CN117683650A - Yarrowia lipolytica engineering strain for high-level production of alpha-linolenic acid, construction method and application - Google Patents
Yarrowia lipolytica engineering strain for high-level production of alpha-linolenic acid, construction method and application Download PDFInfo
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- 235000020661 alpha-linolenic acid Nutrition 0.000 title claims abstract description 76
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Abstract
The invention belongs to the technical field of bioengineering, and discloses a yarrowia lipolytica engineering strain for producing alpha-linolenic acid at a high level, which is obtained by taking yarrowia lipolytica (Yarrowia lipolyticaPo f) as an initial strain, expressing delta 15 desaturase genes and cooperatively and over-expressing acyltransferase. The engineering bacteria of the invention have good stability, high content of intracellular grease alpha-linolenic acid, no time limitation of synthetic route, stable yield and low cost of fermentation substrate.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and relates to a yarrowia lipolytica engineering strain, in particular to a yarrowia lipolytica engineering strain for producing alpha-linolenic acid at a high level, a construction method and application.
Background
Alpha-linolenic acid (ALA, C18:3) Δ9Δ12，Δ15 ) Is a straight chain fatty acid containing eighteen carbon atoms and three double bonds, has a relative molecular mass of 278, is an important constituent of human cell membranes and is a necessary fatty acid for human bodies, and is widely applied in the fields of medical treatment, food, cosmetics and the like. At present, the alpha linolenic acid produced at home and abroad mainly comes from perilla (PerillaFrutescens Britt), but the extraction of the alpha linolenic acid from plants is limited by a plurality of factors, such as long growth period of the plants, high requirement on growth environment, large occupied area, low oil content and the like, so that the increasing market demands of people are difficult to meet.
The microbial fermentation method is not limited by seasons, can realize large-scale production, has the advantages of stable yield, reliable quality, wide sources of materials used for fermentation, low price and the like, and the yarrowia lipolytica (Yarrowia lipolytica) is currently used as a research hotspot for polyunsaturated fatty acid production. Since the naturally occurring fatty acid in yarrowia lipolytica is octadecadienoic acid (LA, C18:2), the fermentative production of alpha-linolenic acid with yarrowia lipolytica requires exogenous introduction of the Δ15 desaturase gene, the mechanism of which is shown in FIG. 1.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a yarrowia lipolytica engineering strain for producing alpha-linolenic acid at a high level, a construction method and application.
The technical scheme adopted for solving the technical problems is as follows:
an engineering strain of yarrowia lipolytica for high-level production of alpha-linolenic acid, which is obtained by taking yarrowia lipolytica (Yarrowia lipolytica Po f) as an original strain, expressing delta 15 desaturase genes and then cooperatively and over-expressing acyltransferase.
Further, the delta 15 desaturase gene is derived from orange Lin Youzhi yeast Lipomyces kononenkoaeLKFAD15, and the sequence of the delta 15 desaturase gene is shown in SEQ ID No. 1;
The acyltransferase is glycerol-3-phosphate linear transferase GPAT and/or lysophosphatidic acid acyltransferase LPAAT and/or diacylglycerol acyltransferase DGAT;
the diacylglycerol acyltransferase is derived from schizochytrium (Sp) ScDGAT, and the ScDGAT gene sequence is shown as SEQ ID NO. 2;
the glycerol-3-phosphate linear transferase is derived from Phaeodactylum tricornutum (Phaeodactylum tricornutum) PtGPAT, and the PtGPAT gene sequence is shown in SEQ ID NO. 3;
the lysophosphatidic acid acyltransferase is derived from PtLPAAT of Phaeodactylum tricornutum (Phaeodactylum tricornutum), and the PtLPAAT gene sequence is shown in SEQ ID NO. 4.
The construction method of the yarrowia lipolytica engineering strain for high-level production of alpha-linolenic acid comprises the following steps:
(1) Construction of recombinant plasmid PUC-Intc-Delta15 DEs
Taking pUC-Intc plasmid as a framework, and inserting an LKFAD15 gene expression cassette into the plasmid framework to obtain a recombinant plasmid PUC-Intc-delta 15DEs;
(2) Construction of genetically engineered strain Polf-D1 for producing alpha-linolenic acid
Transferring the recombinant plasmid pUC-Intc-delta 15DEs into uracil auxotroph yarrowia lipolytica Polf to obtain a yarrowia lipolytica engineering strain Polf-D1;
(3) Preparation of yarrowia lipolytica engineering strain Polf-D1 (URA-)
Inoculating the yarrowia lipolytica engineering strain Polf-D1 into a YPD-5FOA solid culture medium, and after single colony grows out, carrying out streak activation again in the YPD solid culture medium to obtain a yarrowia lipolytica engineering strain Polf-D1 (URA-);
(4) Construction of recombinant plasmid pUC-Intf3-DGAT
Taking pUC-Intf3 plasmid as a framework, and inserting a ScDGAT gene expression cassette into the plasmid framework to obtain a recombinant plasmid pUC-Intf3-DGAT;
(5) Preparation of yarrowia lipolytica engineering strain Polf-D2
Transferring the recombinant plasmid pUC-Intf3-DGAT into a yarrowia lipolytica engineering strain Polf-D1 (URA-) to obtain a yarrowia lipolytica engineering strain Polf-D2;
(6) Construction of recombinant plasmid pUC-Intf3-GPAT
Taking pUC-Intf3 plasmid as a framework, and inserting a PtGPAT gene expression cassette into the plasmid framework to obtain a recombinant plasmid pUC-Intf3-GPAT;
(7) Preparation of yarrowia lipolytica engineering strain Polf-D3
Transferring the recombinant plasmid pUC-Intf3-GPAT into a yarrowia lipolytica engineering strain Polf-D1 (URA-) to obtain a yarrowia lipolytica engineering strain Polf-D3;
(8) Construction of recombinant plasmid pUC-Intf3-LPAAT
Taking pUC-Intf3 plasmid as a framework, and inserting PtLPAAT gene expression cassettes into the plasmid framework to obtain recombinant plasmid pUC-Intf3-LPAAT;
(9) Preparation of yarrowia lipolytica engineering strain Polf-D4
Transferring the recombinant plasmid pUC-Intf3-LPAAT into a yarrowia lipolytica engineering strain Polf-D1 (URA-) to obtain a yarrowia lipolytica engineering strain Polf-D4;
(10) Construction of recombinant plasmid pUC-scp2-GPAT-LPAAT
Taking pUC-scp2 plasmid as a framework, and inserting a PtGPAT gene expression cassette and a ScDGAT gene expression cassette into the plasmid framework to obtain a recombinant plasmid pUC-scp2-GPAT-LPAAT;
(11) Preparation of yarrowia lipolytica engineering strain Polf-D5
Transferring the recombinant plasmid pUC-scp2-GPAT-LPAAT into yarrowia lipolytica engineering strain Polf-D2 (URA-) to obtain yarrowia lipolytica engineering strain Polf-D5.
Further, the construction method of the uracil auxotroph yarrowia lipolytica Polf in the step (2) comprises the following steps: knocking out uracil-encoding genes in yarrowia lipolytica Po1f by CRISPR/Cas9 technology;
specific construction methods are described in journal Journal ofAgricultural and Food Chemistry, 2021, volume 69, 46, pages 13831-13837, under the paper designation "Harnessing Yarrowia lipolytica Peroxisomes as a Subcellular Factory for. Alpha. -Humulene Overproduction".
Further, the construction method of the recombinant plasmid PUC-Intc-delta 15DEs in the step (1) comprises the following steps: the PUC-Intc plasmid with uracil genetic marker is used as a skeleton, and a delta 15 desaturase gene expression cassette is inserted into the plasmid skeleton.
Further, in step (1), the LKFAD15 gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoters or P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (4), the ScDGAT gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (6), the PtGPAT gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (8), the PtLPAAT gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (10), the PtGPAT gene expression cassette and the ScDGAT gene expression cassette promoter are P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators.
Use of an engineered strain of yarrowia lipolytica as described above for the production of alpha-linolenic acid.
A method of producing alpha-linolenic acid by fermentation using a yarrowia lipolytica engineered strain as described above that is fermented to produce alpha-linolenic acid on YNB nitrogen-limited medium;
the formula of the YNB nitrogen limiting medium is as follows: yeast powder 2.5g/L, YNB medium 1.7g/L, nitrogen source 6g/L,115 degrees centigrade sterilization 30 minutes.
Further, the method comprises the following steps:
(1) Preparation of seed liquid
Activating yarrowia lipolytica engineering strains, inoculating the yarrowia lipolytica engineering strains into YPD liquid culture medium, and culturing the yarrowia lipolytica engineering strains to logarithmic growth phase to obtain seed liquid;
(2) Fermentation production of alpha-linolenic acid
Inoculating the seed solution into YNB nitrogen-limiting culture medium according to the volume ratio of 1% -5%, fermenting at 28-30 ℃ and 200-240 rpm, and carrying out ventilation culture for 96-120 h to obtain a fermentation product alpha-linolenic acid;
wherein, the formula of YNB nitrogen limiting medium is: 2.5g/L yeast powder, 1.0-2.5 g/L YNB culture medium, 5-7 g/L nitrogen source, and sterilizing at 115 ℃ for 30 minutes.
Further, in the step (1), the viable count in the seed liquid is 1.2X10 8 ～2.4×10 8 CFU/mL；
In the step (2), the nitrogen source addition amount in the fermentation culture is 6g/L, and the YNB addition amount is 1.7g/L.
The invention has the advantages and positive effects that:
1. the invention takes yarrowia lipolytica Yarrowia lipolytica as an initial strain, firstly expresses delta 15 desaturase genes from Lipomyces kononenkoae in yarrowia lipolytica, then respectively overexpresses three acyl transferases or simultaneously overexpresses engineering strains of two acyl transferases to synthesize alpha-linolenic acid on YNB nitrogen-limited culture medium, the engineering bacteria have good stability, high content of intracellular grease alpha-linolenic acid, no time limitation on synthetic route, stable yield and low cost of fermentation substrate.
2. The strain is a yarrowia lipolytica engineering strain capable of efficiently producing high-content alpha-linolenic acid, firstly overexpressing delta 15 desaturase to produce alpha-linolenic acid, then overexpressing acyltransferase to obviously improve the total oil content, further improving the yield of the alpha-linolenic acid, and ensuring that the total oil content reaches 2.389g/L and the alpha-linolenic acid content reaches 1.249g/L. The method provided by the invention can be used for constructing high-yield oil yeast, has wide application prospects in the fields of renewable energy sources, chemical industry, medical health care and the like, and provides an effective way for commercial production of alpha-linolenic acid by using yarrowia lipolytica.
3. According to the invention, two recombinant plasmids, namely the PUC-Intc-delta 15DEs and the pUC-scp2-GPAT-LPAAT, containing uracil genetic markers are respectively constructed and then are introduced into uracil auxotroph yarrowia lipolytica strains, so that the stability of exogenous genes in the insertion process is improved, and the stability of engineering strains is further improved.
4. According to the invention, the intermediate of the engineering strain is inoculated in a solid culture medium containing 5-FOA for screening, uracil is removed from cells, and the intermediate of the engineering strain is changed into a uracil-deficient strain, so that the recycling of uracil screening markers is realized.
5. The yarrowia lipolytica engineering strain constructed by the invention can synthesize alpha-linolenic acid in YNB nitrogen-limiting medium, and provides an alpha-linolenic acid synthesis way with the advantages of no time limitation, large-scale production, stable yield, reliable quality, wide fermentation substrate source, low cost and the like.
6. The present invention overexpresses a Δ15 extended desaturase and synergistically overexpresses glycerol-3-phosphate acyl-transferase (GPAT) derived from Phaeodactylum tricornutum, lysophosphatidic acid acyltransferase (lysophosphatidate acyl-transferase, LPAAT) derived from Phaeodactylum tricornutum and diacylglycerol acyltransferase (Diacylglycerol acyltransferase, DGAT) derived from schizochytrium. A method for producing alpha-linolenic acid thereby in said host cell, the oil eventually obtained from said cell, and a product produced thereby. The construction method can achieve the aim of simply, effectively and accurately constructing the strain.
Drawings
FIG. 1 is a schematic diagram of a mechanism for producing alpha-linolenic acid and three acyltransferases in the background of the invention; wherein, the upper graph is an alpha-linolenic acid mechanism graph, and the lower graph is a mechanism graph of three acyl transferases;
FIG. 2 is a backbone diagram of the recombinant plasmid PUC-Intc- Δ15DEs in example 1 of the present invention;
FIG. 3 is a skeleton diagram of a recombinant plasmid of example 2 of the present invention; wherein, the recombinant plasmid pUC-Intf3-DGAT, the recombinant plasmid pUC-Intf3-GPAT, the recombinant plasmid pUC-Intf3-LPAAT and the recombinant plasmid pUC-scp2-GPAT-LPAAT skeleton diagram are sequentially arranged from top to bottom;
FIG. 4 is a graph showing the content of alpha-linolenic acid and oil after fermentation of the engineering strain Polf-D1 in example 4 of the present invention;
FIG. 5 is a graph showing the content of alpha-linolenic acid and oil after fermentation of the engineering strain Polf-D2 in example 4 of the present invention;
FIG. 6 is a graph showing the content of alpha-linolenic acid and oil after fermentation of the engineering strain Polf-D3 in example 4 of the present invention;
FIG. 7 is a graph showing the content of alpha-linolenic acid and oil after fermentation of the engineering strain Polf-D4 in example 4 of the present invention;
FIG. 8 is a graph showing the content of alpha-linolenic acid and oil after fermentation of the engineering strain Polf-D5 in example 4 of the present invention.
Detailed Description
The invention will now be further illustrated by reference to the following examples, which are intended to be illustrative, not limiting, and are not intended to limit the scope of the invention.
The various experimental operations involved in the specific embodiments are conventional in the art, and are not specifically noted herein, and may be implemented by those skilled in the art with reference to various general specifications, technical literature or related specifications, manuals, etc. before the filing date of the present invention.
An engineering strain of yarrowia lipolytica for high-level production of alpha-linolenic acid, which is obtained by taking yarrowia lipolytica (Yarrowia lipolytica Po f) as an original strain, expressing delta 15 desaturase genes and then cooperatively and over-expressing acyltransferase.
