ ID No. 2; the nucleotide sequence of the SLC gene encoding phosphatidyltransferase is SEQ ID No. 3; the nucleotide sequence of the PAP gene encoding phosphatidylphosphatase is SEQ ID No. 4; and the nucleotide sequence of the DGAT gene encoding diacylglycerol acyltransferase is SEQ ID No. 5.

[0143] Example 1: Construction of ChDes9-2 engineered bacteria and analysis of its fatty acid and OPO-type ester content

[0144] 1. Construction of ChDes9-2 engineered bacteria

[0145] (1) Design of ChDes9-2 recombination box

[0146] The location of the OLE1 gene on the chromosome was located using the NCBI website. Based on the specific location of the OLE1 gene sequence, an upstream homologous arm was designed 60 bp upstream of OLE1, and a downstream homologous arm was designed 60 bp downstream of OLE1. The Δ9 desaturase gene ChDes9-2 is from the Arctic water flea and is not easily obtained; therefore, the gene sequence of ChDes9-2 and its promoter P were searched on NCBI. PGK Termination of sub-T ADH1 The upstream and downstream 60bp homologous arms of the OLE1 gene and the TRP sequence of the screening gene were sent to Suzhou Genewiz Company for direct synthesis of the gene fragment ChDes9-2 recombination cassette.

[0147] The nucleotide sequence of the ChDes9-2 recombinant cassette is SEQ ID No. 1. Positions 1-60 are the upstream homologous arm of OLE1, positions 61-1044 are the PGK1 promoter, positions 1045-2085 are the ChDes9-2 gene, positions 2086-2250 are the ADH1 terminator, positions 2251-3206 are the TRP selector gene, and positions 3207-3266 are the downstream homologous arm of OLE1.

[0148] (2) Construction of ChDes9-2 engineered bacteria

[0149] The ChDes9-2 recombinant cassette gene fragment described above was introduced into *Saccharomyces cerevisiae* strain YS58 using the lithium acetate conversion method, and then screened and expressed. Details are as follows:

[0150] The host strain used was *Saccharomyces cerevisiae* YS58 (strain preservation number ST12300, purchased from Beijing Keruisibo Biotechnology Co., Ltd.). Yeast transformation was performed using a classic yeast transformation kit (Beijing Coollab Technology Co., Ltd., SK2400). YPD medium was activated twice and cultured to the logarithmic growth phase for preparing competent cells. Cells were collected by freezing and centrifugation, washed twice with water, and then resuspended in 1 / 10 concentration LiAc. A premix was prepared: 240 μl PEG Solution (Beijing Coollab Technology Co., Ltd. SL9510), 36 μl LiAc Solution (Beijing Coollab Technology Co., Ltd. YT0002), 10 μl Carrier DNA (Beijing Coollab Technology Co., Ltd. YT0003), and 5 μl fragment (from the above ChDes9-2 recombinant kit). The mixture was then diluted with sterile water to a final volume of 360 μl. The premixed solution was injected into YS58 competent cells and incubated in water baths at 30℃ and 42℃ for 30 minutes in sequence. After centrifugation, the cells were resuspended in sterile water and spread onto TRP defective selection medium (Beijing Lanboster Biotechnology Co., Ltd. PM2251). Positive transformants were screened after incubation at 30℃ for 2-4 days.

[0151] 2. Analysis of fatty acids and OPO-type ester structures in ChDes9-2 engineered bacteria

[0152] (1) Culture of engineered bacteria

[0153] Seed culture preparation: The original strain YS58 was inoculated into YPD medium, and the ChDes9-2 genetically engineered strain was inoculated into YNB-Trp auxotrophic medium. The cultures were incubated in a constant temperature shaker at 200 rpm and 30℃ for 48 h. After activation culture, the seed culture was obtained.

