CN116179381A - Genetically engineered bacterium for producing OPO structural ester, construction method and application thereof - Google Patents

Genetically engineered bacterium for producing OPO structural ester, construction method and application thereof Download PDF

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CN116179381A
CN116179381A CN202211053882.9A CN202211053882A CN116179381A CN 116179381 A CN116179381 A CN 116179381A CN 202211053882 A CN202211053882 A CN 202211053882A CN 116179381 A CN116179381 A CN 116179381A
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gene encoding
acyltransferase
phosphatidic acid
slc
pap
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刘军锋
杨川
邓利
王芳
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Beijing University of Chemical Technology
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Abstract

The invention relates to a genetic engineering bacterium for producing OPO structural ester, which is a recombinant yeast cell containing a ChDes9-2 gene for encoding stearoyl-CoA delta 9 desaturase; specifically, the genetically engineered strain is obtained by replacing the OLE1 gene encoding delta 9 desaturase with the ChDes9-2 gene encoding stearoyl-CoA delta 9 desaturase in the Saccharomyces cerevisiae strain YS58, and over-expressing GAT1 and/or SLC and/or PAP and/or DGAT genes, and the ratio of OPO structural ester in the Saccharomyces cerevisiae to grease in the Saccharomyces cerevisiae is effectively improved, so that the content of the OPO structural ester in the triglyceride of the Saccharomyces cerevisiae genetically engineered strain reaches 17.2%, and the limitation of OPO structural ester sources is broken through.

Description

Genetically engineered bacterium for producing OPO structural ester, construction method and application thereof
Technical Field
The invention belongs to the technical field of biosynthesis and genetic engineering, and relates to genetic engineering bacteria for producing OPO structural ester, and a construction method and application thereof.
Background
OPO-type structural ester, chemical name 1,3-dioleoyl-2-palmitoyl triglyceride (1, 3-dioleoyl-2-palmitoyl glyceride), is an important component of breast milk fat. Studies have shown that OPO addition to infant formulas increases the content of palmitic acid in the Sn-2 position of the infant formula, and that after milk fat enters the infant, it is first digested initially in the stomach by gastric lipase, during which 10% to 30% of the triglycerides are digested. The fat globules then enter the small intestine by gastric emptying and are hydrolysed by pancreatic lipase to sn-2 monoglycerides and free fatty acids. The sn-2 monoglyceride can be directly absorbed by small intestine to supply power to infant. Thereby reducing constipation and difficult defecation of infants, improving the absorption and utilization of energy and mineral substances of infants, reducing the loss of bone mineral substances such as calcium, magnesium and the like and other nutritional ingredients in bodies, improving the symptom of insufficient calcium and energy intake of infants by the infant formula milk powder, activating immune cells and improving the natural resistance of infants.
OPO structural esters can be synthesized chemically and enzymatically. The chemical synthesis method has the problems of high catalyst toxicity, high reaction temperature, strong reaction randomness, poor selectivity and the like, and is basically not used at present. In recent years, with the development of enzyme engineering technology, the yield of structural ester is improved to a certain extent by enzyme catalytic synthesis, but the problems of higher catalyst cost, poor stability, limited use batch and the like exist in the enzyme; meanwhile, the enzyme catalysis process is greatly restricted by raw materials, and the development of structural ester is limited by the factors such as conversion rate, cost and the like. Therefore, the development of the novel production technology and process of the structural ester reduces the production cost of the structural ester, and has important value and significance for meeting the increasing nutrition and health requirements of people and promoting the development of 'health Chinese strategy'.
Disclosure of Invention
The invention aims to provide a genetically engineered bacterium for producing OPO structural ester, which can produce OPO structural ester with high yield and is easy for industrial production.
The second object of the invention is to provide a construction method of the genetic engineering bacteria for producing OPO structural ester, which can overcome the defects of large raw material constraint, lower conversion rate, higher cost, poorer stability, limited use batch and the like of the OPO structural ester synthesized by an enzyme method, and has important value and significance for meeting the increasing nutrition and health requirements of people and promoting the development of 'health Chinese strategy'.
The invention also aims to provide the application of the genetically engineered bacterium in the production of OPO structural ester, and the genetically engineered bacterium is subjected to fermentation culture to produce the OPO structural ester, so that the genetically engineered bacterium has high conversion rate, low cost and good stability, and is easy for industrial production.
Therefore, the invention firstly provides the genetically engineered bacterium for producing the OPO structural ester.
According to some embodiments of the first aspect of the invention, the OPO-type structural ester-producing genetically engineered bacterium is a recombinant yeast cell comprising a ChDes9-2 gene encoding stearoyl-CoA.DELTA.9 desaturase.
In some embodiments of the invention, the genetically engineered bacterium is a chassis-engineered recombinant yeast cell; preferably, the chassis engineering comprises inhibition of the c16:1 anabolic pathway; further preferred, the inhibition of the C16:1 anabolic pathway is a knockout of the OLE1 gene encoding Δ9 desaturase.
In some preferred embodiments of the invention, the OLE1 gene encoding Δ9 desaturase in the genetically engineered bacterium is replaced with the ChDes9-2 gene encoding stearoyl-coa Δ9 desaturase.
According to some embodiments of the second aspect of the invention, the genetically engineered bacterium is an optimized recombinant yeast cell via a lipid or lipid precursor synthesis pathway.
Preferably, the optimization of the lipid or lipid precursor synthesis pathway comprises overexpressing one or more of the GAT1 gene encoding glycerol-3-phosphate acyltransferase, the SLC gene encoding phosphatidic acid acyltransferase, the PAP gene encoding phosphatidic acid phosphatase, and the DGAT gene encoding diacylglycerol acyltransferase in a recombinant yeast cell; further preferably, the lipid or lipid precursor is a triglyceride or fatty acid; still more preferably, the fatty acid comprises one or more of C16:0, C16:1, C18:0, C18:1.
In some particularly preferred embodiments of the invention, the lipid or lipid precursor synthesis pathway is optimized for overexpression of the GAT1 gene encoding glycerol-3-phosphate acyltransferase or the SLC gene encoding phosphatidic acid acyltransferase or the PAP gene encoding phosphatidic acid phosphatase or the DGAT gene encoding diacylglycerol acyltransferase in a recombinant yeast cell.
In some particularly preferred embodiments of the invention, the lipid or lipid precursor synthesis pathway is optimized to overexpress the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase in recombinant yeast cells.
In some particularly preferred embodiments of the invention, the lipid or lipid precursor synthesis pathway is optimized for overexpression of the GAT1 gene encoding glycerol-3-phosphate acyltransferase, the SLC gene encoding phosphatidic acid acyltransferase, and the PAP gene encoding phosphatidic acid phosphatase in recombinant yeast cells.
In some particularly preferred embodiments of the invention, the lipid or lipid precursor synthesis pathway is optimized for overexpression of the GAT1 gene encoding glycerol-3-phosphate acyltransferase, the SLC gene encoding phosphatidic acid acyltransferase, the PAP gene encoding phosphatidic acid phosphatase and the DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cells.
In the present invention, the yeast cell is selected from the group consisting of: yarrowia lipolytica, rhodosporidium, oleaginous yeast, rhodotorula mucilaginosa, cryptococcus curvatus, candida fermentata, candida lacosaccharomyces, candida iron, candida tropicalis, candida utilis, candida Pityrosporum and saccharomyces cerevisiae, preferably saccharomyces cerevisiae.
The invention also provides a construction method of the genetically engineered bacterium according to the embodiments of the first to third aspects of the invention, which comprises the following steps:
step (A), constructing a recombinant yeast cell A containing a ChDes9-2 gene encoding stearoyl-CoA delta 9 desaturase;
step (Z), optimizing lipid or lipid precursor synthesis pathway.
According to some embodiments of the invention, the step (a) comprises:
Step M, expressing the ChDes9-2 gene encoding stearoyl-CoA delta 9 desaturase in yeast cells;
step N, knocking out the OLE1 gene encoding delta 9 desaturase from yeast cells;
in some preferred embodiments of the invention, step (A) comprises replacing the OLE1 gene encoding Δ9 desaturase with the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase to construct a recombinant yeast cell A comprising the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase.
According to further embodiments of the present invention, the step (Z) includes:
overexpressing the GAT1 gene encoding glycerol-3-phosphate acyltransferase or the SLC gene encoding phosphatidic acid acyltransferase or the PAP gene encoding phosphatidic acid phosphatase or the DGAT gene encoding diacylglycerol acyltransferase in the recombinant yeast cell to obtain a recombinant yeast cell B;
alternatively, a recombinant yeast cell C is obtained by overexpressing the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase in a recombinant yeast cell;
alternatively, a GAT1 gene encoding glycerol-3-phosphate acyltransferase, an SLC gene encoding phosphatidic acid acyltransferase, and a PAP gene encoding phosphatidic acid phosphatase are overexpressed in a recombinant yeast cell to obtain a recombinant yeast cell D;
Alternatively, a GAT1 gene encoding glycerol-3-phosphate acyltransferase, an SLC gene encoding phosphatidic acid acyltransferase, a PAP gene encoding phosphatidic acid phosphatase and a DGAT gene encoding diacylglycerol acyltransferase are overexpressed in a recombinant yeast cell to obtain a recombinant yeast cell E.
The invention also provides application of the genetically engineered bacteria according to the embodiments of the first to third aspects of the invention or the genetically engineered bacteria constructed by the method of the invention in the production of OPO structural ester.
According to the invention, the application comprises the steps of inoculating the genetically engineered bacterium into a fermentation medium for fermentation culture, and then separating and purifying the obtained fermentation culture solution to obtain OPO structural ester; further preferably, the fermentation medium comprises one or more of YPD fermentation medium, YNB-Trp fermentation medium and YNB-Trp/Ura fermentation medium; still more preferably, the temperature of the fermentation culture is 30 ℃; and/or, the fermentation culture time is 72h.