Preferably, the delta 15 desaturase gene is derived from orange Lin Youzhi yeast Lipomyces kononenkoaeLKFAD, and the sequence of the delta 15 desaturase gene is shown as SEQ ID No.1, and specifically comprises the following steps:
ATGTCTGTGGTGGACTTCACCGATACAACGTCGGGATCTGCGATCAACTCATCCAACATATCTCAGCGAGGAAATGGGAGTACAATCGTAGAGACCAAGAAGGGGCCCTCTTCTAATCTTAAAGCCATTGACACCTTCGGTAACGAGTTCAAGGTCCCTGACTACACCATCAAGCAGATTCTAAGTGCAATCCCCAAGCACTGCTACGAACGGTCTCTCGTGCGTTCGCTCGGCTACGTTGCTCGGGACATTACTATGATGTGTCTCATTGGGTACATTGGTCAGAAGACGATTCCCATGGTCGAAATAGCAGGCAAGGAGGGACTGAGTTCTAACATCAGAGGTGGATTGTGGTGTGTCTACTCCTATCTGCTGGGACTGTTTGGCTTTGGTCTCTGGATTCTTGCCCACGAGTGTGGTCATGGAGCCTTCTCTGATTACCAAAATGTCAACGACGTGGTCGGCTGGATCCTCCATTCCTATCTGATCGTTCCGTACTTTAGCTGGAAGTTCTCGCACAGCAAACACCACAAAGCTACAGGACATCTGACTAAGGACATGGTGTTCATCCCCTACACTAAAGAAGAGTTTGTGGAGAAGTCCGGTGTCTCCAAGGTCTCGGAAGTCATGGAGGACTCGCCTATCTGGTCACTGATGGTTCTCATCTTCCAACAGATTGGTGGCCTTCAGCTGTACCTGGCCACCAACGCCACAGGGCAATCCTACCTTGGCCACAGCAAGATTGCCAAATCCCACTACGCCCCAGCCTCACCAGTATTTGACAAGGAGCATTACTGGTATATCATTCTGTCGGATATTGGAATCATCACTACCATCACTGTTGTTTACCAGTGGTACAAGAACTTTGGATTCTTCAACATGTTTGTCAACTGGTTCATGCCCTGGTTGTGGGTTAACCACTGGCTGGTCTTCGTGACCTTTTTGCAGCATACTGACCCTACCATGCCTCATTACCGAGACAACGAATGGACCTTTGCCCGTGGTGCTGCAGCGACGATAGACCGAAACTTCGGCTTCATCGGCCAGCACATTTTCCACGATATTATTGAGACCCACGTGCTTCACCATTATGTATCCCGCATTCCTTTTTACAACGCTCGAGAGGCTACAGACGCCATCAGAAAGGTGATGGGCGAGCATTATCGATACGAGGGAGAGTCTATGTGGTATTCTCTATGGAAGTGCATGCGAATGTGCCAGTACGTGGATGATGCTGACACTGATGCTAAGGGCGTGCTCATGTACAGGAACGTGAATGGAGCAGGCCCGGTTAAGCCCATCGACTGA；
the acyltransferase is glycerol-3-phosphate linear transferase GPAT and/or lysophosphatidic acid acyltransferase LPAAT and/or diacylglycerol acyltransferase DGAT;
the diacylglycerol acyltransferase is derived from schizochytrium (Sp) ScDGAT, and the ScDGAT gene sequence is shown in SEQ ID NO.2, and specifically comprises the following steps:
ATGCAGACACCGTACAGCACGTCGATGGGCTCCGTCTCGAGCTACTCGTCGTCTGGCGACTACGCCATGTCCGAAGCGGAGGGCTCCGAAGTGGACCCTGCCGATGACAGGATGGGCGACTCAATCGGATCGAAACCAAGCTCCTCGTCCGTGACCGGACGCCGGCGACTCACGCAAGAAGAGCGCGACTACTTTTTGCGTCTTGAAAAGGAATGGCGCGAGGAGGACGCATGGGCTGACCAACCCGGGTCCTGGTACTCAATGCTGGCCTGGATGCCGGTCCTCATTGGCCTGCGCGTCTTCAACGTGCTGCTTTCCATCGCCTTTTGGCCTGTCTCCTTTGTGGCGCGGGTCTTCTTCGGCAAAAAGATTCACACGGTCAGTTTCTGGGACGTTCCGCTTTCCCGCCGTAAACAAACTGCCGTGGTGCTCTTGTTCGTCATGCTCCTGCCCATGGTCGTCGTGGTGTACTCGTGGACGCTCGTTCTGCTTCTCTTCCCGCTCACGACCTTTCCCACACTGTGCTACATGGTGTGGATCATTTACGTGGACAAGAGTCCCGAAACAGGCTCGCGCCGACCCTTTTTGCGCTATTGGAAGATGTGGCGCCATTTCGCGAACTATTTCCCGCTTCGACTCATTCGCACCACGCCGCTCGACTCCCGTCGCAAGTACGTCTTTTGCTACCATCCGCACGGAATCATCTCGCTCGGTGCCTTTGGCAACTTTGCGACGGACTCGACCGGATTCTCGCGCAAATTCCCCGGTATCGATTTGCGCGTGCTCACGCTTGCGCTCAACTTTTACTGCCCCCTTTTGCGCGAGTTTCTGCTTTACCTGGGCCTTTGCAGCGCCGCAAAGAAGTCCTGCAACCAAATTTTGCAGCGCGGGCCTGGATCCGCCATCATGCTCGTCGTTGGCGGCGCCGCCGAGTCTCTCGACTCCCAGCCAGGCACCTATCGATTGACGCTCGGCCGAAAGGGGTTCGTGCGCGTTGCCCTTGACAATGGTGCCGATCTAGTGCCGGTGCTTGCCTTTGGCGAGAATGACGTATTTGACACCGTGTACCTCCCACCGAACTCGTGGGCACGAAACGTGCAAGAGTTTGTGCGCAAGAAGCTTGGTTTCGCCACACCAATTTTCAGTGGCCGCGGTATCTTCCAGTACAACATGGGGCTCATGCCTCACCGCCGTCCCATTATTGTCGTTGTTGGAAAACCCATCAAAATGCCCAAAATTCCCGATGAGCTCAAGGGCCGCGCGCTTTCCACCACGGCCGAGGGCGTTGCCCTCGTTGACAAGTACCACGAAAAGTATGTCAAGGCGCTTCGCGAGCTCTGGAACTTGTACAAGGAGCGCTGGGCCGTGCACCGTCAAGGTTCGCTGCTCATTCAAAAGTAA
the glycerol-3-phosphate linear transferase is derived from Phaeodactylum tricornutum (Phaeodactylum tricornutum) PtGPAT, and the PtGPAT gene sequence is shown in SEQ ID NO.3, and specifically comprises the following steps:
ATGGACCTATCGACCGTCCAGAGTGTCGCGGTCCTCATCGGCAAGATTTACGGTATCTACGCTGTCGGAGCCATGACTCTCGCTGCTGCAGTGGCAGTGCGACAGGAGTACAAGATCTGGCAGCCCTCTTCTCTTCCTTCTTTGTCGATTTGGGGTATGATCAAGGTCTTCTGGTTCAACATTGTGTGGTTCGTGCTATGTCTGGTTGGAAGCATTCTCATTTCAGCTAAGTGGTGTCTTCAGCTGGGAAGATCGGACATTGAATACGAGGGCAACCAGTGGGTGGAAGCTACCGTTGCTCGAATCTGTGTCACTCTCTTTGTGGGTCCTGTTCGGGTAGATGGGACTGAGCATCTCCCACCCAACGACAAGAACCCCGCGCCTGTGTATGTGGCCAACCACGAGTCTCAGATTGATGTTGGTGTGGTCTACTACCTGGGCAGACGGTTTAAATGGATCGCCAAGCAGTCAGTTCTGTATCTGCCTGGTGTCGGCCAAATAATGTGGCTGAGTGGCCATGTGCTGATCCAGCGTCAGGGCCGAAACAAGCAATCTGTCTCCAACTTGTTTGAGAAGTCCAATGCCGCAGTTCAATCCGGGGTTCCCATGTTTTTCTTTCCCCAGGGTACCCGTTGGATTGCCAATCGACGAGAGTTCAAGGACGGCGCCTTCAAGGTGGCTCTCGACAACAAGAGTACGTTGATTCCACTCAGCATTGGACTGCCGAGAAACGCCTGGAACTGCGCGTACCCCGTGTCCCTTCTGTGGGGCCGCTCTACTCCTGAGCCCATCACCATCACAGTCCACCCTAGCATCCCCGTCAAGGGAACAGAATCGCGGGACGAGCTCAAGCAGCGATGCTGGGACCAGATCTACTCCGTTCTGGTGCCGCCGGTATCCACCTCTTCGGCCACGGCCGGAGGAAATGATTCAGATAAAAACAAATGA
The lysophosphatidic acid acyltransferase is derived from PtLPAAT of Phaeodactylum tricornutum (Phaeodactylum tricornutum), and the PtLPAAT gene sequence is shown in SEQ ID NO.4, and specifically comprises the following steps:
ATGCGGCATCTGCGAGGTGTGCTTATCACTCTCATCCTGGACTGGGTCTCTCTGTCACGTAGCCTCACTACTCATGCTCCCTACCGACGGACAACCGCCACTCGTTCAACATGCACTGCTCGACAGACGCGCGGATCTCAGCTTGCATCGACCACCCCGTTCGACCGAGACCAGTCGGAATCTTTCATTGTGCCTAACGAGGATGTCAACCCCATCATACGTCTCGGGAAGGACGAGCAGGAGAAGATTGTTAACGGCTTTGGACTATGGTGTGCTGCTGTTTCTGTATTCACAGGACCTCTGTGGGTTGCTGCCATGTCTACACTGCAGGCCATCTACAAGATCAACGCCGATTGGGACCCCAACAGAGCTCTCTACGACAAAACCGGTAAAATCTGGTCCAAGACTTGGCTTACATTGACCGACTCATATCCCACCTTTTCTGGCGACGTGGATAGACTCAAGTCCTCGCAGGGCCCTTGTCTGTACGTTGCCAACCACGCCTCCTGGCTCGACATTCCAGTCATCTGCACCGTGCTGGATCCAGTATTCAAGTTCATTGCCAAGGGTGAGCTCCGAAAGGTGCCTTGCATCGGCCAGCAGCTGGAGGGAGGAAATCACATTTTGATTGACAGAGAAGACAGGCGAAGCCAGCTCCGGACGTTTAAAGATGGTATTGGATGGTTGAAGAAGGGAGTGCCCATCATGGCCTTCCCCGAAGGCATGAGATCGCGAGATGGCAAGCTCATGGACTTCAAGGGCGGTCTGTTCTCCATGGCTGTCAAGACTCAGGTGCCTATTGTCCCCATCACCATTTCTCACACCCATGCCGTCATGCCTAGTAATGCACTGTTTCCTGTCCAGACCGGGGCTGGCAAGTTGCATGTTCACGTGCACGATCCTATTGATACAACTGGCAAAACCGAGGCCGAACTGGGTGCTCTGGTGCGAGCATCCTTTCTTAGTACTCTACCTCTTGGACAGCATCCCAAGCCCGTTGTCCCCGAGATTGAGCAAACAGCCGAGAAAGACCACAAGACGATCCCGATTACGCCCAAGGTGCAGGACACCCACCATCTTCACCAGCAACACATCACCCCGTCCCAAACTTTATCACACTACACGGCGGCGTCTTCCACCATCAACTCGAGTCAAGAGGTCACCAGCAAGAACCGAACAGAGGAGACCACTGTGCCATGA。
the construction method of the yarrowia lipolytica engineering strain for high-level production of alpha-linolenic acid comprises the following steps:
(1) Construction of recombinant plasmid PUC-Intc-Delta15 DEs
The pUC-Intc plasmid (pUC plasmid added to the upper and lower homology arms of the Intc site, the sequence was:
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAGATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTATTTAAATGTGCTGTTCAGAAGCCGGCCGGGTAATGCCACGTAAGAGTCACGTGAATCTAAGGGGAGGGGTGGTTGGGTGGCAACAGACGAGATTAACAGACCAATATAGATCAGGGCTAGGGTACGAGGCCCCTCAAACAAATAATTTTACCGTCCAGTAAATTTTCCACCCTCCTTTGCCCCTTCTACTCGTTCAATACATCCCCATCTCCACCGTTCTCTTCAGATATAGATCTCTTCGCTGAAAGCGTAATGTGGTGTTGTTCAGCCTTGAAGCAGGAAGTGTATTTGGCGGTGTGAAATAGTTCTGACAGCAACGCGAGCCACGCGAAAGGGAACGACAGGCACTCAAAATGACGTGGTAAAGGTCCTGTTTTTTTACACATGATTAAATGAGAAGATTGTGGCTTATTTATATATTTTTTCTTGCAGAAATCACCCCATTTCGACAAAATCATTACTGTTTTGGCGCATGGTACATTCCGCCGTGCGAGTCAAACGCGTCGACGTGTTAGGAGTGCGAGTGCCAGTACAAGTGTCGGTTGTATAACTAAGTACAGGTGGTACGAGTGCGAGTACGTACCTTGTAGTAACCATGATCGTCGTTTGTTGAGTCTGAGAATATTGATTGTCTTCTACTTGTACTTGTACTACCAGTGTAAGTACTACCAGTTTGTACGATAGTCGTATCTATGTACTATCTTTCAGTATTGTACAGCACATACTTTACATGATTATTGATAATGCAAAAAAACTGAAAGAAATATGAGAAATAGAATATTGTAAATATGAGCGATAGAATATTGTAAATATGAGCGATAGAATATTGTATATATTTGTCGTGGTGTGTGCAGATGGTGTTGTCCAGCAAGACAAGTTCTTCAAAGAATAACAGCGTAGTCCATACTCGTACTACATACATCAAGACCCCATACAATCATTGTCTTCGTTATCAGTTCTTGTACCCGTATCTACTGTACATACTTGTATCTTCATTGCCATTGTTGTATCTAAACTACTGGAAGTCTCTGGAGCAAGATTGAGAGTGGTATGTGTCGACCCTTGAGGGCCGATGCAACCTATAGGGGATGCGGTCGGATTGACAGACCGCCTACTGCCAATGTTTGTTACTGCTCTGCTATCCGTCTACGGAGCTACTCCACCAACTCACCAACGTACCCACCATCTGCCAATACGCTACTCCTCCCTAGATCTAAATCGCGAGCTTCGAAAAGTGGGTGAGACATGTCCTCAACTGTCCCCGACCACGCTGGTGTCCGTATGGAATACGTCGTGGCTGTAAGACACAACAATCTCCCCGCTCGTATTCCGACAGATCCACCGGGCCACTCTTGGACGAGCCGCGCGTGCAAAACACAATATGCTCCGAGACCCGACCTTGGCATCTGGATCTTGCTCTTGCGATACGACTACTGTGCCCGTAAAGCTCCCATATAGATACATGCTCGCGCCTCGATAGGCTTATACGGGGGTTGCGTTTTTTTGGGGGTAAAAAAAAGGATACAAAAAAGGGTAAAAAAAATAGTACGTACCTTATTAGCGACGCACGTACGTGGGCTGCGTCCATCAACCCCGCCCATCAATCCATGGAATCTTTGCCGTGAGACACACAAGTTTCAGACTTGCTCCTTTTGAGTCTTCTCATCATTTACGACAGTTCTTTTTCCACCCACAGCCATCCGACTTGTGCATCTCCGTATCCTAGCCGAGCCCATTCGGGGTGCATGACAACTGCGGGATCTTGGGGGTAGAGTGTTTAGCGAAATTAATATATGCAGTATAGTGCAGGATGAGCTAAGAAGCATTCTACAAACAGTTGTATACAGGCACCAGACATTTATATAGTTATTGGGAAACGTCGAGTGGCGTAAGATGGCGGTGCCCTGGAGCCACTCACACATGTATCATAGGTTTAATAAGTCCCGCACGGGTCGTTTCTAATGTAATGTCTCATGTCTAAGAACCACGGTGTATTCACCGAACTATCCTTGAGATACTCATTGCTCATCGCTACGGGCTCGACGCTGGCATTTTTTCTCCGATAATTTCACGGTGGACGTACCCACAGGCCATCCCTTCTGCTTTGGGAACTCGCGTATGCGGC