[0154] Fermentation culture: The YS58 seed culture obtained from activation culture was inoculated into YPD medium, and the ChDes9-2 engineered bacterial seed culture obtained from repeated activation was inoculated into YNB-Trp auxotrophic medium. Strain YS58 was used as the control strain. Fermentation was carried out at 30℃ and 200rpm in shake flasks for 72h to obtain the fermentation products of YS58 and ChDes9-2.

[0155] (2) Extraction of microbial fatty acids

[0156] The saponification method was selected to extract fatty acids from yeast. Details are as follows:

[0157] The fermentation products of the above strains were centrifuged at 6000g for 5 min to collect the cell cells, which were then washed twice with water. 15 mL of a 10% (v / v) KOH-methanol solution (composed of 10 g / 100 mL KOH, 95% methanol, and 5% deionized water) was added and transferred to a ground-glass conical flask. The flask was then placed in an 80℃ water bath (Wuhan Andexin ADX-HH-4, China) for reflux for 2 hours to allow the strong alkali to saponify the intracellular esters. The reflux liquid was collected to obtain fatty acid salts, which were then acidified with hydrochloric acid (4 mL of 6M hydrochloric acid) to obtain fatty acids.

[0158] (3) Treatment of bacterial fatty acids

[0159] Add 4 mL of 1% (volume percentage) sulfuric acid-methanol solution to the fatty acid obtained by saponification in step 2 above, mix well, transfer to a clean stoppered test tube, and react in a water bath at 60°C for 1 h; cool to room temperature, add 5 mL of saturated NaCl solution to precipitate proteins and other impurities; then add 1 mL of n-hexane, collect the organic phase, which is the extracted fatty acid methyl ester; remove the moisture from the sample with anhydrous sodium sulfate.

[0160] (4) Extraction of triglycerides

[0161] Extraction and thin-layer chromatography were used to extract yeast triglycerides. Details are as follows:

[0162] The fermentation products of the above strains were centrifuged at 6000g for 5 minutes to collect the bacterial cells, which were then washed twice with water. The collected bacterial cells were repeatedly freeze-thawed and then ultrasonically disrupted (Kunshan Ultrasonic Instrument Co., Ltd. KQ-500DE, China). The disrupted bacterial suspension was placed in twice the volume of chloroform-methanol (v:v = 2:1) solution, shaken to mix, and the lower layer solution was collected and rotary evaporated to obtain the oil. The obtained oil was dissolved in diethyl ether, and the triglycerides were separated by thin-layer chromatography using n-hexane:diethyl ether:glacial acetic acid (50:50:1, v / v / v) as the developing solvent.

[0163] (5) Triglyceride fatty acid composition

[0164] The fatty acid composition was determined in accordance with GB / T 17376—2008 "Preparation of Fatty Acid Methyl Esters from Animal and Vegetable Oils" and GB / T17377—2008 "Gas Chromatographic Analysis of Fatty Acid Methyl Esters from Animal and Vegetable Oils".

[0165] (6) Triglyceride analysis

[0166] The determination of the Sn-2 fatty acid composition was carried out in accordance with GB / T 24894—2010 "Determination of the 2-position fatty acid composition of triglycerides in animal and vegetable oils". Based on the Sn-1,3-random-2-random distribution theory, the content of Sn-1 and 3 fatty acids in Saccharomyces cerevisiae and the types and contents of triglycerides in Saccharomyces cerevisiae were calculated and analyzed.

[0167] GC detection of fatty acid methyl esters: The fatty acid methyl esters obtained in steps 3 and 6 above were detected and analyzed by gas chromatography using a DB-WAX column of a Shimadzu gas chromatograph (Shimadzu GC-2014 Japan). High-purity nitrogen was used as the detection carrier. A two-stage temperature program was used to separate the fatty acid methyl esters. The initial column temperature was 62℃, held for 5 min, and the temperature was increased at a rate of 3℃ / min. The column temperature was gradually increased from 60℃ to 215℃, and the retention time was 33 min.