The beneficial effects of the invention are mainly as follows: the invention improves the content of OPO structural ester in the saccharomyces cerevisiae, breaks through the limitation of OPO structural ester sources, and provides a new method for realizing the microbial sources of structural ester. In the invention, in the saccharomyces cerevisiae strain YS58, the ChDes9-2 gene and the over-expressed GAT1 and/or SLC and/or PAP and/or DGAT genes are expressed, so that the saccharomyces cerevisiae genetic engineering bacteria are constructed, the ratio of OPO structural ester in the saccharomyces cerevisiae in grease in the saccharomyces cerevisiae is effectively improved, and the content of the OPO structural ester in the saccharomyces cerevisiae genetic engineering bacteria in triglyceride is up to 17.2%.
Drawings
The invention is described in further detail below with reference to the accompanying drawings:
FIG. 1 shows the Saccharomyces cerevisiae triglyceride anabolic pathway.
FIG. 2 shows the effect of expression of the ChDes9-2 gene on fatty acid production by Saccharomyces cerevisiae.
FIG. 3 shows the effect of the ChDes9-2 gene on OPO-type structural ester production by Saccharomyces cerevisiae.
FIG. 4 shows the effect of overexpression of GAT1 gene and SLC gene, and PAP gene and DGAT gene, respectively, on fatty acid production by engineering bacteria.
FIG. 5 shows the effect of overexpression of GAT1 gene and SLC gene, and PAP gene and DGAT gene, respectively, on OPO-type structural ester production by engineering bacteria.
FIG. 6 shows the effect of co-overexpression of SLC gene and PAP gene and co-overexpression of GAT1 gene, SLC gene, PAP gene and DGAT gene on OPO structural ester production by engineering bacteria, respectively.
Detailed Description
In order that the invention may be readily understood, a detailed description of the invention will be provided below with reference to the accompanying drawings and examples. Before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
I terminology
The term "chassis microorganism" also called "chassis microorganism cell" refers to a microorganism cell used as a platform and placed into a functional biological system, so that the cell can have the functions required by human beings and be used for biosynthesis; the chassis microbial cells need to have reduced functions, but have the most basic self-replication and metabolism capabilities, so that the chassis microbial cells can be a blank platform capable of continuously adding functions.
The term "genetically engineered bacterium" as used herein refers to a bacterium, such as Saccharomyces cerevisiae, which produces a desired protein by introducing a desired gene into a host organism (i.e., a host cell or a chassis microorganism or thallus) and expressing the gene. The core technology of genetic engineering is a recombinant technology of DNA, and thus, in the present invention, genetically engineered bacteria are also referred to as recombinant microorganisms, such as recombinant yeast cells.
The term "recombinant" as used herein refers to a transgenic organism constructed by using genetic material of a donor organism or an artificially synthesized gene, cutting the gene by in vitro or ex vivo restriction enzymes, then ligating the gene with a suitable vector to form a recombinant DNA molecule, and introducing the recombinant DNA molecule into a recipient cell or a recipient organism, wherein the organism can exhibit a property of another organism according to a blueprint designed in advance by human.
The term "endogenous gene" as used herein refers to a gene within the genome of a host cell or a cell of the same species as the host cell, for example, the host cell used in the construction of the genetically engineered bacterium of the present invention is the yeast Saccharomyces cerevisiae YS58, and the endogenous gene may be derived from the yeast Saccharomyces cerevisiae YS58.
The term "heterologous gene" as used herein refers to a gene introduced into other species or cells of a host bacterium by genetic engineering in the process of constructing a genetically engineered bacterium, and may be a gene that is artificially optimized, modified or synthesized.
The term "expression" as used herein refers to the expression of a gene in a metabolic pathway, and to the expression of its background promoter.
The term "overexpression" as used herein refers to the expression of a gene in a host cell or genetically engineered bacterium using a strong or super strong promoter.
The term "screening gene" in the present invention refers to a gene, which is a biosynthesis gene with a known function or a known sequence, and can play a role of a specific marker; in the sense of genetic engineering, it is an important marker of recombinant DNA vectors, usually used to check whether transformation is successful or not; in the sense of gene localization, it is a tool for marking a gene of interest, usually for detecting the localization of the gene of interest in a cell. For example, recombinant tryptophan biosynthesis gene (TRP), recombinant uracil biosynthesis gene (URA 3), and the like.
The term "water" as used herein refers to deionized water, distilled water or ultrapure water unless otherwise specified or defined.
II. Embodiment
As described above, the existing enzymatic synthesis method of OPO structural ester has the problems of higher catalyst cost, poor stability, limited use batch and the like; meanwhile, the enzyme catalysis process is greatly restricted by raw materials, and the development of structural ester is limited by the factors such as conversion rate, cost and the like. In view of this, the present inventors have conducted a great deal of research on a method for synthesizing OPO structural esters by a biological method.
The inventor notices that with the continuous development of synthetic biology technology, the intracellular pathway related to oil synthesis and key gene functions are gradually analyzed in recent years, and a theoretical basis is laid for the construction of structural ester-producing engineering bacteria. As shown in FIG. 1, yeast fatty acid synthesis starts with acetyl-CoA carboxylase encoded by ACC1, and long chain fatty acyl-CoA is synthesized by fatty acid synthase FAS catalysis and NADPH consumption extension, and further Triglyceride (TAG), phospholipid, etc. are synthesized. Fatty acyl-CoA synthesizes long chain fatty acids and unsaturated fatty acids by fatty acid elongase Elo and fatty acid desaturase Ole1 on the endoplasmic reticulum membrane, respectively. In the de novo triglyceride synthesis process, acetyl-CoA is catalyzed by acetyl-CoA carboxylase Acc1 to form malonyl-CoA, and the acetyl-CoA and malonyl-CoA react to synthesize C16 and C18 fatty acyl-CoA under the catalysis of fatty acid synthase FAS cycle. Fatty acid is linked to sn-1 position of glycerol-3-phosphate by the reaction catalyzed by glycerol-3-phosphoryl transferase (GPAT) between fatty acyl-CoA and glycerol-3-phosphate to produce lysophosphatidic acid. Lysophosphatidic acid and a molecule of fatty acyl-CoA are then catalyzed by phosphatidic acid acyltransferase (SLC) to link fatty acid to sn-2 to yield phosphatidic acid. Phosphatidic acid is catalyzed by Phosphatidic Acid Phosphatase (PAP) to remove one phosphate group to form diglyceride. Finally, diacylglycerol and fatty acyl-CoA are catalyzed to TAG by diacylglycerol acyltransferase (DGAT).
Based on the above, the present inventors have studied and found that, by constructing a Saccharomyces cerevisiae gene engineering strain in which a ChDes9-2 gene encoding stearoyl-CoA.DELTA.9 desaturase and a GAT1 gene encoding glycerol-3-phosphate acyltransferase and/or a SLC gene encoding phosphatidic acid acyltransferase and/or a PAP gene encoding phosphatidic acid phosphatase and/or a DGAT gene encoding diacylglycerol acyltransferase are expressed and expressed as expression hosts, the ratio of OPO-type structural ester in Saccharomyces cerevisiae to grease in Saccharomyces cerevisiae can be effectively increased, and thus a genetically engineered strain for producing OPO-type structural ester with high efficiency can be constructed and obtained.
Therefore, the genetically engineered bacteria for producing OPO structural ester provided by the invention are constructed by taking saccharomyces cerevisiae as an expression host; the yeast cell is selected from the group consisting of: yarrowia lipolytica, rhodosporidium, oleaginous yeast, rhodotorula mucilaginosa, cryptococcus curvatus, candida fermentata, candida lacosaccharomyces, candida iron, candida tropicalis, candida utilis, candida Pitusa and saccharomyces cerevisiae, preferably saccharomyces cerevisiae; further preferably, saccharomyces cerevisiae YS58 (accession number ST12300, available from Beijing Rui Si Bo Biotech Co., ltd.) is used as the expression host in the present invention.
In an embodiment of the present invention, the OPO-structural ester-producing genetically engineered bacterium is a recombinant yeast cell comprising a ChDes9-2 gene encoding stearoyl-CoA.DELTA.9 desaturase (abbreviated as ChDes9-2 gene), which is also referred to as an OPO-structural ester-producing original genetically engineered bacterium or an OPO-structural ester-producing original recombinant yeast cell in the present invention.
In the present invention, the ChDes9-2 gene encoding stearoyl-CoA.DELTA.9 desaturase is a key gene for maintaining the C18:1 anabolic pathway. In yeast cells, expression of the ChDes9-2 gene encoding stearoyl-CoA.DELTA.9 desaturase with C18:0 as a substrate maintains the C18:1 anabolic pathway.
It will be appreciated by those skilled in the art that the ChDes9-2 gene encoding stearoyl-CoA.DELTA.9 desaturase is a gene that is not found in the chassis strain (i.e., host cell, saccharomyces cerevisiae) used; in the invention, the ChDes9-2 gene for encoding stearoyl-CoA delta 9 desaturase is derived from the flea in the North hucho taimen, the nucleotide sequence of the ChDes9-2 gene is shown in 1045-2085 positions of SEQ ID No.1, and the ChDes9-2 gene is expressed in genetically engineered bacteria.
In some embodiments of the invention, the genetically engineered bacterium is a chassis engineered recombinant yeast cell.
Specifically, the chassis engineering includes inhibiting the expression of endogenous OLE1 gene (abbreviated as OLE1 gene) encoding Δ9 desaturase with C16:0 and C18:0 as substrates, whereby C16:1 anabolic pathway (pathway) of Saccharomyces cerevisiae itself can be inhibited; preferably, the inhibition of the C16:1 anabolic pathway is a knockout of the OLE1 gene encoding Δ9 desaturase.