CGCAATTCCCGGATCTTCCAGTGGTGCATGAACGCATGAGAAAGCCCCCGGAAGATCATCTTCCGGG
GGCTTTTTTTTTGGCGCGCGATACAGACCGGTTCAGACAGGATAAAGAGGAACGCAGAATGTTAGAC
AACACCCGCTTACGCATAGCTATTCAGAAATCAGGCCGTTTAAGCGATGATTCACGAGAATTGCTGGC
CCGCTGCGGCATAAAAATTAATTTACACACTCAGCGCCTGATTGCGATGGCGGAAAACATGCCGATTG
ATATCCTGCGCGTGCGTGATGATGACATTCCGGGTCTGGTAATGGATGGCGTGGTCGATCTCGGTATTA
TCGGCGAAAACGTGCTGGAAGAAGAGCTACTCAACCGCCGCGCACAGGGCGAAGATCCACGCTATTT
AACCCTGCGCCGTCTTGACTTCGGCGGCTGCCGTTTATCGCTGGCAACACCGGTTGACGAAGCCTGG
GACGGCCCGGCCGCGCTGGACGGTAAACGTATCGCTACCTCATATCCGCACCTCCTCAAACGCTACCT
CGACCAGAAAGGCGTCTCTTTTAAATCGTGTCTGTTAAATGGTTCTGTCGAAGTCGCGCCGCGCGCGG
GGCTGGCCGACGCTATCTGCGATTTGGTCTCTACCGGCGCGACGCTTGAAGCTAACGGCCTGCGTGAA
GTCGAAGTTATCTACCGCTCTAAAGCCTGTCTGATTCAGCGCGACGGTGAGATGGCACAGAGCAAGC
AAGAGCTGATCGATAAATTGCTGACCCGTATTCAGGGCGTGATTCAGGCGCGCGAATCGAAATACATC
ATGATGCACGCGCCAAGTGAACGCCTGGAAGAGGTTATCGCCCTGCTGCCAGGCGCCGAAAGGCCGA
CAATTCTGCCGCTGGCAGGCGAGCAACAGCGCGTGGCGATGCACATGGTCAGCAGCGAAACGTTGTT
CTGGGAAACCATGGAGAAACTGAAAGCGCTTGGCGCCAGCTCGATTCTGGTACTGCCGATCGAGAAG
ATGATGGAGTGATCTGACGCCTGATGGCGCTGCGCTTATCAGGCCTACGTAATGCGTTGATATTTTGGG
TTCTGTAGGCCGGATAAGGCGGAACCCTGTGATGGAGTAAAGACCATGAGCTTCAATACCCTGATTGA
CTGGAACAGCTACCGGGGTGTGTTCTGTGGAGCATTCTCACTTTTGGTAAACGACATTGCTTCAAGTG
CAGCGGAATCAAAAAGTATAAAGTGGGCAGCGAGTATACCTGTACAGACTGTAGGCGATAACTCAATC
CAATTACCCCCCACAACATGACTGGCCAAACTGATCTCAAGACTTTATTGAAATCAGCAACACCGATT
CTCAATGAAGGCACATACTTCTTCTGCAACATTCACTTGACGCCTAAAGTTGGTGAGAAATGGACCGA
CAAGACATATTCTGCTATCCACGGACTGTTGCCTGTGTCGGTGGCTACAATACGTGAGTCAGAAGGGC
TGACGGTGGTGGTTCCCAAGGAAAAGGTCGACGAGTATCTGTCTGACTCGTCATTGCCGCCTTTGGA
GTACGACTCCAACTATGAGTGTGCTTGGATCACTTTGACGATACATTCTTCGTTGGAGGCTGTGGGTCT
GACAGCTGCGTTTTCGGCGCGGTTGGCCGACAACAATATCAGCTGCAACGTCATTGCTGGCTTTCATC
ATGATCACATTTTTGTCGGCAAAGGCGACGCCCAGAGAGCCATTGACGTTCTTTCTAATTTGGACCGA
TAGCCGTATAGTCCAGTCTATCTATAAGTTCAACTAACTCGTAACTATTACCATAACATATACTTCACTG
CCCCAGATAAGGTTCCGATAAAAAGTTCTGCAGACTAAATTTATTTCAGTCTCCTCTTCACCACCAAAA
TGCCCTCCTACGAAGCTCGAGCTAACGTCCACAAGTCCGCCTTTGCCGCTCGAGTGCTCAAGCTCGT
GGCAGCCAAGAAAACCAACCTGTGTGCTTCTCTGGATGTTACCACCACCAAGGAGCTCATTGAGCTT
GCCGATAAGGTCGGACCTTATGTGTGCATGATCAAGACCCATATCGACATCATTGACGACTTCACCTAC
GCCGGCACTGTGCTCCCCCTCAAGGAACTTGCTCTTAAGCACGGTTTCTTCCTGTTCGAGGACAGAA
AGTTCGCAGATATTGGCAACACTGTCAAGCACCAGTACAAGAACGGTGTCTACCGAATCGCCGAGTG
GTCCGATATCACCAACGCCCACGGTGTACCCGGAACCGGAATCATTGCTGGCCTGCGAGCTGGTGCC
GAGGAAACTGTCTCTGAACAGAAGAAGGAGGACGTCTCTGACTACGAGAACTCCCAGTACAAGGAG
TTCCTGGTCCCCTCTCCCAACGAGAAGCTGGCCAGAGGTCTGCTCATGCTGGCCGAGCTGTCTTGCA
AGGGCTCTCTGGCCACTGGCGAGTACTCCAAGCAGACCATTGAGCTTGCCCGATCCGACCCCGAGTT
TGTGGTTGGCTTCATTGCCCAGAACCGACCTAAGGGCGACTCTGAGGACTGGCTTATTCTGACCCCCG
GGGTGGGTCTTGACGACAAGGGAGACGCTCTCGGACAGCAGTACCGAACTGTTGAGGATGTCATGTC
TACCGGAACGGATATCATAATTGTCGGCCGAGGTCTGTACGGCCAGAACCGAGATCCTATTGAGGAGG
CCAAGCGATACCAGAAGGCTGGCTGGGAGGCTTACCAGAAGATTAACTGTTAGAGGTTAGACTATGG
ATATGTAATTTAACTGTGTATATAGAGAGCGTGCAAGTATGGAGCGCTTGTTCAGCTTGTATGATGGTC
AGACGACCTGTCTGATCGAGTATGTATGATACTGCACAACCTGTGTATCCGCATGATCTGTCCAATGGG
GCATGTTGTTGTGTTTCTCGATACGGAGATGCTGGGTACAAGTAGCTAATACGATTGAACTACTTATAC
TTATATGAGGCTTGAAGAAAGCTGACTTGTGTATGACTTATTCTCAACTACATCCCCAGTCACAATACC
ACCACTGCACTACCACTACACCAAGCTCCGGATCTTCCAGTGGTGCATGAACGCATGAGAAAGCCCC
CGGAAGATCATCTTCCGGGGGCTTTTTTTTTGGCGCGCGATACAGACCGGTTCAGACAGGATAAAGAG
GAACGCAGAATGTTAGACAACACCCGCTTACGCATAGCTATTCAGAAATCAGGCCGTTTAAGCGATGA
TTCACGAGAATTGCTGGCCCGCTGCGGCATAAAAATTAATTTACACACTCAGCGCCTGATTGCGATGG
CGGAAAACATGCCGATTGATATCCTGCGCGTGCGTGATGATGACATTCCGGGTCTGGTAATGGATGGC
GTGGTCGATCTCGGTATTATCGGCGAAAACGTGCTGGAAGAAGAGCTACTCAACCGCCGCGCACAGG
GCGAAGATCCACGCTATTTAACCCTGCGCCGTCTTGACTTCGGCGGCTGCCGTTTATCGCTGGCAACA
CCGGTTGACGAAGCCTGGGACGGCCCGGCCGCGCTGGACGGTAAACGTATCGCTACCTCATATCCGC
ACCTCCTCAAACGCTACCTCGACCAGAAAGGCGTCTCTTTTAAATCGTGTCTGTTAAATGGTTCTGTC
GAAGTCGCGCCGCGCGCGGGGCTGGCCGACGCTATCTGCGATTTGGTCTCTACCGGCGCGACGCTTG
AAGCTAACGGCCTGCGTGAAGTCGAAGTTATCTACCGCTCTAAAGCCTGTCTGATTCAGCGCGACGGT
GAGATGGCACAGAGCAAGCAAGAGCTGATCGATAAATTGCTGACCCGTATTCAGGGCGTGATTCAGG
CGCGCGAATCGAAATACATCATGATGCACGCGCCAAGTGAACGCCTGGAAGAGGTTATCGCCCTGCT
GCCAGGCGCCGAAAGGCCGACAATTCTGCCGCTGGCAGGCGAGCAACAGCGCGTGGCGATGCACAT
GGTCAGCAGCGAAACGTTGTTCTGGGAAACCATGGAGAAACTGAAAGCGCTTGGCGCCAGCTCGATT
CTGGTACTGCCGATCGAGAAGATGATGGAGTGATCTGACGCCTGATGGCGCTGCGCTTATCAGGCCTA
CGTAATGCGTTGATATTTTGGGTTCTGTAGGCCGGATAAGGCGGAACCCTGTGATGGAGTAAAGACCA
TGAGCTTCAATACCCTGATTGACTGGAACAGCAATTAAGGTACCAAGCTTCTGGGGACCGAGAATTAG
TGTTGATGCTAGCGCAAGGCTTGTTCTTTAGTGGGCATCTACCGCAGCTACTATACACGAGCGCGACGT
AGGTATAGCATGCCAAAGGCGGGTGCGATACCAGATGACGCTATAATATGCCAGATTTGCACTAGCAC
ACATCACTGGAAGCTGTCAATTTGAAAATGTCAATGCAGTCTCACCATTGGACGATCTTGGCACCTCC
TAAACTCTGGCAATGTCTGAGCTCTTTGACACATAAGACATCAAGCAGACGGTCTAAAATCATATGCC
ATGTAAAGAACTACTAGTGCCTGTAGAAGACGCCTGTAGAAGACGCCTGTAGAAGACGCCTGTAGAA
GACGGCTACTTGTATATTCTCACTTGTCGTGCTTCACTCACGGCTCGATGTCCACTGCTTACCTCAGCG
GCAGCGGGCGGAAAAAGCCTACAAACCCCTGTCGGCCGCGTTGCACTTGTTGGGCGACGACCAAATA
TGAGTTGGAAAAGTTTTATCTTCGCCAGAGACGCCGTCCCATTGTTATTATTACAGAGGTCCATGAACA
TGAGCTTTAGATCAGATCTGCACGTGGCGGAGCCGCCCAACCGTGTCTTTTCCCGTCGGTACGAGCTG
ATAAGCTGCACCTCACCAACAATCATGACATGAGGTCTTGTATGCAAGGGAGACGTCGGAGTGCTTCG
GCAGGCTTCGGTTAGGGCTTGGCTGGAGGCTAGCGACTGCAGTGGCGGAGTCCTCGTGGTCTTCCTT
GATGTACCCCGTCGCATGTGAGTCTGCTATGATATATGAAACTAATTCTAATCTAGTTGCGGTTAATTTC
ATATCATGGTCTTTGTTTCTCCCTTATTGAATTCTATTTTTCATATCTTTGGCTGCTCCTCCCCTTTTTCGG
TCTCTCCAGCAATCTTTGGCGAGTCTCGAAACCTGGAGGAAAACCTGTAGACAACCTGCGCGAAACG
ACGGCCAAAAACAGTGAAACTTGAGCCGAAAAAGCATGGAAAATCACACGTGACCATCGCATTTGG
ACTTTCCCATCGTTGCCAGCAACTCCACGTGTTTGGTCACTTGTAGAGGTTTCGGTACTGTAGCTCTAG
CTAGTCCATCGTACGTGTTCACCTGTGGAGACCCGACCCTTGAAGTGCGATACTCTACAGTGCACCAA
GAGTACACCGAAACCAAAAGCAACCGAGTGTTGATCCCTCAATGGTTGGGGGAAATGTTTCACAGAG
CGAGGGCACGAAAGAAGGGGTTGGAAGAAGCAATCACCAGTTTTGCCAACGGTATTTACAGGGTTTT
GTTGCTGTTTGAGTATCGATAGTCCGACACGCAAAAATGAGCAGCACGACAATGGCGAGCCTCCTTTT
TTCGTCCCTCTCTCAAGCCCCCCCGCCCATTCAGACTTTGTGCCAAAAGAACTGCGAAACCAAAGAA
AATAAACGTGTATCCGGGCCGTTTCCTTTCTTAGGGCAAAGCCAAACGGAACGGAGTTGAGGTCAAC
TTGTATGTGATTTGGAGAAGCGCAACAAGGGCGACAACAGTAGGGTTTCGTTTCATAGCTTTTCGGTT
ATCGGTTTTATTTGTTTGATTTTTGTCTCGCACGAGATAGAAGCCATGTTTGGGTCTGAAATGTCGCTC
ATACAGCTCTTCGAATCGGACGTCTTCTTCTTCTTTATAGCTCCCGGTCTTTGTTTTTTGTTCCGTCGTT
TTCGCTAAAAAGAAAAAGTGACATGCTTGTTTTTTGGGCTCCCTTGTTCTGCTCTCTCCAAAAGTGCA
GCGTTAGGGTTGACAGGCATTAGACTCAGCGCTTGGTAACTTCTTAAATAAAGGTGAACCTAGAGAA
AGTTTGGAGGAGAAAAAAAATAACAGTCCACCAGGTGGCGCCATGGGGGTTGAAGAGGGGCCATAT
TCTTGCGGGTATGGGCAGCTGGGGGGTCACAGCATTGATTCCCCCAAACTCCCGTTCCATTCGGCCCA
CTGGAGCGCCCCATTTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC
GCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTG
AGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCT
GCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGC
TCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATA
CGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCC
AGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACA
AAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCC
TGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCC
TTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCT
CCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGT
CTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCA
GAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAG
AACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGAT
CCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA
AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCA
CGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA
AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAG
GCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTA
CGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGACTCCCACGCTCACCGGCT
CCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTAT
CCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGC
GCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCT
CCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTC
GGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCAT
AATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTC
TGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACA
TAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTAC
CGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCA
CCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACAC
GGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATG
AGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAA
AGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGT)
inserting an LKFAD15 gene expression cassette into the plasmid skeleton to obtain a recombinant plasmid PUC-Intc-delta 15DEs;
(2) Construction of genetically engineered strain Polf-D1 for producing alpha-linolenic acid
Transferring the recombinant plasmid pUC-Intc-delta 15DEs into uracil auxotroph yarrowia lipolytica Polf to obtain a yarrowia lipolytica engineering strain Polf-D1;
(3) Preparation of yarrowia lipolytica engineering strain Polf-D1 (URA-)
Inoculating the yarrowia lipolytica engineering strain Polf-D1 into a YPD-5FOA solid culture medium, and after single colony grows out, carrying out streak activation again in the YPD solid culture medium to obtain a yarrowia lipolytica engineering strain Polf-D1 (URA-);
(4) Construction of recombinant plasmid pUC-Intf3-DGAT
The pUC-Intf3 plasmid (pUC plasmid added to the upper and lower homology arms of the Intf3 site, the sequence was:
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAGATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTATTTAAATGTGCTGTTCAGAAGCCGGCCGGGTAATGCCACGTAAGAGTCACGTGAATCTAAGGGGAGGGGTGGTTGGGTGGCAACAGACGAGATTAACAGACCAATATAGATCAGGGCTAGGGTACGAGGCCCCTCAAACAAATAATTTTACCGTCCAGTAAATTTTCCACCCTCCTTTGCCCCTTCTACTCGTTCAATACATCCCCATCTCCACCGTTCTCTTCAGATATAGATCTCTTCGCTGAAAGCGTAATGTGGTGTTGTTCAGCCTTGAAGCAGGAAGTGTATTTGGCGGTGTGAAATAGTTCTGACAGCAACGCGAGCCACGCGAAAGGGAACGACAGGCACTCAAAATGACGTGGTAAAGGTCCTGTTTTTTTACACATGATTAAATGAGAAGATTGTGGCTTATTTATATATTTTTTCTTGCAGAAATCACCCCATTTCGACAAAATCATTACTGTTTTGGCGCATGGTACATTCCGCCGTGCGAGTCAAACGCGTCGACGTGTTAGGAGTGCGAGTGCCAGTACAAGTGTCGGTTGTATAACTAAGTACAGGTGGTACGAGTGCGAGTACGTACCTTGTAGTAACCATGATCGTCGTTTGTTGAGTCTGAGAATATTGATTGTCTTCTACTTGTACTTGTACTACCAGTGTAAGTACTACCAGTTTGTACGATAGTCGTATCTATGTACTATCTTTCAGTATTGTACAGCACATACTTTACATGATTATTGATAATGCAAAAAAACTGAAAGAAATATGAGAAATAGAATATTGTAAATATGAGCGATAGAATATTGTAAATATGAGCGATAGAATATTGTATATATTTGTCGTGGTGTGTGCAGATGGTGTTGTCCAGCAAGACAAGTTCTTCAAAGAATAACAGCGTAGTCCATACTCGTACTACATACATCAAGACCCCATACAATCATTGTCTTCGTTATCAGTTCTTGTACCCGTATCTACTGTACATACTTGTATCTTCATTGCCATTGTTGTATCTAAACTACTGGAAGTCTCTGGAGCAAGATTGAGAGTGGTATGTGTCGACCCTTGAGGGCCGATGCAACCTATAGGGGATGCGGTCGGATTGACAGACCGCCTACTGCCAATGTTTGTTACTGCTCTGCTATCCGTCTACGGAGCTACTCCACCAACTCACCAACGTACCCACCATCTGCCAATACGCTACTCCTCCCTAGATCTAAATCGCGAGCTTCGAAAAGTGGGTGAGACATGTCCTCAACTGTCCCCGACCACGCTGGTGTCCGTATGGAATACGTCGTGGCTGTAAGACACAACAATCTCCCCGCTCGTATTCCGACAGATCCACCGGGCCACTCTTGGACGAGCCGCGCGTGCAAAACACAATATGCTCCGAGACCCGACCTTGGCATCTGGATCTTGCTCTTGCGATACGACTACTGTGCCCGTAAAGCTCCCATATAGATACATGCTCGCGCCTCGATAGGCTTATACGGGGGTTGCGTTTTTTTGGGGGTAAAAAAAAGGATACAAAAAAGGGTAAAAAAAATAGTACGTACCTTATTAGCGACGCACGTACGTGGGCTGCGTCCATCAACCCCGCCCATCAATCCATGGAATCTTTGCCGTGAGACACACAAGTTTCAGACTTGCTCCTTTTGAGTCTTCTCATCATTTACGACAGTTCTTTTTCCACCCACAGCCATCCGACTTGTGCATCTCCGTATCCTAGCCGAGCCCATTCGGGGTGCATGACAACTGCGGGATCTTGGGGGTAGAGTGTTTAGCGAAATTAATATATGCAGTATAGTGCAGGATGAGCTAAGAAGCATTCTACAAACAGTTGTATACAGGCACCAGACATTTATATAGTTATTGGGAAACGTCGAGTGGCGTAAGATGGCGGTGCCCTGGAGCCACTCACACATGTATCATAGGTTTAATAAGTCCCGCACGGGTCGTTTCTAATGTAATGTCTCATGTCTAAGAACCACGGTGTATTCACCGAACTATCCTTGAGATACTCATTGCTCATCGCTACGGGCTCGACGCTGGCATTTTTTCTCCGATAATTTCACGGTGGACGTACCCACAGGCCATCCCTTCTGCTTTGGGAACTCGCGTATGCGGCCGCAATTCCCGGATCTTCCAGTGGTGCATGAACGCATGAGAAAGCCCCCGGAAGATCATCTTCCGGG
GGCTTTTTTTTTGGCGCGCGATACAGACCGGTTCAGACAGGATAAAGAGGAACGCAGAATGTTAGAC
AACACCCGCTTACGCATAGCTATTCAGAAATCAGGCCGTTTAAGCGATGATTCACGAGAATTGCTGGC
CCGCTGCGGCATAAAAATTAATTTACACACTCAGCGCCTGATTGCGATGGCGGAAAACATGCCGATTG
ATATCCTGCGCGTGCGTGATGATGACATTCCGGGTCTGGTAATGGATGGCGTGGTCGATCTCGGTATTA
TCGGCGAAAACGTGCTGGAAGAAGAGCTACTCAACCGCCGCGCACAGGGCGAAGATCCACGCTATTT
AACCCTGCGCCGTCTTGACTTCGGCGGCTGCCGTTTATCGCTGGCAACACCGGTTGACGAAGCCTGG
GACGGCCCGGCCGCGCTGGACGGTAAACGTATCGCTACCTCATATCCGCACCTCCTCAAACGCTACCT
CGACCAGAAAGGCGTCTCTTTTAAATCGTGTCTGTTAAATGGTTCTGTCGAAGTCGCGCCGCGCGCGG
GGCTGGCCGACGCTATCTGCGATTTGGTCTCTACCGGCGCGACGCTTGAAGCTAACGGCCTGCGTGAA
GTCGAAGTTATCTACCGCTCTAAAGCCTGTCTGATTCAGCGCGACGGTGAGATGGCACAGAGCAAGC
AAGAGCTGATCGATAAATTGCTGACCCGTATTCAGGGCGTGATTCAGGCGCGCGAATCGAAATACATC
ATGATGCACGCGCCAAGTGAACGCCTGGAAGAGGTTATCGCCCTGCTGCCAGGCGCCGAAAGGCCGA
CAATTCTGCCGCTGGCAGGCGAGCAACAGCGCGTGGCGATGCACATGGTCAGCAGCGAAACGTTGTT
CTGGGAAACCATGGAGAAACTGAAAGCGCTTGGCGCCAGCTCGATTCTGGTACTGCCGATCGAGAAG
ATGATGGAGTGATCTGACGCCTGATGGCGCTGCGCTTATCAGGCCTACGTAATGCGTTGATATTTTGGG
TTCTGTAGGCCGGATAAGGCGGAACCCTGTGATGGAGTAAAGACCATGAGCTTCAATACCCTGATTGA
CTGGAACAGCTACCGGGGTGTGTTCTGTGGAGCATTCTCACTTTTGGTAAACGACATTGCTTCAAGTG
CAGCGGAATCAAAAAGTATAAAGTGGGCAGCGAGTATACCTGTACAGACTGTAGGCGATAACTCAATC
CAATTACCCCCCACAACATGACTGGCCAAACTGATCTCAAGACTTTATTGAAATCAGCAACACCGATT
CTCAATGAAGGCACATACTTCTTCTGCAACATTCACTTGACGCCTAAAGTTGGTGAGAAATGGACCGA
CAAGACATATTCTGCTATCCACGGACTGTTGCCTGTGTCGGTGGCTACAATACGTGAGTCAGAAGGGC
TGACGGTGGTGGTTCCCAAGGAAAAGGTCGACGAGTATCTGTCTGACTCGTCATTGCCGCCTTTGGA
GTACGACTCCAACTATGAGTGTGCTTGGATCACTTTGACGATACATTCTTCGTTGGAGGCTGTGGGTCT
GACAGCTGCGTTTTCGGCGCGGTTGGCCGACAACAATATCAGCTGCAACGTCATTGCTGGCTTTCATC
ATGATCACATTTTTGTCGGCAAAGGCGACGCCCAGAGAGCCATTGACGTTCTTTCTAATTTGGACCGA
TAGCCGTATAGTCCAGTCTATCTATAAGTTCAACTAACTCGTAACTATTACCATAACATATACTTCACTG
CCCCAGATAAGGTTCCGATAAAAAGTTCTGCAGACTAAATTTATTTCAGTCTCCTCTTCACCACCAAAA
TGCCCTCCTACGAAGCTCGAGCTAACGTCCACAAGTCCGCCTTTGCCGCTCGAGTGCTCAAGCTCGT
GGCAGCCAAGAAAACCAACCTGTGTGCTTCTCTGGATGTTACCACCACCAAGGAGCTCATTGAGCTT
GCCGATAAGGTCGGACCTTATGTGTGCATGATCAAGACCCATATCGACATCATTGACGACTTCACCTAC
GCCGGCACTGTGCTCCCCCTCAAGGAACTTGCTCTTAAGCACGGTTTCTTCCTGTTCGAGGACAGAA
AGTTCGCAGATATTGGCAACACTGTCAAGCACCAGTACAAGAACGGTGTCTACCGAATCGCCGAGTG
GTCCGATATCACCAACGCCCACGGTGTACCCGGAACCGGAATCATTGCTGGCCTGCGAGCTGGTGCC
GAGGAAACTGTCTCTGAACAGAAGAAGGAGGACGTCTCTGACTACGAGAACTCCCAGTACAAGGAG
TTCCTGGTCCCCTCTCCCAACGAGAAGCTGGCCAGAGGTCTGCTCATGCTGGCCGAGCTGTCTTGCA
AGGGCTCTCTGGCCACTGGCGAGTACTCCAAGCAGACCATTGAGCTTGCCCGATCCGACCCCGAGTT
TGTGGTTGGCTTCATTGCCCAGAACCGACCTAAGGGCGACTCTGAGGACTGGCTTATTCTGACCCCCG
GGGTGGGTCTTGACGACAAGGGAGACGCTCTCGGACAGCAGTACCGAACTGTTGAGGATGTCATGTC
TACCGGAACGGATATCATAATTGTCGGCCGAGGTCTGTACGGCCAGAACCGAGATCCTATTGAGGAGG
CCAAGCGATACCAGAAGGCTGGCTGGGAGGCTTACCAGAAGATTAACTGTTAGAGGTTAGACTATGG
ATATGTAATTTAACTGTGTATATAGAGAGCGTGCAAGTATGGAGCGCTTGTTCAGCTTGTATGATGGTC
AGACGACCTGTCTGATCGAGTATGTATGATACTGCACAACCTGTGTATCCGCATGATCTGTCCAATGGG
GCATGTTGTTGTGTTTCTCGATACGGAGATGCTGGGTACAAGTAGCTAATACGATTGAACTACTTATAC
TTATATGAGGCTTGAAGAAAGCTGACTTGTGTATGACTTATTCTCAACTACATCCCCAGTCACAATACC
ACCACTGCACTACCACTACACCAAGCTCCGGATCTTCCAGTGGTGCATGAACGCATGAGAAAGCCCC
CGGAAGATCATCTTCCGGGGGCTTTTTTTTTGGCGCGCGATACAGACCGGTTCAGACAGGATAAAGAG
GAACGCAGAATGTTAGACAACACCCGCTTACGCATAGCTATTCAGAAATCAGGCCGTTTAAGCGATGA
TTCACGAGAATTGCTGGCCCGCTGCGGCATAAAAATTAATTTACACACTCAGCGCCTGATTGCGATGG
CGGAAAACATGCCGATTGATATCCTGCGCGTGCGTGATGATGACATTCCGGGTCTGGTAATGGATGGC
GTGGTCGATCTCGGTATTATCGGCGAAAACGTGCTGGAAGAAGAGCTACTCAACCGCCGCGCACAGG
GCGAAGATCCACGCTATTTAACCCTGCGCCGTCTTGACTTCGGCGGCTGCCGTTTATCGCTGGCAACA
CCGGTTGACGAAGCCTGGGACGGCCCGGCCGCGCTGGACGGTAAACGTATCGCTACCTCATATCCGC
ACCTCCTCAAACGCTACCTCGACCAGAAAGGCGTCTCTTTTAAATCGTGTCTGTTAAATGGTTCTGTC
GAAGTCGCGCCGCGCGCGGGGCTGGCCGACGCTATCTGCGATTTGGTCTCTACCGGCGCGACGCTTG
AAGCTAACGGCCTGCGTGAAGTCGAAGTTATCTACCGCTCTAAAGCCTGTCTGATTCAGCGCGACGGT
GAGATGGCACAGAGCAAGCAAGAGCTGATCGATAAATTGCTGACCCGTATTCAGGGCGTGATTCAGG
CGCGCGAATCGAAATACATCATGATGCACGCGCCAAGTGAACGCCTGGAAGAGGTTATCGCCCTGCT
GCCAGGCGCCGAAAGGCCGACAATTCTGCCGCTGGCAGGCGAGCAACAGCGCGTGGCGATGCACAT
GGTCAGCAGCGAAACGTTGTTCTGGGAAACCATGGAGAAACTGAAAGCGCTTGGCGCCAGCTCGATT
CTGGTACTGCCGATCGAGAAGATGATGGAGTGATCTGACGCCTGATGGCGCTGCGCTTATCAGGCCTA
CGTAATGCGTTGATATTTTGGGTTCTGTAGGCCGGATAAGGCGGAACCCTGTGATGGAGTAAAGACCA
TGAGCTTCAATACCCTGATTGACTGGAACAGCTTCTGGGGACCGAGAATTAGTGTTGATGCTAGCGCA
AGGCTTGTTCTTTAGTGGGCATCTACCGCAGCTACTATACACGAGCGCGACGTAGGTATAGCATGCCA