[0168] The standard is a mixed standard of fatty acid methyl esters (Shanghai Yuanye Biotechnology Co., Ltd., B25881).

[0169] The results are as follows Figure 2As shown, the main fatty acids in *Saccharomyces cerevisiae* are C16:0, C16:1, C18:0, and C18:1, with C16:1 and C18:1 being the most abundant. The results show that after replacing the OLE1 gene in strain YS58 with the ChDes9-2 gene, the C16:0 content in strain ChDes9-2 increased by 10%, the C16:1 content decreased by 25%, and the C18:1 content increased by 13% compared to strain YS58. This indicates that replacing the OLE1 gene with the ChDes9-2 gene not only inhibited the synthesis of C16:1 in the cells but also promoted the synthesis and accumulation of C18:1 and C16:0.

[0170] The results are as follows Figure 3 As shown, after replacing the OLE1 gene of the original strain YS58 with the ChDes9-2 gene, the proportion of OPO-type structural esters in the triglycerides of the engineered strain ChDes9-2 increased by 5% compared with the original strain YS58, indicating that the replacement of the OLE1 gene by ChDes9-2 promoted the synthesis of OPO-type structural esters.

[0171] Example 2: Effects of overexpression of the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, or the DGAT gene encoding diacylglycerol acyltransferase in ChDes9-2 engineered bacteria on fatty acid and OPO-type ester content.

[0172] 1. Construction of engineered bacteria overexpressing GAT1, SLC, PAP, or DGAT

[0173] (1) Construct GAT1, SLC, PAP, or DGAT overexpression vectors

[0174] The GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidylacyltransferase, the PAP gene encoding phosphatidylphosphatase, and the DGAT gene encoding diacylglycerol acyltransferase all originated from *Saccharomyces cerevisiae* YS58. Therefore, using the *Saccharomyces cerevisiae* YS58 genome as a template, PCR amplification was performed to obtain the GAT1, SLC, PAP, and DGAT gene fragments. The GAT1 and DGAT gene fragments were then ligated into the plasmid pSP-GM2. https: / / www.addgene.org / The expression vectors pSP-GM2-GAT1 and pSP-GM2-DGAT were obtained by connecting the restriction sites BamHI and KpnI in plasmid pSP-GM2. The SLC and PAP gene fragments were then connected to the restriction sites NotI and SpeI in plasmid pSP-GM2 to obtain the expression vectors pSP-GM2-SLC and pSP-GM2-PAP.

[0175] The nucleotide sequence of the gene GAT1 is shown in SEQ ID No. 3; the nucleotide sequence of the SLC gene encoding phosphatidyl acyltransferase is shown in SEQ ID No. 4; the nucleotide sequence of the gene PAP is shown in SEQ ID No. 5; and the nucleotide sequence of the DGAT gene encoding diacylglycerol acyltransferase is shown in SEQ ID No. 6.

[0176] Table 1. Primers used in this embodiment

[0177]

[0178] (2) Constructing engineered bacteria GAT1-ChDes9-2, SLC-ChDes9-2, PAP-ChDes9-2 and DGAT-ChDes9-2

[0179] The expression plasmids pSP-GM2-GAT1, pSP-GM2-SLC, pSP-GM2-PAP, and pSP-GM2-DGAT were transformed into the host bacteria, namely the ChDes9-2 engineered bacteria obtained in Example 1, to obtain the Saccharomyces cerevisiae engineered bacteria GAT1-ChDes9-2, SLC-ChDes9-2, PAP-ChDes9-2, and DGAT-ChDes9-2.

[0180] 2. Effects of overexpression of GAT1, SLC, PAP, or DGAT genes on the proportion of bacterial fatty acids and OPO-type esters.