In some preferred embodiments of the invention, the OLE1 gene encoding Δ9 desaturase in the genetically engineered bacterium is replaced with the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase, thereby inhibiting endogenous expression of the Δ9 desaturase-encoding OLE1 gene with C16:0 and C18:0 as substrates, while simultaneously expressing the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase with C18:0 as substrate, increasing the fatty acid duty cycle of recombinant bacteria C18:1 and C16:0; the corresponding recombinant Saccharomyces cerevisiae cells are referred to as OPO-type structural ester-producing recombinant yeast cells A in the present invention.
In some particularly preferred embodiments of the invention, replacement of the OLE1 gene encoding Δ9 desaturase with the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase is accomplished by homologous recombination techniques. By introducing the gene encoding stearoyl-CoA.DELTA.9 desaturase ChDes9-2 from Metridia extremely north into said oleaginous Saccharomyces cerevisiae in the form of recombinant DNA fragments, the OLE1 gene encoding Δ9 desaturase in Saccharomyces cerevisiae is replaced with the gene encoding stearoyl-CoA.DELTA.9 desaturase ChDes9-2, inhibiting the anabolic pathway of C16:1 while maintaining the anabolic pathway of C18:1, facilitating the accumulation of C16:0 and C18:1; in the invention, the nucleotide sequence of the recombinant DNA fragment containing the ChDes9-2 gene is shown as SEQ ID No. 1.
Positions 1-60 of SEQ ID No.1 are the upstream homology 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 screening gene, and positions 3207-3266 are the downstream homology arm of OLE 1.
In order to increase the OPO-type structural ester production capacity of the starting recombinant yeast, in some preferred embodiments of the invention the lipid or lipid precursor synthesis pathway of the resulting OPO-type structural ester-producing starting recombinant yeast cells is optimized to obtain optimized recombinant yeast cells via the lipid or lipid precursor synthesis pathway, also referred to herein as OPO-type structural ester-producing recombinant yeast cells via the lipid or lipid precursor synthesis pathway.
Preferably, the lipid or lipid precursor synthesis pathway is optimized in the present invention by overexpressing one or more of the endogenous GAT1 gene encoding glycerol-3-phosphate acyltransferase (abbreviated as GAT1 gene), the SLC gene encoding phosphatidic acid acyltransferase (abbreviated as SLC gene), the PAP gene encoding phosphatidic acid phosphatase (abbreviated as PAP gene) and the DGAT gene encoding diacylglycerol acyltransferase (abbreviated as DGAT gene) in a recombinant yeast cell; further preferably, the lipid or lipid precursor is a triglyceride or fatty acid; still more preferably, the fatty acid comprises one or more of C16:0, C16:1, C18:0, C18:1.
It will be appreciated by those skilled in the art that the lipid or lipid precursor comprises both the triglyceride and fatty acid products, and thus the fatty acid composition in Saccharomyces cerevisiae is first regulated in the present invention to regulate the triglyceride. The term triglyceride herein refers to a mixed triglyceride in Saccharomyces cerevisiae including OPO-type structural esters.
In the invention, the nucleotide sequence of the GAT1 gene for encoding the glycerol-3-phosphate acyltransferase is shown as SEQ ID No. 2; the nucleotide sequence of the SLC gene for encoding phosphatidic acid acyltransferase is shown as SEQ ID No. 3; the nucleotide sequence of the PAP gene for encoding phosphatidic acid phosphatase is shown as SEQ ID No. 4; the nucleotide sequence of the DGAT gene for encoding the diacylglycerol acyltransferase is shown as SEQ ID No. 5.
In some particularly preferred embodiments of the invention, the lipid or lipid precursor synthesis pathway is optimized for overexpression of the GAT1 gene encoding glycerol-3-phosphate acyltransferase or the SLC gene encoding phosphatidic acid acyltransferase or the PAP gene encoding phosphatidic acid phosphatase or the DGAT gene encoding diacylglycerol acyltransferase in a recombinant yeast cell; the corresponding recombinant Saccharomyces cerevisiae cells are referred to as OPO-type structural ester-producing recombinant yeast cells B in the present invention.
In some particularly preferred embodiments of the invention, the optimization of the lipid or lipid precursor synthesis pathway is overexpression of the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase in recombinant yeast cells; the corresponding recombinant Saccharomyces cerevisiae cells are referred to as OPO-type structural ester-producing recombinant yeast cells C in the present invention.
In some particularly preferred embodiments of the invention, the lipid or lipid precursor synthesis pathway is optimized for overexpression of the GAT1 gene encoding glycerol-3-phosphate acyltransferase, the SLC gene encoding phosphatidic acid acyltransferase, and the PAP gene encoding phosphatidic acid phosphatase in recombinant yeast cells; the corresponding recombinant Saccharomyces cerevisiae cells are referred to as OPO-type structural ester-producing recombinant yeast cells D in the present invention.
In some particularly preferred embodiments of the invention, the lipid or lipid precursor synthesis pathway is optimized for overexpression of the GAT1 gene encoding glycerol-3-phosphate acyltransferase, the SLC gene encoding phosphatidic acid acyltransferase, the PAP gene encoding phosphatidic acid phosphatase and the DGAT gene encoding diacylglycerol acyltransferase in a recombinant yeast cell; the corresponding recombinant Saccharomyces cerevisiae cells are referred to herein as OPO-type structural ester-producing recombinant yeast cells E.
Based on the above, it will be understood that the above-described OPO-structural ester-producing recombinant yeast cells B to E are obtained by optimizing the synthetic route of lipid or lipid precursor based on OPO-structural ester-producing recombinant yeast cell A.
Research results show that the genetically engineered bacteria for producing OPO structural ester provided by the invention contain more than 10% of lipid by dry weight of yeast cells after fermentation culture.
In order to achieve the above embodiment, the present invention further provides a method for constructing an OPO structural ester-producing genetically engineered bacterium according to the embodiment of the present invention, including:
step (A), constructing a recombinant yeast cell A containing a ChDes9-2 gene encoding stearoyl-CoA delta 9 desaturase;
step (Z), optimizing lipid or lipid precursor synthesis pathway.
According to some embodiments of the invention, the step (a) comprises:
step M, expressing the ChDes9-2 gene encoding stearoyl-CoA delta 9 desaturase in yeast cells;
step N, knocking out the OLE1 gene encoding delta 9 desaturase from yeast cells;
in some preferred embodiments of the invention, step (A) comprises replacing the OLE1 gene encoding Δ9 desaturase with the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase to construct a recombinant yeast cell A comprising the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase (also referred to herein as OPO-type structural ester-producing genetically engineered bacterium A).
Specifically, in step (A), expression of endogenous C16:0 and C18:0 substrates encoding Δ9 desaturase OLE1 gene is inhibited while expressing C18:0 substrates stearoyl-CoA Δ9 desaturase ChDes9-2 can be gene-replaced by homologous recombination.
Wherein, the nucleotide sequence of the coding gene of the stearoyl-CoA delta 9 desaturase ChDes9-2 taking C18:0 as a substrate is shown in 1045-2085 of SEQ ID No. 1.
In a specific embodiment of the invention, this is achieved by homologous recombination techniques. By introducing the Δ9 desaturase ChDes9-2 gene from the North hucho taiwanensis into the oleaginous Saccharomyces cerevisiae in the form of recombinant DNA fragments, the Saccharomyces cerevisiae OLE1 gene is replaced by the ChDes9-2 gene, the anabolic pathway of C16:1 is inhibited, and the anabolic pathway of C18:1 is maintained, so that the accumulation of C16:0 and C18:1 is facilitated; the nucleotide sequence of the recombinant DNA fragment is shown as SEQ ID No. 1.
Positions 1-60 of SEQ ID No.1 are the upstream homology 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 screening gene, and positions 3207-3266 are the downstream homology arm of OLE 1.
According to further embodiments of the present invention, the step (Z) includes:
step (B), overexpressing GAT1 gene encoding glycerol-3-phosphate acyltransferase or SLC gene encoding phosphatidic acid acyltransferase or PAP gene encoding phosphatidic acid phosphatase or DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cell to obtain recombinant yeast cell B;
alternatively, step (C), over-expressing the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase in a recombinant yeast cell to obtain a recombinant yeast cell C;
alternatively, step (D), over-expressing the GAT1 gene encoding glycerol-3-phosphate acyltransferase, the SLC gene encoding phosphatidic acid acyltransferase, and the PAP gene encoding phosphatidic acid phosphatase in a recombinant yeast cell to obtain a recombinant yeast cell D;
alternatively, step (E), over-expressing the GAT1 gene encoding glycerol-3-phosphate acyltransferase, the SLC gene encoding phosphatidic acid acyltransferase, the PAP gene encoding phosphatidic acid phosphatase and the DGAT gene encoding diacylglycerol acyltransferase in the recombinant yeast cell to obtain a recombinant yeast cell E.