AAGGCGGGTGCGATACCAGATGACGCTATAATATGCCAGATTTGCACTAGCACACATCACTGGAAGCT
GTCAATTTGAAAATGTCAATGCAGTCTCACCATTGGACGATCTTGGCACCTCCTAAACTCTGGCAATG
TCTGAGCTCTTTGACACATAAGACATCAAGCAGACGGTCTAAAATCATATGCCATGTAAAGAACTACT
AGTGCCTGTAGAAGACGCCTGTAGAAGACGCCTGTAGAAGACGCCTGTAGAAGACGGCTACTTGTAT
ATTCTCACTTGTCGTGCTTCACTCACGGCTCGATGTCCACTGCTTACCTCAGCGGCAGCGGGCGGAAA
AAGCCTACAAACCCCTGTCGGCCGCGTTGCACTTGTTGGGCGACGACCAAATATGAGTTGGAAAAGT
TTTATCTTCGCCAGAGACGCCGTCCCATTGTTATTATTACAGAGGTCCATGAACATGAGCTTTAGATCA
GATCTGCACGTGGCGGAGCCGCCCAACCGTGTCTTTTCCCGTCGGTACGAGCTGATAAGCTGCACCTC
ACCAACAATCATGACATGAGGTCTTGTATGCAAGGGAGACGTCGGAGTGCTTCGGCAGGCTTCGGTT
AGGGCTTGGCTGGAGGCTAGCGACTGCAGTGGCGGAGTCCTCGTGGTCTTCCTTGATGTACCCCGTC
GCATGTGAGTCTGCTATGATATATGAAACTAATTCTAATCTAGTTGCGGTTAATTTCATATCATGGTCTTT
GTTTCTCCCTTATTGAATTCTATTTTTCATATCTTTGGCTGCTCCTCCCCTTTTTCGGTCTCTCCAGCAAT
CTTTGGCGAGTCTCGAAACCTGGAGGAAAACCTGTAGACAACCTGCGCGAAACGACGGCCAAAAAC
AGTGAAACTTGAGCCGAAAAAGCATGGAAAATCACACGTGACCATCGCATTTGGACTTTCCCATCGTT
GCCAGCAACTCCACGTGTTTGGTCACTTGTAGAGGTTTCGGTACTGTAGCTCTAGCTAGTCCATCGTA
CGTGTTCACCTGTGGAGACCCGACCCTTGAAGTGCGATACTCTACAGTGCACCAAGAGTACACCGAA
ACCAAAAGCAACCGAGTGTTGATCCCTCAATGGTTGGGGGAAATGTTTCACAGAGCGAGGGCACGA
AAGAAGGGGTTGGAAGAAGCAATCACCAGTTTTGCCAACGGTATTTACAGGGTTTTGTTGCTGTTTG
AGTATCGATAGTCCGACACGCAAAAATGAGCAGCACGACAATGGCGAGCCTCCTTTTTTCGTCCCTCT
CTCAAGCCCCCCCGCCCATTCAGACTTTGTGCCAAAAGAACTGCGAAACCAAAGAAAATAAACGTGT
ATCCGGGCCGTTTCCTTTCTTAGGGCAAAGCCAAACGGAACGGAGTTGAGGTCAACTTGTATGTGATT
TGGAGAAGCGCAACAAGGGCGACAACAGTAGGGTTTCGTTTCATAGCTTTTCGGTTATCGGTTTTATT
TGTTTGATTTTTGTCTCGCACGAGATAGAAGCCATGTTTGGGTCTGAAATGTCGCTCATACAGCTCTTC
GAATCGGACGTCTTCTTCTTCTTTATAGCTCCCGGTCTTTGTTTTTTGTTCCGTCGTTTTCGCTAAAAAG
AAAAAGTGACATGCTTGTTTTTTGGGCTCCCTTGTTCTGCTCTCTCCAAAAGTGCAGCGTTAGGGTTG
ACAGGCATTAGACTCAGCGCTTGGTAACTTCTTAAATAAAGGTGAACCTAGAGAAAGTTTGGAGGAG
AAAAAAAATAACAGTCCACCAGGTGGCGCCATGGGGGTTGAAGAGGGGCCATATTCTTGCGGGTATG
GGCAGCTGGGGGGTCACAGCATTGATTCCCCCAAACTCCCGTTCCATTCGGCCCACTGGAGCGCCCC
ATTTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCC
ACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACA
TTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAAT
CGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCG
CTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCAC
AGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTA
AAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACG
CTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC
CTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGC
GTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGG
CTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA
ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTA
TGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTG
GTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAA
ACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTC
AAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATT
TTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCA
ATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCA
GCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAG
GGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGACTCCCACGCTCACCGGCTCCAGATTTATC
AGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATC
CAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT
GCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAA
CGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT
CGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTAC
TGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTG
TATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTT
TAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGA
TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCT
GGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTG
AATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACA
TATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCT
GACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC)
As a skeleton, inserting a ScDGAT gene expression cassette into the plasmid skeleton to obtain a recombinant plasmid pUC-Intf3-DGAT;
(5) Preparation of yarrowia lipolytica engineering strain Polf-D2
Transferring the recombinant plasmid pUC-Intf3-DGAT into a yarrowia lipolytica engineering strain Polf-D1 (URA-) to obtain a yarrowia lipolytica engineering strain Polf-D2;
(6) Construction of recombinant plasmid pUC-Intf3-GPAT
Taking pUC-Intf3 plasmid as a framework, and inserting a PtGPAT gene expression cassette into the plasmid framework to obtain a recombinant plasmid pUC-Intf3-GPAT;
(7) Preparation of yarrowia lipolytica engineering strain Polf-D3
Transferring the recombinant plasmid pUC-Intf3-GPAT into a yarrowia lipolytica engineering strain Polf-D1 (URA-) to obtain a yarrowia lipolytica engineering strain Polf-D3;
(8) Construction of recombinant plasmid pUC-Intf3-LPAAT
Taking pUC-Intf3 plasmid as a framework, and inserting PtLPAAT gene expression cassettes into the plasmid framework to obtain recombinant plasmid pUC-Intf3-LPAAT;
(9) Preparation of yarrowia lipolytica engineering strain Polf-D4
Transferring the recombinant plasmid pUC-Intf3-LPAAT into a yarrowia lipolytica engineering strain Polf-D1 (URA-) to obtain a yarrowia lipolytica engineering strain Polf-D4;
(10) Construction of recombinant plasmid pUC-scp2-GPAT-LPAAT
The pUC-scp2 plasmid (pUC plasmid added to the upper and lower homology arms of the scp2 site, the sequence was:
GCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAGATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTATTTAAATGTGCTGTTCAGAAGCCGGCCGGGTAATGCCACGTAAGAGTCACGTGAATCTAAGGGGAGGGGTGGTTGGGTGGCAACAGACGAGATTAACAGACCAATATAGATCAGGGCTAGGGTACGAGGCCCCTCAAACAAATAATTTTACCGTCCAGTAAATTTTCCACCCTCCTTTGCCCCTTCTACTCGTTCAATACATCCCCATCTCCACCGTTCTCTTCAGATATAGATCTCTTCGCTGAAAGCGTAATGTGGTGTTGTTCAGCCTTGAAGCAGGAAGTGTATTTGGCGGTGTGAAATAGTTCTGACAGCAACGCGAGCCACGCGAAAGGGAACGACAGGCACTCAAAATGACGTGGTAAAGGTCCTGTTTTTTTACACATGATTAAATGAGAAGATTGTGGCTTATTTATATATTTTTTCTTGCAGAAATCACCCCATTTCGACAAAATCATTACTGTTTTGGCGCATGGTACATTCCGCCGTGCGAGTCAAACGCGTCGACGTGTTAGGAGTGCGAGTGCCAGTACAAGTGTCGGTTGTATAACTAAGTACAGGTGGTACGAGTGCGAGTACGTACCTTGTAGTAACCATGATCGTCGTTTGTTGAGTCTGAGAATATTGATTGTCTTCTACTTGTACTTGTACTACCAGTGTAAGTACTACCAGTTTGTACGATAGTCGTATCTATGTACTATCTTTCAGTATTGTACAGCACATACTTTACATGATTATTGATAATGCAAAAAAACTGAAAGAAATATGAGAAATAGAATATTGTAAATATGAGCGATAGAATATTGTAAATATGAGCGATAGAATATTGTATATATTTGTCGTGGTGTGTGCAGATGGTGTTGTCCAGCAAGACAAGTTCTTCAAAGAATAACAGCGTAGTCCATACTCGTACTACATACATCAAGACCCCATACAATCATTGTCTTCGTTATCAGTTCTTGTACCCGTATCTACTGTACATACTTGTATCTTCATTGCCATTGTTGTATCTAAACTACTGGAAGTCTCTGGAGCAAGATTGAGAGTGGTATGTGTCGACCCTTGAGGGCCGATGCAACCTATAGGGGATGCGGTCGGATTGACAGACCGCCTACTGCCAATGTTTGTTACTGCTCTGCTATCCGTCTACGGAGCTACTCCACCAACTCACCAACGTACCCACCATCTGCCAATACGCTACTCCTCCCTAGATCTAAATCGCGAGCTTCGAAAAGTGGGTGAGACATGTCCTCAACTGTCCCCGACCACGCTGGTGTCCGTATGGAATACGTCGTGGCTGTAAGACACAACAATCTCCCCGCTCGTATTCCGACAGATCCACCGGGCCACTCTTGGACGAGCCGCGCGTGCAAAACACAATATGCTCCGAGACCCGACCTTGGCATCTGGATCTTGCTCTTGCGATACGACTACTGTGCCCGTAAAGCTCCCATATAGATACATGCTCGCGCCTCGATAGGCTTATACGGGGGTTGCGTTTTTTTGGGGGTAAAAAAAAGGATACAAAAAAGGGTAAAAAAAATAGTACGTACCTTATTAGCGACGCACGTACGTGGGCTGCGTCCATCAACCCCGCCCATCAATCCATGGAATCTTTGCCGTGAGACACACAAGTTTCAGACTTGCTCCTTTTGAGTCTTCTCATCATTTACGACAGTTCTTTTTCCACCCACAGCCATCCGACTTGTGCATCTCC
GTATCCTAGCCGAGCCCATTCGGGGTGCATGACAACTGCGGGATCTTGGGGGTAGAGTGTTTAGCGAA
ATTAATATATGCAGTATAGTGCAGGATGAGCTAAGAAGCATTCTACAAACAGTTGTATACAGGCACCAG
ACATTTATATAGTTATTGGGAAACGTCGAGTGGCGTAAGATGGCGGTGCCCTGGAGCCACTCACACAT
GTATCATAGGTTTAATAAGTCCCGCACGGGTCGTTTCTAATGTAATGTCTCATGTCTAAGAACCACGGT
GTATTCACCGAACTATCCTTGAGATACTCATTGCTCATCGCTACGGGCTCGACGCTGGCATTTTTTCTCC
GATAATTTCACGGTGGACGTACCCACAGGCCATCCCTTCTGCTTTGGGAACTCGCGTATGCGGCCGCA
ATTCCCGGATCTTCCAGTGGTGCATGAACGCATGAGAAAGCCCCCGGAAGATCATCTTCCGGGGGCTT
TTTTTTTGGCGCGCGATACAGACCGGTTCAGACAGGATAAAGAGGAACGCAGAATGTTAGACAACAC
CCGCTTACGCATAGCTATTCAGAAATCAGGCCGTTTAAGCGATGATTCACGAGAATTGCTGGCCCGCT
GCGGCATAAAAATTAATTTACACACTCAGCGCCTGATTGCGATGGCGGAAAACATGCCGATTGATATCC
TGCGCGTGCGTGATGATGACATTCCGGGTCTGGTAATGGATGGCGTGGTCGATCTCGGTATTATCGGCG
AAAACGTGCTGGAAGAAGAGCTACTCAACCGCCGCGCACAGGGCGAAGATCCACGCTATTTAACCCT
GCGCCGTCTTGACTTCGGCGGCTGCCGTTTATCGCTGGCAACACCGGTTGACGAAGCCTGGGACGGC
CCGGCCGCGCTGGACGGTAAACGTATCGCTACCTCATATCCGCACCTCCTCAAACGCTACCTCGACCA