[0181] (1) Culture of engineered bacteria

[0182] Seed culture preparation: Recombinant strain ChDes9-2 was inoculated into YNB-Trp auxotrophic medium, and genetically engineered strains GAT1-ChDes9-2, SLC-ChDes9-2, PAP-ChDes9-2, and DGAT-ChDes9-2 were inoculated into YNB-Trp / Ura auxotrophic medium, respectively. The cultures were incubated in a constant temperature shaker at 200 rpm and 30℃ for 48 h. After activation culture, seed culture was obtained.

[0183] Fermentation culture: The ChDes9-2 seed culture obtained from activation culture was inoculated into YNB-Trp auxotrophic medium. The genetically engineered bacterial seed cultures of GAT1-ChDes9-2, SLC-ChDes9-2, PAP-ChDes9-2, and DGAT-ChDes9-2, obtained by repeated activation, were inoculated into YNB-Trp / Ura auxotrophic medium, respectively. Strain ChDes9-2 served as the control strain. Fermentation was carried out at 30℃ and 200 rpm in shake flasks for 72 h to obtain the fermentation products of ChDes9-2, GAT1-ChDes9-2, SLC-ChDes9-2, PAP-ChDes9-2, and DGAT-ChDes9-2.

[0184] (2) Extraction of bacterial fatty acids is carried out in the same manner as in Example 1.

[0185] (3) The treatment of bacterial fatty acids is the same as that in Example 1.

[0186] (4) The extraction of triglycerides was carried out in the same manner as in Example 1.

[0187] (5) The triglyceride fatty acid composition is the same as that in Example 1.

[0188] (6) Triglyceride analysis was performed using the same method as in Example 1.

[0189] The results are as follows Figure 4 As shown, compared with the integrated strain ChDes9-2, the total fatty acid types and contents of engineered strains GAT1-ChDes9-2, SLC-ChDes9-2, PAP-ChDes9-2 and DGAT-ChDes9-2 did not change significantly, indicating that the overexpression of GAT1, SLC or PAP or DGAT genes did not cause significant changes in the total fatty acids of the bacteria.

[0190] The results are as follows Figure 5As shown, compared to the triglyceride composition of the integrative strain ChDes9-2, the proportion of OPO-type structural esters in total triglycerides increased by 3% in the engineered strain GAT1-ChDes9-2. The proportion of OPO-type structural esters in total triglycerides increased by 4% in the engineered strain SLC-ChDes9-2, by 3% in the engineered strain PAP-ChDes9-2, and decreased by 2% in the engineered strain DGAT-ChDes9-2. This indicates that the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidylacyltransferase, and the PAP gene encoding phosphatidylphosphatase in *Saccharomyces cerevisiae* can promote the synthesis of OPO-type structural esters, with the SLC gene encoding phosphatidylacyltransferase having the greatest effect on OPO-type structural ester synthesis. The DGAT gene encoding diacylglycerol acyltransferase has a certain inhibitory effect on the synthesis of OPO-type structural esters.

[0191] Example 3: Effects of overexpression of the SLC gene encoding phosphatidyl acyltransferase and the PAP gene encoding phosphatidyl phosphatase on the content of OPO-type esters in ChDes9-2 engineered bacteria.

[0192] 1. Construction of genetically engineered bacteria that overexpress the SLC gene encoding phosphatidyl acyltransferase and the PAP gene encoding phosphatidyl phosphatase.

[0193] (1) Construction of pSP-GM2-SLC-PAP expression vector

[0194] The SLC gene and PAP gene encoding phosphatidyl acyltransferase were derived from Saccharomyces cerevisiae YS58. Therefore, using the Saccharomyces cerevisiae YS58 genome as a template, the SLC gene fragment was amplified by PCR. The obtained SLC gene fragment was then ligated between the BamHⅠ and KpnⅠ restriction sites of the plasmid pSP-GM2-PAP obtained in Example 2 to obtain the expression vector pSP-GM2-SLC-PAP.

[0195] The nucleotide sequence of the SLC gene is shown in SEQ ID No. 3; the nucleotide sequence of the PAP gene is shown in SEQ ID No. 4.