Further, in the step (B), the overexpression of the GAT1 gene encoding glycerol-3-phosphate acyltransferase of saccharomyces cerevisiae may be achieved by introducing a gene encoding the GAT1 gene encoding glycerol-3-phosphate acyltransferase into saccharomyces cerevisiae; overexpression of the SLC gene encoding phosphatidic acid acyltransferase of Saccharomyces cerevisiae may be achieved by introducing into Saccharomyces cerevisiae a gene encoding the SLC gene encoding phosphatidic acid acyltransferase; overexpression of the PAP gene encoding phosphatidic acid phosphatase of Saccharomyces cerevisiae can be achieved by introducing a gene encoding the PAP gene encoding phosphatidic acid phosphatase into Saccharomyces cerevisiae; overexpression of the DGAT gene encoding diacylglycerol acyltransferase of Saccharomyces cerevisiae can be achieved by introducing a gene encoding the DGAT gene encoding diacylglycerol acyltransferase into Saccharomyces cerevisiae;
Wherein the nucleotide sequence of the GAT1 gene of the saccharomyces cerevisiae encoding glycerol-3-phosphate acyltransferase is shown as SEQ ID No. 2; the nucleotide sequence of the SLC gene of the saccharomyces cerevisiae coding phosphatidic acid acyltransferase is shown as SEQ ID No. 3; the nucleotide sequence of the PAP gene of the saccharomyces cerevisiae coding phosphatidic acid phosphatase is shown as SEQ ID No. 4; the nucleotide sequence of the DGAT gene of the Saccharomyces cerevisiae coding diacylglycerol acyltransferase is shown as SEQ ID No. 5.
In a specific embodiment of the invention, overexpression of the GAT1 gene encoding glycerol-3-phosphate acyltransferase or the SLC gene encoding phosphatidic acid acyltransferase or the PAP gene encoding phosphatidic acid phosphatase or the DGAT gene encoding diacylglycerol acyltransferase is achieved by constructing a plasmid that drives expression of the gene of interest by a strong promoter. Performing PCR amplification by taking a Saccharomyces cerevisiae YS58 genome as a template to obtain a GAT1 gene fragment, connecting the GAT1 gene fragment to a plasmid pSP-GM2 to obtain a plasmid pSP-GM2-GAT1, and transferring the plasmid pSP-GM2-GAT1 into the strain obtained in the step (A) by a lithium acetate transformation method, wherein the nucleotide sequence of the GAT1 is shown as SEQ ID No. 3; performing PCR amplification by taking a Saccharomyces cerevisiae YS58 genome as a template to obtain an SLC gene fragment, connecting the SLC gene fragment to a plasmid pSP-GM2 to obtain a plasmid pSP-GM2-SLC, and transferring the plasmid pSP-GM2-SLC into the strain obtained in the step (A) by a lithium acetate conversion method, wherein the nucleotide sequence of the SLC is shown as SEQ ID No. 4; performing PCR amplification by taking a Saccharomyces cerevisiae YS58 genome as a template to obtain a PAP gene fragment, connecting the PAP gene fragment to a plasmid pSP-GM2 to obtain a plasmid pSP-GM2-PAP, and transferring the plasmid pSP-GM2-PAP into the strain obtained in the step (A) by a lithium acetate transformation method, wherein the nucleotide sequence of the PAP is shown as SEQ ID No. 5; and (3) performing PCR amplification by taking a Saccharomyces cerevisiae YS58 genome as a template to obtain a PAP gene fragment, connecting the PAP gene fragment to a plasmid pSP-GM2 to obtain a plasmid pSP-GM2-PAP, and transferring the plasmid pSP-GM2-PAP into the strain obtained in the step (A) by a lithium acetate transformation method, wherein the nucleotide sequence of the PAP is shown as SEQ ID No. 5.
In the step (C), the simultaneous overexpression of the SLC gene encoding phosphatidic acid acyltransferase of Saccharomyces cerevisiae and the PAP gene encoding phosphatidic acid phosphatase of Saccharomyces cerevisiae may be achieved by simultaneously introducing the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase into Saccharomyces cerevisiae.
In a specific embodiment of the invention, overexpression of the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase is achieved by constructing a plasmid that drives expression of the gene of interest by a strong promoter. And (C) performing PCR amplification by taking a Saccharomyces cerevisiae YS58 genome as a template to obtain a PAP gene fragment, and connecting the PAP gene fragment to the plasmid pSP-GM2-SLC obtained in the step (B) to obtain the plasmid pSP-GM2-SLC-PAP. Transferring the plasmid pSP-GM2-SLC-PAP into the strain obtained in the step (A) by a lithium acetate transformation method. The nucleotide sequence of the SLC is shown as SEQ ID No. 3; the nucleotide sequence of the PAP coding gene is shown as SEQ ID No. 4.
In the step (D), the simultaneous overexpression of the GAT1 gene encoding glycerol-3-phosphate acyltransferase and the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase in Saccharomyces cerevisiae can be achieved by simultaneously introducing the GAT1 gene encoding glycerol-3-phosphate acyltransferase and the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase into Saccharomyces cerevisiae.
In a specific embodiment of the invention, overexpression of the GAT1 gene encoding glycerol-3-phosphate acyltransferase, the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase is achieved by constructing a plasmid that drives expression of the gene of interest by a strong promoter. And (3) taking a Saccharomyces cerevisiae YS58 genome as a template, carrying out PCR amplification to obtain a GAT1 gene fragment, and connecting the GAT1 gene fragment to the pSP-GM2-SLC-PAP plasmid obtained in the step (C) to obtain a plasmid pSP-GM2-GAT1-SLC-PAP. Transferring the obtained plasmid pSP-GM2-GAT1-SLC-PAP into the strain obtained in the step (A) by using a lithium acetate transformation method. The nucleotide sequence of the GAT1 is shown as SEQ ID No. 2; the nucleotide sequence of the SLC is shown as SEQ ID No. 3; the nucleotide sequence of PAP is shown as SEQ ID No. 4.
In the step (E), the simultaneous overexpression of the GAT1 gene encoding glycerol-3-phosphate acyltransferase and the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase and the DGAT gene encoding diacylglycerol acyltransferase of Saccharomyces cerevisiae can be achieved by simultaneously introducing the GAT1 gene encoding glycerol-3-phosphate acyltransferase and the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase and the DGAT gene encoding diacylglycerol acyltransferase into Saccharomyces cerevisiae.
In a specific embodiment of the invention, overexpression of the GAT1 gene encoding glycerol-3-phosphate acyltransferase, the SLC gene encoding phosphatidic acid acyltransferase, the PAP gene encoding phosphatidic acid phosphatase and the DGAT gene encoding diacylglycerol acyltransferase is achieved by constructing a plasmid that drives expression of the gene of interest by a strong promoter. And (3) taking a Saccharomyces cerevisiae YS58 genome as a template, carrying out PCR amplification to obtain a DGAT gene fragment, and connecting the DGAT gene fragment to the pSP-GM2-GAT1-SLC-PAP plasmid obtained in the step (D) to obtain a plasmid pSP-GM2-GAT1-SLC-PAP-DGAT. Transferring the obtained plasmid pSP-GM2-GAT1-SLC-PAP-DGAT into the strain obtained in the step (A) by using a lithium acetate transformation method. The nucleotide sequence of the GAT1 is shown as SEQ ID No. 2; the nucleotide sequence of the SLC is shown as SEQ ID No. 3; the nucleotide sequence of PAP is shown as SEQ ID No. 4; the nucleotide sequence of the DGAT is shown as SEQ ID No. 5.
In some specific preferred embodiments of the present invention, the above-described construction method of OPO-type structural ester-producing genetically engineered bacterium comprising step (A) can be understood as a method for increasing the ratio of fatty acids in Saccharomyces cerevisiae C16:0 and C18:1 by replacing the OLE1 gene encoding Δ9 desaturase with the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase in the method of step (A), thereby constructing recombinant yeast cell A (also referred to as OPO-type structural ester-producing genetically engineered bacterium A herein) comprising the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase, thereby achieving an increase in the ratio of fatty acids in Saccharomyces cerevisiae C16:0 and C18:1.
In other specific preferred embodiments of the present invention, the above-described construction method of the OPO-structural ester-producing genetically engineered strain comprising steps (A) and (S2) may be understood as a method for increasing the ratio of fatty acids in Saccharomyces cerevisiae C16:0 and C18:1, wherein the method in step (A) is followed by replacing the OLE1 gene encoding Δ9 desaturase with the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase, thereby improving the ratio of fatty acids in Saccharomyces cerevisiae C16:0 and C18:1, and then the method in steps (B), (C), (D) and (E) are followed, respectively, thereby increasing the ratio of fatty acids in Saccharomyces cerevisiae C16:0 and C18:1.
From the above, it can be seen that the genetically engineered bacteria for producing OPO structural ester provided by the invention is obtained by firstly performing the modification of step (A) in Saccharomyces cerevisiae to obtain an initial genetically engineered bacteria A, and then further performing the modification of step (B) and/or (C) and/or (D) and/or (E) based on the initial genetically engineered bacteria (A). Wherein step (A) inhibits expression of an endogenous OLE1 gene encoding Δ9 desaturase having C16:0 and C18:0 as substrates, while expressing stearoyl-CoA Δ9 desaturase ChDes9-2 having C18:0 as substrate; step (B) overexpressing the GAT1 gene encoding glycerol-3-phosphate acyltransferase or the SLC gene encoding phosphatidic acid acyltransferase or the PAP gene encoding phosphatidic acid phosphatase or the DGAT gene encoding diacylglycerol acyltransferase of Saccharomyces cerevisiae while expressing stearoyl-CoA.DELTA.9 desaturase ChDes9-2 with C18:0 as a substrate; step (C) of overexpressing the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase of Saccharomyces cerevisiae while expressing stearoyl-CoA.DELTA.9 desaturase ChDes9-2 with C18:0 as a substrate; step (D) overexpressing the GAT1 gene encoding glycerol-3-phosphate acyltransferase and the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase of Saccharomyces cerevisiae while expressing stearoyl-CoA.DELTA.9 desaturase ChDes9-2 with C18:0 as a substrate; step (E) overexpressing the GAT1 gene encoding glycerol-3-phosphate acyltransferase and the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase and the DGAT gene encoding diacylglycerol acyltransferase of Saccharomyces cerevisiae while expressing stearoyl-CoA.DELTA.9 desaturase ChDes9-2 using C18:0 as a substrate. The invention improves the content of OPO structural ester in the saccharomyces cerevisiae and breaks through the limitation of OPO structural ester sources.