GAAAGGCGTCTCTTTTAAATCGTGTCTGTTAAATGGTTCTGTCGAAGTCGCGCCGCGCGCGGGGCTGG
CCGACGCTATCTGCGATTTGGTCTCTACCGGCGCGACGCTTGAAGCTAACGGCCTGCGTGAAGTCGAA
GTTATCTACCGCTCTAAAGCCTGTCTGATTCAGCGCGACGGTGAGATGGCACAGAGCAAGCAAGAGC
TGATCGATAAATTGCTGACCCGTATTCAGGGCGTGATTCAGGCGCGCGAATCGAAATACATCATGATGC
ACGCGCCAAGTGAACGCCTGGAAGAGGTTATCGCCCTGCTGCCAGGCGCCGAAAGGCCGACAATTCT
GCCGCTGGCAGGCGAGCAACAGCGCGTGGCGATGCACATGGTCAGCAGCGAAACGTTGTTCTGGGA
AACCATGGAGAAACTGAAAGCGCTTGGCGCCAGCTCGATTCTGGTACTGCCGATCGAGAAGATGATG
GAGTGATCTGACGCCTGATGGCGCTGCGCTTATCAGGCCTACGTAATGCGTTGATATTTTGGGTTCTGT
AGGCCGGATAAGGCGGAACCCTGTGATGGAGTAAAGACCATGAGCTTCAATACCCTGATTGACTGGA
ACAGCTACCGGGGTGTGTTCTGTGGAGCATTCTCACTTTTGGTAAACGACATTGCTTCAAGTGCAGCG
GAATCAAAAAGTATAAAGTGGGCAGCGAGTATACCTGTACAGACTGTAGGCGATAACTCAATCCAATT
ACCCCCCACAACATGACTGGCCAAACTGATCTCAAGACTTTATTGAAATCAGCAACACCGATTCTCAA
TGAAGGCACATACTTCTTCTGCAACATTCACTTGACGCCTAAAGTTGGTGAGAAATGGACCGACAAG
ACATATTCTGCTATCCACGGACTGTTGCCTGTGTCGGTGGCTACAATACGTGAGTCAGAAGGGCTGAC
GGTGGTGGTTCCCAAGGAAAAGGTCGACGAGTATCTGTCTGACTCGTCATTGCCGCCTTTGGAGTAC
GACTCCAACTATGAGTGTGCTTGGATCACTTTGACGATACATTCTTCGTTGGAGGCTGTGGGTCTGAC
AGCTGCGTTTTCGGCGCGGTTGGCCGACAACAATATCAGCTGCAACGTCATTGCTGGCTTTCATCATG
ATCACATTTTTGTCGGCAAAGGCGACGCCCAGAGAGCCATTGACGTTCTTTCTAATTTGGACCGATAG
CCGTATAGTCCAGTCTATCTATAAGTTCAACTAACTCGTAACTATTACCATAACATATACTTCACTGCCC
CAGATAAGGTTCCGATAAAAAGTTCTGCAGACTAAATTTATTTCAGTCTCCTCTTCACCACCAAAATGC
CCTCCTACGAAGCTCGAGCTAACGTCCACAAGTCCGCCTTTGCCGCTCGAGTGCTCAAGCTCGTGGC
AGCCAAGAAAACCAACCTGTGTGCTTCTCTGGATGTTACCACCACCAAGGAGCTCATTGAGCTTGCC
GATAAGGTCGGACCTTATGTGTGCATGATCAAGACCCATATCGACATCATTGACGACTTCACCTACGCC
GGCACTGTGCTCCCCCTCAAGGAACTTGCTCTTAAGCACGGTTTCTTCCTGTTCGAGGACAGAAAGTT
CGCAGATATTGGCAACACTGTCAAGCACCAGTACAAGAACGGTGTCTACCGAATCGCCGAGTGGTCC
GATATCACCAACGCCCACGGTGTACCCGGAACCGGAATCATTGCTGGCCTGCGAGCTGGTGCCGAGG
AAACTGTCTCTGAACAGAAGAAGGAGGACGTCTCTGACTACGAGAACTCCCAGTACAAGGAGTTCC
TGGTCCCCTCTCCCAACGAGAAGCTGGCCAGAGGTCTGCTCATGCTGGCCGAGCTGTCTTGCAAGGG
CTCTCTGGCCACTGGCGAGTACTCCAAGCAGACCATTGAGCTTGCCCGATCCGACCCCGAGTTTGTGG
TTGGCTTCATTGCCCAGAACCGACCTAAGGGCGACTCTGAGGACTGGCTTATTCTGACCCCCGGGGTG
GGTCTTGACGACAAGGGAGACGCTCTCGGACAGCAGTACCGAACTGTTGAGGATGTCATGTCTACCG
GAACGGATATCATAATTGTCGGCCGAGGTCTGTACGGCCAGAACCGAGATCCTATTGAGGAGGCCAAG
CGATACCAGAAGGCTGGCTGGGAGGCTTACCAGAAGATTAACTGTTAGAGGTTAGACTATGGATATGT
AATTTAACTGTGTATATAGAGAGCGTGCAAGTATGGAGCGCTTGTTCAGCTTGTATGATGGTCAGACGA
CCTGTCTGATCGAGTATGTATGATACTGCACAACCTGTGTATCCGCATGATCTGTCCAATGGGGCATGT
TGTTGTGTTTCTCGATACGGAGATGCTGGGTACAAGTAGCTAATACGATTGAACTACTTATACTTATATG
AGGCTTGAAGAAAGCTGACTTGTGTATGACTTATTCTCAACTACATCCCCAGTCACAATACCACCACT
GCACTACCACTACACCAAGCTCCGGATCTTCCAGTGGTGCATGAACGCATGAGAAAGCCCCCGGAAG
ATCATCTTCCGGGGGCTTTTTTTTTGGCGCGCGATACAGACCGGTTCAGACAGGATAAAGAGGAACGC
AGAATGTTAGACAACACCCGCTTACGCATAGCTATTCAGAAATCAGGCCGTTTAAGCGATGATTCACG
AGAATTGCTGGCCCGCTGCGGCATAAAAATTAATTTACACACTCAGCGCCTGATTGCGATGGCGGAAA
ACATGCCGATTGATATCCTGCGCGTGCGTGATGATGACATTCCGGGTCTGGTAATGGATGGCGTGGTCG
ATCTCGGTATTATCGGCGAAAACGTGCTGGAAGAAGAGCTACTCAACCGCCGCGCACAGGGCGAAGA
TCCACGCTATTTAACCCTGCGCCGTCTTGACTTCGGCGGCTGCCGTTTATCGCTGGCAACACCGGTTG
ACGAAGCCTGGGACGGCCCGGCCGCGCTGGACGGTAAACGTATCGCTACCTCATATCCGCACCTCCTC
AAACGCTACCTCGACCAGAAAGGCGTCTCTTTTAAATCGTGTCTGTTAAATGGTTCTGTCGAAGTCGC
GCCGCGCGCGGGGCTGGCCGACGCTATCTGCGATTTGGTCTCTACCGGCGCGACGCTTGAAGCTAAC
GGCCTGCGTGAAGTCGAAGTTATCTACCGCTCTAAAGCCTGTCTGATTCAGCGCGACGGTGAGATGGC
ACAGAGCAAGCAAGAGCTGATCGATAAATTGCTGACCCGTATTCAGGGCGTGATTCAGGCGCGCGAA
TCGAAATACATCATGATGCACGCGCCAAGTGAACGCCTGGAAGAGGTTATCGCCCTGCTGCCAGGCG
CCGAAAGGCCGACAATTCTGCCGCTGGCAGGCGAGCAACAGCGCGTGGCGATGCACATGGTCAGCA
GCGAAACGTTGTTCTGGGAAACCATGGAGAAACTGAAAGCGCTTGGCGCCAGCTCGATTCTGGTACT
GCCGATCGAGAAGATGATGGAGTGATCTGACGCCTGATGGCGCTGCGCTTATCAGGCCTACGTAATGC
GTTGATATTTTGGGTTCTGTAGGCCGGATAAGGCGGAACCCTGTGATGGAGTAAAGACCATGAGCTTC
AATACCCTGATTGACTGGAACAGCTTCTGGGGACCGAGAATTAGTGTTGATGCTAGCGCAAGGCTTGT
TCTTTAGTGGGCATCTACCGCAGCTACTATACACGAGCGCGACGTAGGTATAGCATGCCAAAGGCGGG
TGCGATACCAGATGACGCTATAATATGCCAGATTTGCACTAGCACACATCACTGGAAGCTGTCAATTTG
AAAATGTCAATGCAGTCTCACCATTGGACGATCTTGGCACCTCCTAAACTCTGGCAATGTCTGAGCTC
TTTGACACATAAGACATCAAGCAGACGGTCTAAAATCATATGCCATGTAAAGAACTACTAGTGCCTGT
AGAAGACGCCTGTAGAAGACGCCTGTAGAAGACGCCTGTAGAAGACGGCTACTTGTATATTCTCACTT
GTCGTGCTTCACTCACGGCTCGATGTCCACTGCTTACCTCAGCGGCAGCGGGCGGAAAAAGCCTACA
AACCCCTGTCGGCCGCGTTGCACTTGTTGGGCGACGACCAAATATGAGTTGGAAAAGTTTTATCTTCG
CCAGAGACGCCGTCCCATTGTTATTATTACAGAGGTCCATGAACATGAGCTTTAGATCAGATCTGCACG
TGGCGGAGCCGCCCAACCGTGTCTTTTCCCGTCGGTACGAGCTGATAAGCTGCACCTCACCAACAATC
ATGACATGAGGTCTTGTATGCAAGGGAGACGTCGGAGTGCTTCGGCAGGCTTCGGTTAGGGCTTGGC
TGGAGGCTAGCGACTGCAGTGGCGGAGTCCTCGTGGTCTTCCTTGATGTACCCCGTCGCATGTGAGTC
TGCTATGATATATGAAACTAATTCTAATCTAGTTGCGGTTAATTTCATATCATGGTCTTTGTTTCTCCCTTA
TTGAATTCTATTTTTCATATCTTTGGCTGCTCCTCCCCTTTTTCGGTCTCTCCAGCAATCTTTGGCGAGT
CTCGAAACCTGGAGGAAAACCTGTAGACAACCTGCGCGAAACGACGGCCAAAAACAGTGAAACTTG
AGCCGAAAAAGCATGGAAAATCACACGTGACCATCGCATTTGGACTTTCCCATCGTTGCCAGCAACT
CCACGTGTTTGGTCACTTGTAGAGGTTTCGGTACTGTAGCTCTAGCTAGTCCATCGTACGTGTTCACCT
GTGGAGACCCGACCCTTGAAGTGCGATACTCTACAGTGCACCAAGAGTACACCGAAACCAAAAGCA
ACCGAGTGTTGATCCCTCAATGGTTGGGGGAAATGTTTCACAGAGCGAGGGCACGAAAGAAGGGGTT
GGAAGAAGCAATCACCAGTTTTGCCAACGGTATTTACAGGGTTTTGTTGCTGTTTGAGTATCGATAGT
CCGACACGCAAAAATGAGCAGCACGACAATGGCGAGCCTCCTTTTTTCGTCCCTCTCTCAAGCCCCC
CCGCCCATTCAGACTTTGTGCCAAAAGAACTGCGAAACCAAAGAAAATAAACGTGTATCCGGGCCGT
TTCCTTTCTTAGGGCAAAGCCAAACGGAACGGAGTTGAGGTCAACTTGTATGTGATTTGGAGAAGCG
CAACAAGGGCGACAACAGTAGGGTTTCGTTTCATAGCTTTTCGGTTATCGGTTTTATTTGTTTGATTTT
TGTCTCGCACGAGATAGAAGCCATGTTTGGGTCTGAAATGTCGCTCATACAGCTCTTCGAATCGGACG
TCTTCTTCTTCTTTATAGCTCCCGGTCTTTGTTTTTTGTTCCGTCGTTTTCGCTAAAAAGAAAAAGTGA
CATGCTTGTTTTTTGGGCTCCCTTGTTCTGCTCTCTCCAAAAGTGCAGCGTTAGGGTTGACAGGCATTA
GACTCAGCGCTTGGTAACTTCTTAAATAAAGGTGAACCTAGAGAAAGTTTGGAGGAGAAAAAAAATA
ACAGTCCACCAGGTGGCGCCATGGGGGTTGAAGAGGGGCCATATTCTTGCGGGTATGGGCAGCTGGG
GGGTCACAGCATTGATTCCCCCAAACTCCCGTTCCATTCGGCCCACTGGAGCGCCCCATTTAAATAGC
TTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATA
CGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTT
GCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCG
CGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTC
GTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGG
ATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCG
TTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAG
GTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT
CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCT
CATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGA
ACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGAC
ACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGC
TACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTC
TGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGG
TAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCT
TTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG
ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATA
TATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTA
TTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCT
GGCCCCAGTGCTGCAATGATACCGCGACTCCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCA
GCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATT
GTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACA
GGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCG
AGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAA
GTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATC
CGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGAC
CGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTC
ATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGAT
GTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAA
AAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATAC
TCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGT
ATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC)
Inserting a PtGPAT gene expression cassette and a ScDGAT gene expression cassette into the plasmid skeleton to obtain a recombinant plasmid pUC-scp2-GPAT-LPAAT;
(11) Preparation of yarrowia lipolytica engineering strain Polf-D5
Transferring the recombinant plasmid pUC-scp2-GPAT-LPAAT into yarrowia lipolytica engineering strain Polf-D2 (URA-) to obtain yarrowia lipolytica engineering strain Polf-D5.