[0196] Table 2. Primers used in this embodiment

[0197]

[0198] (2) Construction of engineered Saccharomyces cerevisiae SLC-PAP-ChDes9-2

[0199] The expression plasmid pSP-GM2-SLC-PAP was transformed into the host bacterium, namely the ChDes9-2 engineered bacterium obtained in Example 1, to obtain the Saccharomyces cerevisiae engineered bacterium SLC-PAP-ChDes9-2.

[0200] 2. Effects of simultaneous overexpression of SLC and PAP genes on fatty acid and OPO-type ester content.

[0201] (1) The culture of engineered bacteria was carried out in the same way as in Example 2. The fermentation culture strain was SLC-PAP-ChDes9-2, and the recombinant strain ChDes9-2 was used as a control.

[0202] (2) Extraction of bacterial fatty acids is carried out in the same manner as in Example 1.

[0203] (3) The treatment of bacterial fatty acids is the same as that in Example 1.

[0204] (4) The extraction of triglycerides was carried out in the same manner as in Example 1.

[0205] (5) The triglyceride fatty acid composition is the same as that in Example 1.

[0206] (6) Triglyceride analysis was performed using the same method as in Example 1.

[0207] The results are as follows Figure 6 As shown, compared with the triglyceride composition of engineered strain ChDes9-2, the proportion of OPO-type structural esters in the total triglycerides of engineered strain SLC-PAP-ChDes9-2 increased by 7%, indicating that overexpression of Saccharomyces cerevisiae glycerol-3-phosphoacyltransferase SLC and the PAP gene encoding phosphatidylphosphatase can catalyze the synthesis of OPO-type structural esters more efficiently.

[0208] Example 4: Effects of overexpression of the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, and the PAP gene encoding phosphatidylphosphatase on the content of OPO-type esters in ChDes9-2 engineered bacteria.

[0209] 1. Construction of engineered bacteria overexpressing the gene oil-3-phosphoacyltransferase GAT1, the SLC gene encoding phosphatidyltransferase, and the PAP gene encoding phosphatidylphosphatase.

[0210] (1) Construction of pSP-GM2-GAT1-SLC-PAP expression vector

[0211] The GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidylacyltransferase, and the PAP gene encoding phosphatidylphosphatase all originated from *Saccharomyces cerevisiae* YS58. Therefore, using the *Saccharomyces cerevisiae* YS58 genome as a template, the GAT1 gene fragment was amplified by PCR. The GAT1 gene fragment was then ligated between the NheⅠ and KpnⅠ restriction sites of the plasmid pSP-GM2-SLC-PAP described in Example 3 to obtain the plasmid pSP-GM2-GAT1-SLC-PAP.

[0212] The nucleotide sequence of the gene fragment GAT1 is shown in SEQ ID No. 2; the nucleotide sequence of the SLC is shown in SEQ ID No. 3; and the nucleotide sequence of the PAP is shown in SEQ ID No. 4.

[0213] Table 3. Primers used in this embodiment

[0214]

[0215] (2) Construction of engineered Saccharomyces cerevisiae GAT1-SLC-PAP-ChDes9-2

[0216] The expression plasmid pSP-GM2-GAT1-SLC-PAP was transformed into the host bacterium, namely the ChDes9-2 engineered bacterium obtained in Example 1, to obtain the Saccharomyces cerevisiae engineered bacterium GAT1-SLC-PAP-ChDes9-2.

[0217] 2. Effects of overexpression of GAT1, SLC, and PAP genes on OPO-type ester structures in engineered bacteria

[0218] (1) The culture of engineered bacteria was carried out in the same way as in Example 2. The fermentation culture strain was GAT1-SLC-PAP-ChDes9-2, and the recombinant strain ChDes9-2 was used as a control.

[0219] (2) Extraction of bacterial fatty acids is carried out in the same manner as in Example 1.

[0220] (3) The treatment of bacterial fatty acids is the same as that in Example 1.