It will be appreciated by those skilled in the art that the above-described steps (A) to (B), (C), (D) or (E) for constructing an OPO-structural ester-producing genetically engineered bacterium can also be understood as a genetically modified host organism, and thus the OPO-structural ester-producing genetically engineered bacterium is also referred to as an OPO-structural ester-producing genetically modified yeast cell in the present invention.
The genetic modification is an engineered genetic modification comprising modulation of protein expression, in particular, an increase or decrease in expression of the genetically modified protein.
For example, a genetically modified yeast cell A obtained after modification in step (A) that has increased stearoyl-CoA Δ9 desaturase (ChDes 9-2) protein activity levels and decreased Δ9 desaturase (OLE 1) protein activity levels with C16:0 and C18:0 as substrates relative to an otherwise identical yeast cell lacking the genetic modification.
A genetically modified yeast cell B obtained after modification in step (B) which has increased glycerol-3-phosphate acyltransferase (GAT 1) or phosphatidic acid acyltransferase (SLC) or Phosphatidic Acid Phosphatase (PAP) or diacylglycerol acyltransferase (DGAT) protein activity level relative to an otherwise identical yeast cell lacking the genetic modification.
A genetically modified yeast cell C obtained after modification in step (C) that increases the level of phosphatidic acid acyltransferase (SLC) and Phosphatidic Acid Phosphatase (PAP) protein activity relative to an otherwise identical yeast cell lacking the genetic modification.
A genetically modified yeast cell D obtained after modification in step (D) that has increased levels of glycerol-3-phosphate acyltransferase (GAT 1), phosphatidic acid acyltransferase (SLC) and Phosphatidic Acid Phosphatase (PAP) protein activity relative to an otherwise identical yeast cell lacking the genetic modification.
A genetically modified yeast cell E obtained after modification in step (E) that increases the level of glycerol-3-phosphate acyltransferase (GAT 1), phosphatidic acid acyltransferase (SLC), phosphatidic Acid Phosphatase (PAP) and diacylglycerol acyltransferase (DGAT) protein activity relative to an otherwise identical yeast cell lacking the genetic modification.
It will also be appreciated by those skilled in the art that the above-described OPO-structural ester-producing genetically modified yeast cells B-E are each obtained by optimizing the lipid or lipid precursor synthesis pathway of genetically modified yeast cell A, and thus that the OPO-structural ester-producing genetically modified yeast cells B-E have increased stearoyl-CoA Δ9 desaturase (ChDes 9-2) protein activity levels and that the genetic modification has decreased Δ9 desaturase (OLE 1) protein activity levels with C16:0 and C18:0 as substrates relative to an otherwise identical yeast cell lacking the genetic modification.
Research results show that the genetically engineered bacteria for producing OPO structural ester provided by the invention contain more than 10% of lipid by dry weight of yeast cells after fermentation culture.
The application of the genetically engineered bacterium or the genetically engineered bacterium constructed by the method in the production of OPO structural ester can be understood as a method for producing OPO structural ester by using the genetically engineered bacterium or the genetically engineered bacterium constructed by the method, and can be further understood as a method for preparing or producing C16:0 and C18:1 by using the genetically engineered bacterium or the genetically engineered bacterium constructed by the method.
Based on the above, the construction method of the genetically engineered bacteria for producing OPO structural ester provided by the invention can be understood as the application of the genetically engineered bacteria for producing OPO structural ester in the embodiment of the invention in improving the ratio of C16:0 and C18:1 fatty acids in Saccharomyces cerevisiae;
or, the genetically engineered bacterium for producing OPO structural ester in the embodiment of the invention is applied to improving the fatty acid ratio of C16:0 of saccharomyces cerevisiae on the sn-2 position of triglyceride;
or, the genetically engineered bacterium for producing OPO structural ester in the embodiment of the invention is applied to improving the ratio of fatty acid in the sn-1 and 3 positions of triglyceride of C18:1 of Saccharomyces cerevisiae;
Or, the genetically engineered bacteria for producing OPO structural ester in the embodiment of the invention are applied to the preparation or production of OPO structural ester.
In some embodiments of the invention, the genetically engineered bacteria are inoculated into a fermentation medium for fermentation culture, and then the obtained fermentation culture solution is separated and purified to obtain OPO structural ester.
Specifically, the genetically engineered bacteria are inoculated into a fermentation medium, and fermentation culture is carried out, wherein the engineering bacteria provided by the invention are inoculated into the YNB auxotroph medium, and shake flask culture and activation are carried out at 30 ℃ and 200rpm to obtain seed liquid; and then inoculating the seed solution to the YNB auxotroph medium for culturing for 72 hours at 30 ℃ under shaking at 200rpm, and collecting the fermentation product.
In some specific embodiments of the present invention, the method for preparing or producing C16:0 and C18:1 using the genetically engineered bacterium described above or the genetically engineered bacterium constructed by the method described above comprises the steps of:
(1) Fermenting and culturing the recombinant strain in the first aspect in YNB auxotroph medium, and collecting fermentation product.
In a specific embodiment of the present invention, the steps are specifically: inoculating the engineering bacteria to the YNB auxotroph culture medium, and culturing and activating at 30 ℃ and 200rpm by shaking to obtain seed liquid; the seed solution was then inoculated into the YNB auxotroph medium at 30℃and shaking flask at 200rpm for 72 hours.
(2) The fatty acid in the fermentation product is extracted by a saponification method, so that C16:0 and C18:1 are obtained.
In a specific embodiment of the present invention, the steps are specifically: placing the fermentation product in a methanol solution containing 10% (volume percent) KOH; then condensing and refluxing for 2 hours at 80 ℃, collecting condensed and refluxed liquid to obtain fatty acid salt, and acidifying by hydrochloric acid (adding 4mL of hydrochloric acid with the concentration of 6M) to obtain fatty acid.
In other specific embodiments of the present invention, the method for preparing or producing OPO-type structural ester using the genetically engineered bacterium described above or the genetically engineered bacterium constructed by the above method comprises the steps of:
(1) Fermenting and culturing the engineering bacteria in the first aspect in YNB auxotroph culture medium, and collecting fermentation products.
In a specific embodiment of the present invention, the steps are specifically: inoculating the engineering bacteria to the YNB auxotroph culture medium, and culturing and activating at 30 ℃ and 200rpm by shaking to obtain seed liquid; and then inoculating the seed solution to the YNB auxotroph medium for culturing for 72 hours at 30 ℃ under shaking at 200rpm, and collecting the fermentation product.
(2) Extracting grease in the fermentation product by an extraction method to obtain OPO structural ester.
In a specific embodiment of the present invention, the steps are specifically: repeatedly freezing and thawing the collected thalli four times, then performing ultrasonic crushing, placing the crushed thalli suspension into chloroform-methanol (v: v=2:1) solution, shaking and mixing uniformly, sucking the lower solution, and performing rotary evaporation to obtain the grease. The obtained oil was dissolved in diethyl ether, and then purified with n-hexane: diethyl ether: glacial acetic acid (50:50:1, v/v/v) was used as a developing agent and the triglycerides were isolated by thin layer chromatography.
III, correlation analysis and detection method
(1) Triglyceride fatty acid composition detection.
The fatty acid composition is determined according to GB/T17376-2008 animal and vegetable oil fatty acid methyl ester preparation and GB/T17377-2008 animal and vegetable oil fatty acid methyl ester gas chromatographic analysis.
(2) Triglyceride structural analysis.
The composition of Sn-2 fatty acid is measured according to GB/T24894-2010 'measurement of 2-fatty acid component of animal and vegetable fat and oil triglyceride molecule', and the content of Sn-1,3 fatty acid of Saccharomyces cerevisiae and the composition and content of triglyceride in Saccharomyces cerevisiae are calculated and analyzed according to the theory of Sn-1, 3-random-2-random distribution.
In the invention, in the Saccharomyces cerevisiae YS58, firstly, the endogenous OLE1 gene is replaced by the ChDes9-2 gene, so that the anabolic pathway of C16:1 is inhibited while the anabolic pathway of C18:1 in the Saccharomyces cerevisiae is maintained, and the ratio of C16:0 to C18:1 fatty acid in the Saccharomyces cerevisiae is improved. And then over-expressing GAT1 and/or SLC and/or PAP and/or DGAT genes endogenous in the Saccharomyces cerevisiae on the basis, constructing Saccharomyces cerevisiae engineering bacteria, and improving the ratio of OPO structural ester in grease in the Saccharomyces cerevisiae.
IV, examples
The present invention will be specifically described below by way of specific examples. The experimental methods described below, unless otherwise specified, are all laboratory conventional methods and employ conventional experimental equipment. The experimental materials described below, unless otherwise specified, are commercially available.
The following media were used in the examples, wherein the percentages are by mass:
final concentration composition of LB medium: 0.5% of yeast powder, 1% of peptone, 0.5% of sodium chloride and 1.7% of agar (for solid culture medium), wherein the solvent is deionized water, and the pH value is natural. % are expressed in g/100ml.
YPD medium final concentration composition: 1% of yeast powder, 2% of peptone, 2% of glucose and 1.7% of agar (for solid culture medium), wherein the solvent is deionized water, and the pH value is natural. % are expressed in g/100ml.
Final concentration composition of YNB-Trp fermentation Medium: 0.17% of an amino-free yeast nitrogen source (Shanghai Yeast Biotechnology Co., ltd., S24483), 0.5% of ammonium sulfate, 0.129%Dropout Supplement-TRP (Peking Coolibo technology Co., ltd., PM 2270), 2% of glucose, deionized water as a solvent, pH6.0. The addition of D-S amino acid is determined by the selection marker of the expression vector, and 1.7% agar is added to the solid medium based on the above components. % are expressed in g/100ml.