Preferably, the method for constructing the uracil auxotroph yarrowia lipolytica Polf in the step (2) comprises the following steps: knocking out uracil-encoding genes in yarrowia lipolytica Po1f by CRISPR/Cas9 technology;
specific construction methods are described in journal Journal ofAgricultural and Food Chemistry, 2021, volume 69, 46, pages 13831-13837, under the paper designation "Harnessing Yarrowia lipolytica Peroxisomes as a Subcellular Factory for. Alpha. -Humulene Overproduction".
Preferably, the construction method of the recombinant plasmid PUC-Intc-delta 15DEs in the step (1) comprises the following steps: the PUC-Intc plasmid with uracil genetic marker is used as a skeleton, and a delta 15 desaturase gene expression cassette is inserted into the plasmid skeleton.
Preferably, in step (1), the LKFAD15 gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoters or P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (4), the ScDGAT gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of promoters and terminatorIs T derived from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (6), the PtGPAT gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (8), the PtLPAAT gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (10), the PtGPAT gene expression cassette and the ScDGAT gene expression cassette promoter are P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators.
Use of an engineered strain of yarrowia lipolytica as described above for the production of alpha-linolenic acid.
A method of producing alpha-linolenic acid by fermentation using a yarrowia lipolytica engineered strain as described above that is fermented to produce alpha-linolenic acid on YNB nitrogen-limited medium;
the formula of the YNB nitrogen limiting medium is as follows: yeast powder 2.5g/L, YNB medium 1.7g/L, nitrogen source 6g/L,115 degrees centigrade sterilization 30 minutes.
Preferably, the method comprises the following steps:
(1) Preparation of seed liquid
Activating yarrowia lipolytica engineering strains, inoculating the yarrowia lipolytica engineering strains into YPD liquid culture medium, and culturing the yarrowia lipolytica engineering strains to logarithmic growth phase to obtain seed liquid;
(2) Fermentation production of alpha-linolenic acid
Inoculating the seed solution into YNB nitrogen-limiting culture medium according to the volume ratio of 1% -5%, fermenting at 28-30 ℃ and 200-240 rpm, and carrying out ventilation culture for 96-120 h to obtain a fermentation product alpha-linolenic acid;
wherein, the formula of YNB nitrogen limiting medium is: 2.5g/L yeast powder, 1.0-2.5 g/L YNB culture medium, 5-7 g/L nitrogen source, and sterilizing at 115 ℃ for 30 minutes.
Preferably, in the step (1), the viable count in the seed liquid is 1.2X10 8 ～2.4×10 8 CFU/mL；
In the step (2), the nitrogen source addition amount in the fermentation culture is 6g/L, and the YNB addition amount is 1.7g/L.
Specifically, the preparation and detection of the correlation are as follows:
in this example, although yarrowia lipolytica was used as the starting strain, conventional uracil auxotroph yarrowia lipolytica was used as the starting strain for transformation to perform the test in the example.
Yarrowia lipolytica as used herein was itself capable of synthesizing C16 and C18 long chain fatty acids, for which purpose delta 15 desaturases from Lipomyces kononenkoae (LKFAD 15) were converted to uracil auxotrophs yarrowia lipolytica Po1f was constructed.
EXAMPLE 1 construction of yarrowia lipolytica engineering Strain Polf-D1 engineering Strain for producing alpha-linolenic acid
1. Construction of uracil auxotrophs yarrowia lipolytica Polf
The part is uracil auxotroph yarrowia lipolytica Polf construction, and the construction method comprises the following steps: the gene sequence of uracil-encoding yarrowia lipolytica Polf (URA-) in uracil auxotrophs is obtained by knocking out the gene sequence of uracil-encoding yarrowia lipolytica Po1f by CRISPR/Cas9 technology, wherein 1200bp is selected from the upstream and downstream of the gene sequence of uracil-encoding gene as homology arms.
Specific construction methods are described in journal Journal ofAgricultural and Food Chemistry, 2021, volume 69, 46, pages 13831-13837, under the paper designation "Harnessing Yarrowia lipolytica Peroxisomes as a Subcellular Factory for. Alpha. -Humulene Overproduction".
2. Construction of recombinant plasmid PUC-Intc-Delta15 DEs
S1, synthesizing an optimized delta 15 desaturase gene from Lipomyces kononenkoae (LKFAD 15) to obtain a codon optimized delta 15 desaturase gene, wherein the nucleotide sequence of the codon optimized delta 15 desaturase gene is shown as a sequence table SEQ ID No. 1.
S2, amplification of target genes
S21, performing PCR amplification by taking the delta 15 desaturase gene synthesized in the step S1 as a template and taking LKFAD15-F (the nucleotide sequence is shown as SEQ ID NO. 5) and LKFAD15-R (the nucleotide sequence is shown as SEQ ID NO. 6) as primers to obtain an amplified fragment LKFAD15;
s22, performing PCR amplification by taking yarrowia lipolytica genome as a template and YAT-F and YAT-R as primers to obtain a promoter PYAT, wherein the gene sequence of the promoter PYAT is shown as SEQ ID NO. 7;
s23, performing PCR amplification by taking yarrowia lipolytica genome as a template and CY-F and CY-R as primers to obtain a terminator TCYC1t, wherein the gene sequence of the terminator TCYC1t is shown as SEQ ID NO. 8;
the procedure for PCR was as follows: denaturation at 98℃for 10s, annealing at 56℃for 10s, and extension at 72℃for 1min (extension time=target fragment length/1 kb, unit min), 35 cycles were repeated.
S3, enzyme cutting is carried out on the PUC-Intc plasmid with uracil genetic markers through BamH1 enzyme, the three amplified genes are connected into an enzyme-cut PUC-Intc plasmid skeleton, and ClonExpress MultiS One Step CloningKit is adopted for one-step cloning, so that recombinant plasmid PUC-Intc-delta 15DEs with uracil genetic markers is constructed;
S4, purifying and recovering each fragment by AxyPrepTM DNAGel Extraction Kit (purchased from Corning life sciences Co., ltd.);
s5, transforming the circular recombinant plasmid into competent cells of escherichia coli DH5a, screening the grown transformant by an ampicillin-resistant flat plate, and verifying by colony PCR and sequencing to obtain a positive recombinant plasmid PUC-Intc-delta 15DEs, wherein a skeleton diagram of the recombinant plasmid PUC-Intc-delta 15DEs is shown in figure 2.
Wherein,
(1) the LKFAD15-F gene sequence is shown in SEQ ID NO.5, and specifically comprises the following steps:
GTGAAGTCCACCACAGACATTTTGAATGATTCTTATACTC
(2) the LKFAD15-R gene sequence is shown in SEQ ID NO.6, and specifically comprises the following steps:
CGGTTAAGCCCATCGACTGATCATGTAATTAGTTATGTCA
③P YAT the gene sequence of the promoter is shown in SEQ ID NO.7, and specifically comprises the following steps:
GTACGTAGCAACAACAGTGTACGCAGTACTATAGAGGAACAATTGCCCCGGAGAAGACGGCCAGGCCGCCTAGATGACAAATTCAACAACTCACAGCTGACTTTCTGCCATTGCCACTAGGGGGGGGCCTTTTTATATGGCCAAGCCAAGCTCTCCACGTCGGTTGGGCTGCACCCAACAATAAATGGGTAGGGTTGCACCAACAAAGGGATGGGATGGGGGGTAGAAGATACGAGGATAACGGGGCTCAATGGCACAAATAAGAACGAATACTGCCATTAAGACTCGTGATCCAGCGACTGACACCATTGCATCATCTAAGGGCCTCAAAACTACCTCGGAACTGCTGCGCTGATCTGGACACCACAGAGGTTCCGAGCACTTTAGGTTGCACCAAATGTCCCACCAGGTGCAGGCAGAAAACGCTGGAACAGCGTGTACAGTTTGTCTTAGCAAAAAGTGAAGGCGCTGAGGTCGAGCAGGGTGGTGTGACTTGTTATAGCCTTTAGAGCTGCGAAAGCGCGTATGGATTTGGCTCATCAGGCCAGATTGAGGGTCTGTGGACACATGTCATGTTAGTGTACTTCAATCGCCCCCTGGATATAGCCCCGACAATAGGCCGTGGCCTCATTTTTTTGCCTTCCGCACATTTCCATTGCTCGGTACCCACACCTTGCTTCTCCTGCACTTGCCAACCTTAATACTGGTTTACATTGACCAACATCTTACAAGCGGGGGGCTTGTCTAGGGTATATATAAACAGTGGCTCTCCCAATCGGTTGCCAGTCTCTTTTTTCCTTTCTTTCCCCACAGATTCGAAATCTAAACTACACATCACACAATGCCTGTTACTGACGTCCTTAAGCGAAAGTCCGGTGTCATCGTCGGCGACGATGTCCGAGCCGTGAGTATCCACGACAAGATCAGTGTCGAGACGACGCGTTTTGTGTAATGACACAATCCGAAAGTCGCTAGCAACACACACTCTCTACACAAACTAACCCAGCTCTTC
④T CYC1t the gene sequence of the terminator is shown in SEQ ID NO.8, and specifically comprises the following steps:
TCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCTCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGC；
wherein, the PCR enzyme used in the PCR reaction is PrimeSTAR MaxDNA polymerase of TAKARA, the PCR amplification system is shown in Table 1, the BamH1 cleavage system is shown in Table 2, and the one-step cloning system is shown in Table 3.
TABLE 1 PCR amplification System
| Reagent(s) | Usage amount |
| PrimeSTARMax(2×) | 25μL |
| Primer LKFAD15-F | 2μL |
| Primer LKFAD15-F | 2μL |
| Template | 1μL |
| ddH2O | 20μL |
TABLE 2 PUC-57-inTF3 plasmid enzyme digestion system
| Component (A) | Recombination reactions |
| Plasmid vector | 2μg |
| BamH1 enzyme | 2μL |
| 10xQCutBuffer | 5μL |
| ddH2O | To50μL |
TABLE 3 one-step cloning System
| Component (A) | Recombination reactions |
| Linearization carrier | XμL |
| N inserts | Y1+Y2+…YnμL |
| 2×ClonExpressMix | 5μL |
| ddH2O | To10μL |
X= (0.02 Xbase pair of cloning vector) ng (0.03 pmol)
Y= (0.02. Times.base pair per fragment) ng (0.03 pmol)
3. Construction of yarrowia lipolytica engineering strain Polf-D1
The construction method of the yarrowia lipolytica genetic engineering strain expressing the delta 15 desaturase gene comprises the following steps in sequence:
s1 activation of uracil auxotrophs yarrowia lipolytica Po1f
Taking 5 mu L of uracil auxotroph strain obtained in the step 1 of the example 1, namely uracil auxotroph yarrowia lipolytica Polf streaked on YPD solid medium for activation, and growing single colony;
s2 preparation of uracil auxotroph yarrowia lipolytica Po1f competence
Selecting single colony to 5mL YPD liquid culture medium, culturing in a shaking table at 28 ℃ and 220rpm until the absorption value of bacterial liquid at 600nm is 0.8-1.0;
taking 1mL of bacterial liquid, centrifuging at 4 ℃ and 4000rpm for 3min, collecting bacterial cells, and preparing competence according to the operation method of a yeast transformation kit Frozen-EZ YeastTransformation IITM, wherein the specific method comprises the following steps: re-suspending with 1mLEZ1, centrifuging at 4000rpm at 4deg.C for 3min, and collecting thallus again; the supernatant was discarded, and 100. Mu.L of EZ2 suspension cells were used, and 50. Mu.L of each was dispensed and stored at-80℃to obtain yarrowia lipolytica Po1f competent cells.
S3 construction of yarrowia lipolytica Po1f-D1
Taking out yarrowia lipolytica Po1f competent cells stored at-80deg.C, and placing on ice for 10min; 1. Mu.g of the recombinant plasmid PUC-Intc-Delta15 DEs with uracil genetic marker obtained in the step 2 of example 1 was added into competent cells of yarrowia lipolytica Po1f, gently stirred and mixed, then 500. Mu.L of Frozen-EZ Solution3 (commercially available) was added, mixed uniformly, incubated in a shaker at 28℃and 220rpm for 60min, spread on YNB solid medium for screening, and the grown transformant was verified to be the correct engineering strain Polf-D1,
wherein, the proportion of YPD solid culture medium is shown in Table 4; the proportion of YPD liquid culture medium is shown in Table 5; the proportions of YNB solid medium are shown in Table 6.
TABLE 4 YPD solid Medium ratio Table
| Name of the name | Yeast paste | Peptone | Glucose | Agar-agar |
| Content (g/L) | 10 | 20 | 20 | 20 |
TABLE 5 YPD liquid Medium ratio Table
| Name of the name | Yeast paste | Peptone | Glucose |
| Content (g/L) | 10 | 20 | 20 |
TABLE 6 YNB solid Medium proportioning Table
| Name of the name | YNB (without amino acid and ammonium sulfate) | Ammonium sulfate | Glucose | Agar-agar |
| Content (g/L) | 1.7 | 5 | 20 | 25 |
EXAMPLE 2 construction of recombinant plasmid for increasing lipid content
1. Construction of recombinant plasmid pUC-Intf3-DGAT
S1, amplification of target gene fragment
S11, synthesizing an optimized ScDGAT gene derived from Schizochytrium Sp (ScDGAT)) to obtain a codon optimized ScDGAT gene, wherein the nucleotide sequence of the codon optimized ScDGAT gene is shown as a sequence table SEQ ID No. 2.