[0221] (4) The extraction of triglycerides was carried out in the same manner as in Example 1.

[0222] (5) The triglyceride fatty acid composition is the same as that in Example 1.

[0223] (6) Triglyceride analysis was performed using the same method as in Example 1.

[0224] The results are as follows Figure 6As shown, compared with the triglyceride composition of the integrated bacterium ChDes9-2, the OPO-type structural ester in the engineered bacterium GAT1-SLC-PAP-ChDes9-2 reached 17%, indicating that overexpression of GAT1, SLC and PAP can enhance the synthesis of OPO-type structural esters.

[0225] Example 5: Effects of overexpression of the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, and the DGAT gene encoding diacylglycerol acyltransferase on the content of OPO-type esters in the engineered ChDes9-2 bacteria.

[0226] 1. Construction of genetically engineered bacteria that overexpress the GAT1 gene (encoding glycerol-3-phosphoacyltransferase), the SLC gene (encoding phosphatidyltransferase), the PAP gene (encoding phosphatidylphosphatase), and the DGAT gene (encoding diacylglycerol acyltransferase).

[0227] (1) Constructing the pSP-GM2-GAT1-SLC-PAP-DGAT expression vector

[0228] The GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidylacyltransferase, the PAP gene encoding phosphatidylphosphatase, and the DGAT gene encoding diacylglycerol acyltransferase all originated from *Saccharomyces cerevisiae* YS58. Therefore, using the *Saccharomyces cerevisiae* YS58 genome as a template, the DGAT gene fragment was amplified by PCR. The DGAT gene fragment was ligated between the restriction sites SpeⅠ and SacⅠ of the plasmid pSP-GM2-GAT1-SLC-PAP described in Example 4 to obtain the plasmid pSP-GM2-GAT1-SLC-PAP-DGAT.

[0229] The nucleotide sequence of the gene fragment GAT1 is shown in SEQ ID No. 2; the nucleotide sequence of the SLC is shown in SEQ ID No. 3; the nucleotide sequence of the PAP is shown in SEQ ID No. 4; the nucleotide sequence of the PAP is shown in SEQ ID No. 4.

[0230] Table 4. Primers used in this embodiment

[0231]

[0232] (2) Construction of the genetically engineered Saccharomyces cerevisiae strain GAT1-SLC-PAP-DGAT-ChDes9-2

[0233] The expression plasmid pSP-GM2-GAT1-SLC-PAP-DGAT was transformed into the host bacterium, namely the ChDes9-2 engineered bacterium obtained in Example 1, to obtain the engineered bacterium GAT1-SLC-PAP-DGAT-ChDes9-2.

[0234] 2. Effects of overexpression of GAT1, SLC, PAP and DGAT genes on OPO-type ester structures

[0235] (1) The culture of engineered bacteria was carried out in the same way as in Example 2. The fermentation culture strain was GAT1-SLC-PAP-DGAT-ChDes9-2, and the recombinant strain ChDes9-2 was used as a control.

[0236] (2) Extraction of bacterial fatty acids is carried out in the same manner as in Example 1.

[0237] (3) The treatment of bacterial fatty acids is the same as that in Example 1.

[0238] (4) The extraction of triglycerides was carried out in the same manner as in Example 1.

[0239] (5) The triglyceride fatty acid composition is the same as that in Example 1.

[0240] (6) Triglyceride analysis was performed using the same method as in Example 1.

[0241] The results are as follows Figure 6 As shown, compared to the engineered strain ChDes9-2, the proportion of OPO-type structural esters in the total triglycerides of the engineered strain GAT1-SLC-PAP-DGAT-ChDes9-2 was only 14%, which was lower than that of the engineered strain GAT1-SLC-PAP-ChDes9-2. This indicates that when GAT1, SLC, PAP and DGAT are expressed simultaneously, the DGAT gene encoding diacylglycerol acyltransferase has a relative inhibitory effect on the synthesis of OPO-type structural esters, and cannot catalyze the synthesis of OPO-type structural esters to the maximum extent in the ChDes9-2 strain.