Final concentration composition of YNB-Ura/Trp fermentation medium: 0.17% of an amino-free yeast nitrogen source (Shanghai Yeast Biotechnology Co., ltd., S24483), 0.5% of ammonium sulfate, 0.12%Dropout Supplement-Ura/Trp (Beijing Cool Bo Tech Co., ltd., PM 2270), 2% of glucose, deionized water as a solvent, pH6.0. The addition of D-S amino acid is determined by the selection marker of the expression vector, and 1.7% agar is added to the solid medium based on the above components. % are expressed in g/100ml.
In the following examples: the nucleotide sequence of the ChDes9-2 is 1045-2085 of SEQ ID No. 1; the nucleotide sequence of the GAT1 gene encoding glycerol-3-phosphate acyltransferase is SEQ ID No.2; the nucleotide sequence of the SLC gene for encoding phosphatidic acid acyltransferase is SEQ ID No.3; the nucleotide sequence of PAP gene for encoding phosphatidic acid phosphatase is SEQ ID No.4; the nucleotide sequence of the DGAT gene encoding diacylglycerol acyltransferase is SEQ ID No.5.
Example 1: construction of CHDes9-2 engineering bacteria and analysis of fatty acid and OPO type structural ester content thereof
1. Construction of ChDes9-2 engineering bacteria
(1) Design of the ChDes9-2 recombination cassette
The position of the gene OLE1 on the chromosome is searched through NCBI website, and an upstream homology arm is designed at 60bp position on the upstream side of the OLE1 and a downstream homology arm is designed at 60bp position on the downstream side according to the specific position of the OLE1 gene sequence. The Δ9 desaturase gene ChDes9-2 is not readily available from the North hucho taimen, and therefore the gene sequence of ChDes9-2 and promoter P will be referred to on NCBI PGK Terminator T ADH1 The 60bp homology arm on the upstream and downstream of the OLE1 gene and the screening gene TRP sequence were delivered to the Suzhou Jinweizhi company to directly synthesize a gene fragment ChDes9-2 recombination cassette.
The nucleotide sequence of the gene fragment ChDes9-2 recombination box is SEQ ID No.1. Wherein, the 1 st to 60 th positions are the upstream homology arm of OLE1, the 61 st to 1044 th positions are PGK1 promoter, the 1045 th to 2085 th positions are ChDes9-2 gene, the 2086 th to 2250 th positions are ADH1 terminator, the 2251 th to 3206 th positions are TRP screening genes, and the 3207 th to 3266 th positions are the downstream homology arm of OLE 1.
(2) Construction of ChDes9-2 engineering bacteria
The ChDes9-2 recombination cassette gene fragment is introduced into a Saccharomyces cerevisiae YS58 strain by a lithium acetate transformation method for screening and expression. The method comprises the following steps:
the host strain is Saccharomyces cerevisiae YS58 (with the strain deposit number of ST12300, purchased from Beijing Rui Si Bo Biotechnology Co., ltd.), and the classical yeast transformation kit (SK 2400, beijing Cool Bo Biotechnology Co., ltd.) is used for yeast transformation. YPD medium was cultured to the log phase after two activations for preparation of competent cells, and cells were collected by freeze centrifugation and suspended with LiAc at 1/10 concentration after two water washes. Preparing a premix: 240 μl of PEG Solution (Beijing cool pacing technology Co., ltd. SL 9510), 36 μl of LiAc Solution (Beijing cool pacing technology Co., ltd. YT 0002), 10 μl of Carrier DNA (Beijing cool pacing technology Co., ltd. YT 0003), 5 μl of fragments (ChDes 9-2 recombination cassette described above) and a final volume of 360 μl were filled with sterile water. The premix was injected into YS58 competent cells, followed by centrifugation in a water bath at 30℃and 42℃for 30 minutes, followed by resuspension of the cells with sterile water and plating onto TRP-deficient screening medium (PM 2251, beijing Lan Bosi Tex Biol Co., ltd.) and culturing at 30℃for 2-4 days to screen positive transformants.
2. Fatty acid and OPO structural ester analysis of CHDes9-2 engineering bacteria
(1) Engineering bacteria culture
Obtaining seed liquid: inoculating an original strain YS58 into a YPD culture medium, inoculating the ChDes9-2 genetically engineered bacterium into an YNB-Trp auxotroph culture medium, culturing for 48 hours in a constant temperature shaking table at 200rpm and 30 ℃, and obtaining seed liquid after activating culture.
Fermentation culture: inoculating YS58 seed solution obtained by activation culture into YPD culture medium, and inoculating ChDes9-2 engineering bacteria seed solution obtained by repeated activation twice into YNB-Trp auxotroph culture medium. Wherein the strain YS58 is a control strain, and the strain is subjected to shake flask fermentation culture at 30 ℃ and 200rpm for 72 hours to obtain a YS58 fermentation product and a ChDes9-2 fermentation product.
(2) Extraction of thallus fatty acid
The yeast fatty acid is extracted by saponification method. The method comprises the following steps:
the fermentation products of the above strains were collected by centrifugation for 5min at 6000g and washed twice with 15mL of 10% (volume percent) KOH-methanol solution (10 g/100mL KOH, 95% methanol and 5% deionized water) and transferred to a grind flask. Then placing in a water bath kettle (WUHan An Dexin ADX-HH-4 China) at 80 ℃ to condense and reflux for 2 hours, allowing the strong base to perform saponification reaction with intracellular esters, collecting condensed reflux liquid to obtain fatty acid salt, and acidifying with hydrochloric acid (adding 4mL of hydrochloric acid with the concentration of 6M) to obtain fatty acid.
(3) Treatment of bacterial fatty acids
Adding 4mL of 1% (volume percentage) sulfuric acid-methanol solution into the fatty acid obtained by the saponification method in the step 2, uniformly mixing, transferring into a clean test tube with a plug, and reacting in a water bath kettle at 60 ℃ for 1h; cooling at room temperature, and adding 5mL of saturated NaCl solution to separate out impurities such as protein; then adding 1mL of n-hexane, and collecting an organic phase to obtain fatty acid methyl ester through extraction; the water content of the sample was removed with anhydrous sodium sulfate.
(4) Extraction of triglycerides
Extracting yeast triglyceride by extraction method and thin layer chromatography. The method comprises the following steps:
and (3) collecting thalli after 6000g and 5min centrifugation of fermentation products of the strains, washing twice, repeatedly freezing and thawing the collected thalli, then performing ultrasonic crushing (KQ-500 DE China of Kunshan ultrasonic instruments Co., ltd.), placing the crushed thalli suspension into chloroform methanol (v: v=2:1) solution with twice volume, shaking and mixing uniformly, sucking the lower solution, and performing rotary evaporation to obtain the grease. The obtained oil was dissolved in diethyl ether, and then purified with n-hexane: diethyl ether: glacial acetic acid (50:50:1, v/v/v) was used as a developing agent and the triglycerides were isolated by thin layer chromatography.
(5) Triglyceride fatty acid composition
The fatty acid composition is determined according to GB/T17376-2008 animal and vegetable oil fatty acid methyl ester preparation and GB/T17377-2008 animal and vegetable oil fatty acid methyl ester gas chromatographic analysis.
(6) Triglyceride analysis
The composition of Sn-2 fatty acid is measured according to GB/T24894-2010 (measurement of 2-fatty acid component of animal and vegetable oil and fat triglyceride molecule), and the Sn-1, 3-fatty acid content of Saccharomyces cerevisiae and the type and content of triglyceride in Saccharomyces cerevisiae are calculated and analyzed according to the theory of Sn-1, 3-random-2-random distribution.
GC detection of fatty acid methyl ester: the fatty acid methyl esters obtained in the above steps 3 and 6 were subjected to gas phase detection and analysis using a DB-WAX column of Shimadzu gas phase analyzer (Shimadzu GC-2014 Japan), and the fatty acid methyl esters were separated by a two-stage temperature-raising program using high purity nitrogen as a detection carrier, with an initial column temperature of 62℃for 5 minutes, a temperature-raising rate of 3℃per minute, and a column temperature gradually raised from 60℃to 215℃with a retention time of 33 minutes.
The standard was a fatty acid methyl ester mixed standard (Shanghai Yuan Yes Biotechnology Co., ltd., B25881).
As a result, as shown in FIG. 2, the main fatty acids of Saccharomyces cerevisiae are C16:0, C16:1, C18:0, C18:1, with the most abundant amounts being C16:1 and C18:1. As shown by the implementation results, after the OLE1 gene in the YS58 strain is replaced by the ChDes9-2 gene for expression, the ChDes9-2 strain has 10 percent of increased C16:1 content and 13 percent of increased C18:1 content compared with the YS58 strain C16:0 content. It was revealed that the replacement of the OLE1 gene with the ChDes9-2 gene not only inhibited the synthesis of bacterial C16:1 but also promoted the synthesis and accumulation of C18:1 and C16:0.
As shown in FIG. 3, after the original strain YS58OLE1 gene is replaced by the ChDes9-2 gene, the ratio of OPO structural ester in triglyceride of the engineering bacterium ChDes9-2 is increased by 5% relative to the original strain YS58, which shows that the replacement of the OLE1 gene by the ChDes9-2 promotes the synthesis of OPO structural ester.
Example 2: effects of overexpression of GAT1 Gene encoding Glycerol-3-phosphate Acyltransferase or SLC Gene encoding phosphatidic acid Acyltransferase or PAP Gene encoding phosphatidic acid phosphatase or DGAT Gene encoding diacylglycerol Acyltransferase in ChDes9-2 engineering bacterium on fatty acid and OPO structural ester content
1. Construction of engineering bacteria over-expressing GAT1 or SLC or PAP or DGAT
(1) Construction of GAT1 or SLC or PAP or DGAT overexpression vectors
The GAT1 gene encoding glycerol-3-phosphoryl transferase and the SLC gene encoding phosphatidic acid acylase and the PAP gene encoding phosphatidic acid phosphatase and the DGAT gene encoding diacylglycerol acylase are both derived from Saccharomyces cerevisiae YS58, so that the GAT1, SLC, PAP and DGAT gene fragments are obtained by PCR amplification by using the Saccharomyces cerevisiae YS58 genome as a template. GAT1 and DGAT gene fragments were ligated to plasmid pSP-GM2, respectivelyhttps://www.addgene.org/64740 Between BamHI and KpnI cleavage sites to obtain expression vectors pSP-GM2-GAT1 and pSP-GM2-DGAT, and the SLC and PAP gene fragments are respectively connected between NotI and SpeI cleavage sites of plasmid pSP-GM2 to obtain expression vectors pSP-GM2-SLC and pSP-GM2-PAP.
The nucleotide sequence of the gene GAT1 is SEQ ID No.3; the nucleotide sequence of the SLC gene of the gene coding phosphatidic acid acyl transferase is shown in SEQ ID No. 4; the nucleotide sequence of the gene PAP is shown as SEQ ID No. 5; the nucleotide sequence of the DGAT gene of the gene coding diacylglycerol acyltransferase is shown as SEQ ID No. 6.
TABLE 1 primers used in this example
Figure BDA0003824268610000181
(2) Construction of engineering bacteria GAT1-ChDes9-2, SLC-ChDes9-2, PAP-ChDes9-2 and DGAT-ChDes9-2
The expression plasmids pSP-GM2-GAT1, pSP-GM2-SLC, pSP-GM2-PAP and pSP-GM2-DGAT are respectively transferred into host bacteria, namely the ChDes9-2 engineering bacteria obtained in the first embodiment, so as to obtain Saccharomyces cerevisiae engineering bacteria GAT1-ChDes9-2, SLC-ChDes9-2, PAP-ChDes9-2 and DGAT-ChDes9-2.
2. Influence of overexpression of GAT1 or SLC or PAP or DGAT Gene on the ratio of mycofatty acids and OPO-type structural esters
(1) Engineering bacteria culture
Obtaining seed liquid: recombinant bacteria ChDes9-2 are inoculated into YNB-Trp auxotroph medium, GAT1-ChDes9-2, SLC-ChDes9-2, PAP-ChDes9-2 and DGAT-ChDes9-2 genetic engineering bacteria are respectively inoculated into YNB-Trp/Ura auxotroph medium, and are cultured for 48 hours in a constant temperature shaking table at 200rpm and 30 ℃ to obtain seed liquid after activation culture.
Fermentation culture: inoculating the ChDes9-2 seed solution obtained by the activation culture into YNB-Trp auxotroph culture medium, and inoculating GAT1-ChDes9-2, SLC-ChDes9-2, PAP-ChDes9-2 and DGAT-ChDes9-2 gene engineering bacteria seed solution obtained by repeated activation twice into YNB-Trp/Ura auxotroph culture medium respectively. Wherein the strain ChDes9-2 is a control strain, and the strain ChDes9-2 is subjected to shake flask fermentation culture at 30 ℃ and 200rpm for 72 hours to obtain a ChDes9-2 fermentation product, a GAT1-ChDes9-2, a SLC-ChDes9-2, a PAP-ChDes9-2 and a DGAT-ChDes9-2 fermentation product.
(2) The extraction of the cell fatty acid was the same as in example 1.
(3) The treatment of the cell fatty acid was the same as in example 1.
(4) The extraction of triglycerides was the same as in example 1.
(5) Triglyceride fatty acid composition was the same as in example 1.
(6) Triglyceride analysis was performed in the same manner as in example 1.
As shown in FIG. 4, compared with the integrated bacteria ChDes9-2, the total fatty acid types and the total fatty acid content of engineering bacteria GAT1-ChDes9-2, SLC-ChDes9-2, PAP-ChDes9-2 and DGAT-ChDes9-2 are not obviously changed, which indicates that the overexpression of GAT1, SLC or PAP or DGAT genes does not cause the obvious change of the total fatty acid of the bacterial body.
As shown in FIG. 5, the ratio of OPO structural ester in engineering bacteria GAT1-ChDes9-2 to total triglyceride is improved by 3% relative to the composition of triglyceride in the integrated bacteria ChDes 9-2. The ratio of OPO structural ester in engineering bacteria SLC-ChDes9-2 in total triglyceride is increased by 4%, the ratio of OPO structural ester in engineering bacteria PAP-ChDes9-2 in total triglyceride is increased by 3%, and the ratio of OPO structural ester in engineering bacteria DGAT-ChDes9-2 in total triglyceride is reduced by 2%. It was demonstrated that Saccharomyces cerevisiae GAT1 gene encoding glycerol-3-phosphate acyltransferase, SLC gene encoding phosphatidic acid acyltransferase and PAP gene encoding phosphatidic acid phosphatase can promote synthesis of OPO type structural esters, wherein SLC gene encoding phosphatidic acid acyltransferase has the greatest effect on synthesis of OPO type structural esters. The DGAT gene encoding diacylglycerol acyltransferase has a certain inhibition effect on the synthesis of OPO type structural ester.
Example 3: effect of overexpression of SLC Gene encoding phosphatidic acid acyltransferase and PAP Gene encoding phosphatidic acid phosphatase on OPO-type structural ester content in CHDes9-2 engineering bacterium
1. Construction of SLC Gene encoding phosphatidic acid acyltransferase and PAP Gene engineering bacterium encoding phosphatidic acid phosphatase by overexpressing Gene
(1) Construction of pSP-GM2-SLC-PAP expression vector
The SLC gene and the PAP gene encoding phosphatidic acid acyltransferase are derived from Saccharomyces cerevisiae YS58, so that the Saccharomyces cerevisiae YS58 genome is used as a template, PCR amplification is performed to obtain SLC gene fragments, and the obtained SLC gene fragments are connected between BamHI and KpnI of plasmid pSP-GM2-PAP enzyme cleavage site obtained in example 2 to obtain an expression vector pSP-GM2-SLC-PAP.
The nucleotide sequence of the gene SLC is shown as SEQ ID No. 3; the nucleotide sequence of the gene PAP is shown as SEQ ID No. 4.
TABLE 2 primers used in this example
Figure BDA0003824268610000201
(2) Construction of Saccharomyces cerevisiae engineering bacteria SLC-PAP-ChDes9-2
Transferring the expression plasmid pSP-GM2-SLC-PAP into host bacteria, namely the ChDes9-2 engineering bacteria obtained in the first embodiment, to obtain the Saccharomyces cerevisiae engineering bacteria SLC-PAP-ChDes9-2.
2. Simultaneous overexpression of SLC and PAP genes on fatty acid and OPO-type structural ester content
(1) Engineering bacteria are cultured, and the strain SLC-PAP-ChDes9-2 is fermented and cultured by the same method as that of the embodiment 2, and meanwhile recombinant bacteria ChDes9-2 are used as a control.
(2) The extraction of the cell fatty acid was the same as in example 1.
(3) The treatment of the cell fatty acid was the same as in example 1.
(4) The extraction of triglycerides was the same as in example 1.
(5) Triglyceride fatty acid composition was the same as in example 1.
(6) Triglyceride analysis was performed in the same manner as in example 1.
As shown in FIG. 6, compared with the composition of triglyceride in engineering bacteria CHDes9-2, the ratio of OPO structural ester in the engineering bacteria SLC-PAP-CHDes9-2 triglyceride in total triglyceride is improved by 7%, which shows that the overexpression of Saccharomyces cerevisiae glycerol-3-phosphate acyltransferase SLC and PAP gene for encoding phosphatidic acid phosphatase can catalyze the synthesis of OPO structural ester more efficiently.
Example 4: effect of overexpression of GAT1 Gene encoding Glycerol-3-phosphate Acyltransferase, SLC Gene encoding phosphatidic acid Acyltransferase and PAP Gene encoding phosphatidic acid phosphatase in ChDes9-2 engineering bacterium on OPO-type structural ester content
1. Construction of overexpression Gene oil-3-Phosphoryl acid transferase GAT1, SLC Gene encoding Phosphatidic acid acyltransferase and PAP Gene engineering bacterium encoding Phosphatidic acid phosphatase
(1) Construction of pSP-GM2-GAT1-SLC-PAP expression vector
The GAT1 gene encoding glycerol-3-phosphate acyltransferase, the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase are both derived from Saccharomyces cerevisiae YS58, so that the genome of Saccharomyces cerevisiae YS58 is used as a template for PCR amplification to obtain GAT1 gene fragments. The GAT1 gene fragment was ligated between the cleavage sites NheI and KpnI of the plasmid pSP-GM2-SLC-PAP described in example 3 to give the plasmid pSP-GM2-GAT1-SLC-PAP.
The nucleotide sequence of the gene fragment GAT1 is shown in SEQ ID No. 2; the nucleotide sequence of the SLC is shown as SEQ ID No. 3; the nucleotide sequence of PAP is shown as SEQ ID No. 4.
TABLE 3 primers used in this example
Figure BDA0003824268610000211
(2) Construction of Saccharomyces cerevisiae engineering GAT1-SLC-PAP-ChDes9-2
Transferring the expression plasmid pSP-GM2-GAT1-SLC-PAP into host bacteria, namely the ChDes9-2 engineering bacteria obtained in the first embodiment, to obtain Saccharomyces cerevisiae engineering bacteria GAT1-SLC-PAP-ChDes9-2.
2. Influence of overexpression of GAT1, SLC and PAP genes on OPO structural ester of engineering bacteria
(1) Engineering bacteria are cultured, and the fermentation culture strain GAT1-SLC-PAP-ChDes9-2 is used as a control in the same way as the culture method of the embodiment 2.