[0242] It should be noted that the embodiments described above are merely preferred embodiments of the present invention, used to explain the present invention, and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications having the same function.

Claims

1. A genetically engineered bacterium that produces OPO-type structural esters, which is a recombinant yeast cell containing the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase; The genetically engineered bacteria are recombinant yeast cells with modified chassis; the chassis modification includes the inhibition of the C16:1 anabolic pathway; the inhibition of the C16:1 anabolic pathway is the knockout of the OLE1 gene encoding Δ9 desaturase. The OLE1 gene encoding Δ9 desaturase in the genetically engineered bacteria is replaced by the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase; the nucleotide sequence of the ChDes9-2 is positions 1045-2085 of SEQ ID No. 1; The genetically engineered bacteria are recombinant yeast cells optimized through lipid or lipid precursor synthesis pathways. The optimization of the lipid or lipid precursor synthesis pathway involves overexpressing the SLC gene encoding phosphatidyl acyltransferase and the PAP gene encoding phosphatidyl phosphatase in recombinant yeast cells. Alternatively, the lipid or lipid precursor synthesis pathway may be optimized by overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, and the PAP gene encoding phosphatidylphosphatase in recombinant yeast cells. Alternatively, the lipid or lipid precursor synthesis pathway may be optimized by overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, and the DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cells. The lipid or lipid precursor is a triglyceride.

2. The genetically engineered bacterium according to claim 1, characterized in that, The yeast cells are selected from the group consisting of the following yeast cells: Yeastra lipolytica, Rhodotorula rubrum, lipophilic yeast, lipophilic yeast, Rhodotorula glutinis, Cryptococcus curvifolius, Candida curvifolius, Myxosporium fermentum, Candida lakoffii, Candida rubrum, Candida tropicalis, Candida utilis, Myxosporium dermatophyte, and Saccharomyces cerevisiae.

3. The genetically engineered bacteria according to claim 2, characterized in that, The yeast cells are Saccharomyces cerevisiae.

4. A method for constructing a genetically engineered bacterium as described in any one of claims 1-3, comprising: Step (A): Construct recombinant yeast cell A containing the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase; Step (Z) optimizes lipid or lipid precursor synthesis pathways; Step (A) includes replacing the OLE1 gene encoding Δ9 desaturase with the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase to construct a recombinant yeast cell A containing the ChDes9-2 gene encoding stearoyl-CoAΔ9 desaturase. Step (Z) includes: Recombinant yeast cells B were obtained by overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, or the DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cells. Alternatively, recombinant yeast cells C can be obtained by overexpressing the SLC gene encoding phosphatidyl transferase and the PAP gene encoding phosphatidyl phosphatase in recombinant yeast cells. Alternatively, recombinant yeast cells D can be obtained by overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, and the PAP gene encoding phosphatidylphosphatase in recombinant yeast cells. Alternatively, recombinant yeast cells E can be obtained by overexpressing the GAT1 gene encoding glycerol-3-phosphoacyltransferase, the SLC gene encoding phosphatidyltransferase, the PAP gene encoding phosphatidylphosphatase, and the DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cells.

5. The application of the genetically engineered bacteria as described in any one of claims 1-3 or the genetically engineered bacteria constructed by the construction method as described in claim 4 in the production of OPO-type structural esters.

6. The application according to claim 5, characterized in that, The application includes inoculating the genetically engineered bacteria into a fermentation medium, carrying out fermentation culture, and then separating and purifying the obtained fermentation culture broth to obtain an OPO-type structural ester.

7. The application according to claim 6, characterized in that, The fermentation medium includes one or more of YPD fermentation medium, YNB-Trp fermentation medium and YNB-Trp / Ura fermentation medium; the fermentation temperature is 30℃; and / or the fermentation time is 72h.

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