(2) The extraction of the cell fatty acid was the same as in example 1.
(3) The treatment of the cell fatty acid was the same as in example 1.
(4) The extraction of triglycerides was the same as in example 1.
(5) Triglyceride fatty acid composition was the same as in example 1.
(6) Triglyceride analysis was performed in the same manner as in example 1.
As shown in FIG. 6, the OPO structural ester in the engineering bacterium GAT1-SLC-PAP-ChDes9-2 reached 17% relative to the triglyceride composition in the integrative bacterium ChDes9-2, which indicates that over-expression of GAT1, SLC and PAP can enhance synthesis of OPO structural ester.
Example 5: effects of overexpression of GAT1 Gene encoding Glycerol-3-phosphate acyltransferase, SLC Gene encoding phosphatidic acid acyltransferase, PAP Gene encoding phosphatidic acid phosphatase and DGAT Gene encoding diacylglycerol acyltransferase in ChDes9-2 engineering bacterium on OPO type structural ester content of engineering bacterium
1. Construction of GAT1 Gene encoding Glycerol-3-phosphate Acyltransferase, SLC Gene encoding phosphatidic acid Acyltransferase, PAP Gene encoding phosphatidic acid phosphatase and DGAT Gene engineering bacterium encoding diacylglycerol Acyltransferase
(1) Construction of pSP-GM2-GAT1-SLC-PAP-DGAT expression vector
The GAT1 gene encoding glycerol-3-phosphoryl transferase, the SLC gene encoding phosphatidic acid acylase, the PAP gene encoding phosphatidic acid phosphatase and the DGAT gene encoding diacylglycerol acylase are both derived from Saccharomyces cerevisiae YS58, so that the genome of Saccharomyces cerevisiae YS58 is used as a template for PCR amplification to obtain a DGAT gene fragment. The DGAT gene fragment was ligated between the plasmid pSP-GM2-GAT1-SLC-PAP cleavage site SpeI and SacI described in example 4 to give the plasmid pSP-GM2-GAT1-SLC-PAP-DGAT.
The nucleotide sequence of the gene fragment GAT1 is shown in SEQ ID No. 2; the nucleotide sequence of the SLC is shown as SEQ ID No. 3; the nucleotide sequence of PAP is shown as SEQ ID No. 4; the nucleotide sequence of PAP is shown as SEQ ID No. 4.
TABLE 4 primers used in this example
Figure BDA0003824268610000221
(2) Construction of Saccharomyces cerevisiae Gene engineering bacterium GAT1-SLC-PAP-DGAT-ChDes9-2
Transferring the expression plasmid pSP-GM2-GAT1-SLC-PAP-DGAT into host bacteria, namely the ChDes9-2 engineering bacteria obtained in the first embodiment, to obtain engineering bacteria GAT1-SLC-PAP-DGAT-ChDes9-2.
2. Effects of overexpression of GAT1, SLC, PAP and DGAT genes on OPO-type structural esters
(1) Engineering bacteria are cultured, and the fermentation culture strain GAT1-SLC-PAP-DGAT-ChDes9-2 is used as a control in the same way as the culture method of the embodiment 2.
(2) The extraction of the cell fatty acid was the same as in example 1.
(3) The treatment of the cell fatty acid was the same as in example 1.
(4) The extraction of triglycerides was the same as in example 1.
(5) Triglyceride fatty acid composition was the same as in example 1.
(6) Triglyceride analysis was performed in the same manner as in example 1.
As shown in FIG. 6, compared with engineering bacteria CHDes9-2, the ratio of OPO structural ester in engineering bacteria GAT1-SLC-PAP-DGAT-CHDes9-2 triglyceride is only 14%, which is lower than that of engineering bacteria GAT1-SLC-PAP-CHDes9-2, and shows that when GAT1, SLC, PAP and DGAT are expressed simultaneously, the DGAT gene encoding diacylglycerol acyltransferase has relative inhibition on synthesis of OPO structural ester, and the OPO structural ester cannot be catalyzed to the greatest extent in the strain of CHDes 9-2.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are used for explaining the present invention, not to be construed as limiting the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.

Claims (10)

1. A genetically engineered bacterium for producing OPO structural esters is a recombinant yeast cell containing a ChDes9-2 gene encoding stearoyl-CoA delta 9 desaturase.
2. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium is a chassis-engineered recombinant yeast cell; preferably, the chassis engineering comprises inhibition of the c16:1 anabolic pathway; further preferred, the inhibition of the C16:1 anabolic pathway is a knockout of the OLE1 gene encoding Δ9 desaturase.
3. The genetically engineered bacterium of claim 2, wherein OLE1 gene encoding Δ9 desaturase in the genetically engineered bacterium is replaced with ChDes9-2 gene encoding stearoyl-coa Δ9 desaturase.
4. A genetically engineered bacterium of claim 2 or 3, wherein the genetically engineered bacterium is a recombinant yeast cell optimized for lipid or lipid precursor synthesis pathways; preferably, the optimization of the lipid or lipid precursor synthesis pathway comprises overexpressing one or more of the GAT1 gene encoding glycerol-3-phosphate acyltransferase, the SLC gene encoding phosphatidic acid acyltransferase, the PAP gene encoding phosphatidic acid phosphatase, and the DGAT gene encoding diacylglycerol acyltransferase in a recombinant yeast cell; further preferably, the lipid or lipid precursor is a triglyceride or fatty acid; still more preferably, the fatty acid comprises one or more of C16:0, C16:1, C18:0, C18:1.
5. The genetically engineered bacterium of claim 4, wherein,
the lipid or lipid precursor synthesis pathway is optimized for over-expressing the GAT1 gene encoding glycerol-3-phosphate acyltransferase or the SLC gene encoding phosphatidic acid acyltransferase or the PAP gene encoding phosphatidic acid phosphatase or the DGAT gene encoding diacylglycerol acyltransferase in a recombinant yeast cell;
alternatively, the lipid or lipid precursor synthesis pathway is optimized to overexpress the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase in recombinant yeast cells;
alternatively, the lipid or lipid precursor synthesis pathway is optimized to overexpress the GAT1 gene encoding glycerol-3-phosphate acyltransferase, the SLC gene encoding phosphatidic acid acyltransferase, and the PAP gene encoding phosphatidic acid phosphatase in recombinant yeast cells;
alternatively, the lipid or lipid precursor synthesis pathway is optimized for overexpression of the GAT1 gene encoding glycerol-3-phosphate acyltransferase, the SLC gene encoding phosphatidic acid acyltransferase, the PAP gene encoding phosphatidic acid phosphatase, and the DGAT gene encoding diacylglycerol acyltransferase in recombinant yeast cells.
6. The recombinant yeast cell of any one of claims 1-5, wherein the yeast cell is selected from the group consisting of: yarrowia lipolytica, rhodosporidium, oleaginous yeast, rhodotorula mucilaginosa, cryptococcus curvatus, candida fermentata, candida lacosaccharomyces, candida iron, candida tropicalis, candida utilis, candida Pityrosporum and saccharomyces cerevisiae, preferably saccharomyces cerevisiae.
7. A method of constructing the genetically engineered bacterium of any one of claims 1 to 6, comprising:
step (A), constructing a recombinant yeast cell A containing a ChDes9-2 gene encoding stearoyl-CoA delta 9 desaturase;
step (Z), optimizing lipid or lipid precursor synthesis pathway.
8. The method of claim 7, wherein step (a) comprises:
step M, expressing the ChDes9-2 gene encoding stearoyl-CoA delta 9 desaturase in yeast cells;
step N, knocking out the OLE1 gene encoding delta 9 desaturase from yeast cells;
preferably, step (A) comprises replacing the OLE1 gene encoding Δ9 desaturase with the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase to construct a recombinant yeast cell A comprising the ChDes9-2 gene encoding stearoyl-CoA Δ9 desaturase.
9. The method of claim 8, wherein step (a) comprises:
overexpressing the GAT1 gene encoding glycerol-3-phosphate acyltransferase or the SLC gene encoding phosphatidic acid acyltransferase or the PAP gene encoding phosphatidic acid phosphatase or the DGAT gene encoding diacylglycerol acyltransferase in the recombinant yeast cell to obtain a recombinant yeast cell B;
Alternatively, a recombinant yeast cell C is obtained by overexpressing the SLC gene encoding phosphatidic acid acyltransferase and the PAP gene encoding phosphatidic acid phosphatase in a recombinant yeast cell;
alternatively, a GAT1 gene encoding glycerol-3-phosphate acyltransferase, an SLC gene encoding phosphatidic acid acyltransferase, and a PAP gene encoding phosphatidic acid phosphatase are overexpressed in a recombinant yeast cell to obtain a recombinant yeast cell D;
alternatively, a GAT1 gene encoding glycerol-3-phosphate acyltransferase, an SLC gene encoding phosphatidic acid acyltransferase, a PAP gene encoding phosphatidic acid phosphatase and a DGAT gene encoding diacylglycerol acyltransferase are overexpressed in a recombinant yeast cell to obtain a recombinant yeast cell E.
10. Use of the genetically engineered bacterium according to any one of claims 1 to 5 or the genetically engineered bacterium constructed by the construction method according to any one of claims 6 to 9 for producing OPO-type structural esters; preferably, the application comprises the steps of inoculating the genetically engineered bacteria into a fermentation medium for fermentation culture, and then separating and purifying the obtained fermentation culture solution to obtain OPO structural ester; further preferably, the fermentation medium comprises one or more of YPD fermentation medium, YNB-Trp fermentation medium and YNB-Trp/Ura fermentation medium; still more preferably, the temperature of the fermentation culture is 30 ℃; and/or, the fermentation culture time is 72h.
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