S12, carrying out PCR amplification by using the ScDGAT gene synthesized in the step S11 as a template and using a primer ScDGAT-F (with a nucleotide sequence shown as SEQ ID NO. 9) and a primer ScDGAT-R (with a nucleotide sequence shown as SEQ ID NO. 10) as primers to obtain an amplified fragment ScDGAT;
s14, performing PCR amplification by taking yarrowia lipolytica genome as a template and YAT-F and YAT-R genes as primers to obtain a promoter P YAT ；
S15, performing PCR amplification by taking yarrowia lipolytica genome as a template and CY-F and CY-R genes as primers to obtain a terminator T CYC1t ；
Wherein the enzymes and systems used for PCR amplification are as shown in Table 1, and the procedure for PCR is as follows: denaturation at 98℃for 10s, annealing at 56℃for 10s, and extension at 72℃for 1min (extension time=target fragment length/1 kb, unit min), 35 cycles were repeated.
S2 construction of recombinant plasmid pUC-Intf3-DGAT
S21, carrying out enzyme digestion on a plasmid pUC-Intf3 with uracil genetic markers through BamH1 enzyme, connecting amplified fragments of ScDGAT, a promoter PYAT and a codon TCYC1t into an digested pUC-Intf3 plasmid skeleton, and carrying out one-step cloning by adopting ClonExpress MultiS One Step Cloning Kit to construct a recombinant plasmid pUC-Intf3-DGAT;
S22, purifying and recovering each fragment by AxyPrepTM DNAGel Extraction Kit (purchased from Corning life sciences Co., ltd.);
s23, transforming the circular recombinant plasmid into escherichia coli DH5a competent cells, screening the grown transformant by an ampicillin-resistant flat plate, and verifying by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-Intf3-DGAT. The recombinant plasmid carries uracil genetic markers, and a skeleton diagram of the pUC-Intf3-DGAT plasmid is shown in FIG. 3.
Wherein (1) the gene sequence of ScDGAT-F is shown as SEQ ID NO.9, specifically:
GAGTATAAGAATCATTCAAATTACTTTTGAATGAGCAGCG
(2) the gene sequence of the ScDGAT-R is shown as SEQ ID NO.10, and specifically comprises the following steps:
TGACATAACTAATTACATGAATGCAGACACCGTACAGCAC
wherein the system of one-step cloning is shown in Table 3, and the BamH1 cleavage system of the plasmid pUC-Intf3-DGAT is shown in Table 7
TABLE 7 plasmid pUC-Intf3-DGAT cleavage System
| Component (A) | Recombination reactions |
| Plasmid vector | 2μg |
| BamH1 enzyme | 2μL |
| 10xQCutBuffer | 5μL |
| ddH 2 O | To50μL |
2. Construction of recombinant plasmid pUC-Intf3-GPAT
S1, amplification of target gene fragment
S11, synthesizing an optimized PtGPAT gene derived from Phaeodactylum tricornutum (PtGPAT) to obtain a codon optimized PtGPAT gene, wherein the nucleotide sequence of the codon optimized PtGPAT gene is shown as a sequence table SEQ ID No. 3.
S12, carrying out PCR amplification by taking the PtGPAT gene synthesized in the step S11 as a template and taking a primer PtGPAT-F (with a nucleotide sequence shown as SEQ ID NO. 11) and a primer PtGPAT-R (with a nucleotide sequence shown as SEQ ID NO. 12) as primers to obtain an amplified fragment PtGPAT;
S14, performing PCR amplification by taking yarrowia lipolytica genome as a template and YAT-F and YAT-R groups as primers to obtain a promoter P YAT ；
S15, performing PCR amplification by taking yarrowia lipolytica genome as a template and CY-F and CY-R groups as primers to obtain a terminator T CYC1t ；
Wherein the enzymes and systems used for PCR amplification are as shown in Table 1, and the procedure for PCR is as follows: denaturation at 98℃for 10s, annealing at 56℃for 10s, and extension at 72℃for 1min (extension time=target fragment length/1 kb, unit min), 35 cycles were repeated.
S2 construction of recombinant plasmid pUC-Intf3-GPAT
S21, carrying out enzyme digestion on a plasmid pUC-Intf3 with uracil genetic markers through BamH1 enzyme, connecting amplified fragments of PtGPAT, a promoter PYAT and a codon TCYC1t into an digested pUC-Intf3 plasmid skeleton, and carrying out one-step cloning by adopting ClonExpress MultiS One Step Cloning Kit to construct a recombinant plasmid pUC-Intf3-GPAT;
s22, purifying and recovering each fragment by AxyPrepTM DNAGel Extraction Kit (purchased from Corning life sciences Co., ltd.);
s23, transforming the circular recombinant plasmid into escherichia coli DH5a competent cells, screening the grown transformant by an ampicillin-resistant flat plate, and verifying by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-Intf3-GPAT. The recombinant plasmid carries uracil genetic markers, and a skeleton diagram of the pUC-Intf3-GPAT plasmid is shown in FIG. 3.
Wherein, the gene sequence of (1) PtGPAT-F is shown as SEQ ID NO.11, specifically:
GAGTATAAGAATCATTCAAAATGGACCTATCGACCGTCCA
(2) the gene sequence of PtGPAT-R is shown as SEQ ID NO.12, and specifically comprises:
GTGACATAACTAATTACATGATCATTTGTTTTTATCTGAATC
wherein the system of one-step cloning is shown in Table 3, and the BamH1 cleavage system of the plasmid pUC-Intf3-GPAT is shown in Table 7
3. Construction of recombinant plasmid pUC-Intf3-LPAAT
S1, amplification of target gene fragment
S11, synthesizing an optimized PtLPAAT gene derived from Phaeodactylum tricornutum (PtLPAAT) to obtain a codon optimized PtLPAAT gene, wherein the nucleotide sequence of the codon optimized PtLPAAT gene is shown as a sequence table SEQ ID No. 4.
S12, taking the PtLPAAT gene synthesized in the step S11 as a template, and carrying out PCR amplification by using a primer PtLPAAT-F (the nucleotide sequence of which is shown as SEQ ID NO. 13) and a primer PtLPAAT-R (the nucleotide sequence of which is shown as SEQ ID NO. 14) to obtain an amplified fragment PtLPAAT;
s14, performing PCR amplification by taking yarrowia lipolytica genome as a template and YAT-F and YAT-R groups as primers to obtain a promoter P YAT ；
S15, performing PCR amplification by taking yarrowia lipolytica genome as a template and CY-F and CY-R groups as primers to obtain a terminator T CYC1t ；
Wherein the enzymes and systems used for PCR amplification are as shown in Table 1, and the procedure for PCR is as follows: denaturation at 98℃for 10s, annealing at 56℃for 10s, and extension at 72℃for 1min (extension time=target fragment length/1 kb, unit min), 35 cycles were repeated.
S2 construction of recombinant plasmid pUC-Intf3-LPAAT
S21, carrying out enzyme digestion on a plasmid pUC-Intf3 with uracil genetic markers through BamH1 enzyme, connecting amplified fragments of PtLPAAT, a promoter PYAT and a codon TCYC1t into an digested pUC-Intf3 plasmid skeleton, and carrying out one-step cloning by adopting ClonExpress MultiS One Step Cloning Kit to construct a recombinant plasmid pUC-Intf3-LPAAT;
s22, purifying and recovering each fragment by AxyPrepTM DNAGel Extraction Kit (purchased from Corning life sciences Co., ltd.);
s23, transforming the circular recombinant plasmid into escherichia coli DH5a competent cells, screening the grown transformant by an ampicillin-resistant flat plate, and verifying by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-Intf3-LPAAT. The recombinant plasmid carries uracil genetic markers, and the skeleton diagram of the pUC-Intf3-LPAAT plasmid is shown in FIG. 3.
Wherein, the gene sequence of (1) PtLPAAT-F is shown as SEQ ID NO.13, specifically:
GAGTATAAGAATCATTCAAAATGCGGCATCTGCGAGGTGT
(2) the gene sequence of PtLPAAT-R is shown as SEQ ID NO.14, and specifically comprises the following steps:
GTGACATAACTAATTACATGATCATGGCACAGTGGTCTCCTC
the system of one-step cloning is shown in Table 3, and the BamH1 cleavage system of the plasmid pUC-Intf3-LPAAT is shown in Table 7.
4. Construction of recombinant plasmid pUC-scp2-GPAT-LPAAT
S1, amplification of target gene fragment
S11, synthesizing optimized PtGPAT gene fragment from Phaeodactylum tricornutum (PtGPAT) and PtLPAAT gene fragment from Phaeodactylum tricornutum (PtLPAAT), to obtain PtGPAT gene fragment and PtLPAAT gene fragment.
S12, carrying out PCR amplification by using the PtGPAT fragment and the PtLPAAT fragment synthesized in the step S11 as templates and using primers P-PtLPAAT-F (with a nucleotide sequence shown as SEQ ID NO. 15), P-PtLPAAT-F (with a nucleotide sequence shown as SEQ ID NO. 16), P-PtGPAT-F (with a nucleotide sequence shown as SEQ ID NO. 17) and P-PtGPAT-R (with a nucleotide sequence shown as SEQ ID NO. 18) as primers to obtain amplified fragments P-PtGPAT and P-PtLPAAT;
s14, performing PCR amplification by taking yarrowia lipolytica genome as a template and YAT-F and YAT-R genes as primers to obtain a promoter P YAT ；
S15, performing PCR amplification by taking yarrowia lipolytica genome as a template and CY-F and CY-R genes as primers to obtain a terminator T CYC1t ；
S16, performing PCR amplification by using yarrowia lipolytica genome as a template and EXP-F (with a nucleotide sequence shown as SEQ ID NO. 19) and EXP-R (with a nucleotide sequence shown as SEQ ID NO. 20) as primers to obtain a promoter P EXP ；
S17, PCR amplification is carried out by taking yarrowia lipolytica genome as a template, and XPR2T-F (nucleotide sequence shown as SEQ ID NO. 21) and XPR2T-R (nucleotide sequence shown as SEQ ID NO. 22) genes as primers to obtain a promoter T XPR2t ；
Wherein the enzymes and systems used for PCR amplification are as shown in Table 1, and the procedure for PCR is as follows: denaturation at 98℃for 10s, annealing at 56℃for 10s, and extension at 72℃for 1min (extension time=target fragment length/1 kb, unit min), 35 cycles were repeated.
S2 construction of recombinant plasmid pUC-scp2-GPAT-LPAAT
S21, enzyme cutting a plasmid pUC-Intf3 with uracil genetic marker by BamH1 enzyme, connecting amplified fragments of P-PtGPAT, P-PtLPAAT, promoter PYAT, codon TCYC1t, promoter PEXP and codon TXPR2t into an enzyme-cut pUC-scp2 plasmid skeleton, and adopting ClonExpress MultiS One Step Cloning Kit to perform one-step cloning to construct a recombinant plasmid
pUC-scp2-GPAT-LPAAT；
S22, purifying and recovering each fragment by AxyPrepTM DNAGel Extraction Kit (purchased from Corning life sciences Co., ltd.);
s23, transforming the circular recombinant plasmid into escherichia coli DH5a competent cells, screening the grown transformant by an ampicillin-resistant flat plate, and verifying by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-scp2-GPAT-LPAAT. The recombinant plasmid has uracil genetic marker, and the skeleton diagram of pUC-scp2-GPAT-LPAAT plasmid is shown in FIG. 3.
Wherein,
(1) the gene sequence of the P-PtLPAAT-F is shown as SEQ ID NO.15, and specifically comprises the following steps:
AGTATAAGAATCATTCAAAATGGACCTATCGACCGTCCAG
The gene sequence of the P-PtLPAAT-R is shown as SEQ ID NO.16, and specifically comprises the following steps:
TGACATAACTAATTACATGATCATTTGTTTTTATCTGAAT
(2) the gene sequence of the P-PtGPAT-F is shown as SEQ ID NO.17, and specifically comprises the following steps:
CACAAGACATATCTACAGCAATGGACCTATCGACCGTCCA
the gene sequence of the P-PtGPAT-R is shown as SEQ ID NO.18, and specifically comprises the following steps:
ACAAGTTCCGTAGTTGGATCTCATTTGTTTTTATCTGAAT
the gene sequence of EXP-F is shown as SEQ ID NO.19, and specifically comprises the following steps:
TGCTGTAGATATGTCTTGTG
the gene sequence of EXP-R is shown as SEQ ID NO.20, and specifically comprises the following steps:
GGAGTTTGGCGCCCGTTTTT
the gene sequence of XPR2t-F is shown in SEQ ID NO.21, and is specifically as follows:
GACACGGGCATCTCACTTGC
the gene sequence of XPR2t-R is shown in SEQ ID NO.22, and is specifically as follows:
GATCCAACTACGGAACTTGT
5. among them, the system of one-step cloning is shown in Table 3, and the BamH1 cleavage system of the plasmid pUC-scp2-GPAT-LPAAT is shown in Table 7.
Example 3 construction of yarrowia lipolytica Polf-D2, polf-D3, polf-D4, polf-D5 engineering strains for the production of alpha-linolenic acid this section is a construction method of a genetically engineered strain for the production of alpha-linolenic acid comprising the following steps, carried out in sequence:
s1, activation of engineering strain Polf-D1
Taking 5 mu L of the engineering strain Polf-D1 to streak on a YPD-5FOA solid culture medium, removing uracil by cells under the action of 5FOA, recycling a screening mark, and streaking and activating again in the YPD solid culture medium after a single colony grows out to obtain the engineering strain Polf-D1 (URA-);
S2 preparation of uracil auxotroph yarrowia lipolytica Po1f competence
Selecting single colony to 5mL YPD liquid culture medium, culturing in a shaking table at 28 ℃ and 220rpm until the absorption value of bacterial liquid at 600nm is 0.8-1.0;
taking 1mL of bacterial liquid, centrifuging at 4 ℃ and 4000rpm for 3min, collecting bacterial cells, and preparing competence according to the operation method of a yeast transformation kit Frozen-EZ Yeast Transformation IITM, wherein the specific method comprises the following steps: re-suspending with 1mLEZ1, centrifuging at 4 ℃ and 4000rpm for 3min, and collecting the thalli again; removing supernatant, suspending thallus with 100 μl EZ2, packaging 50 μl each, and preserving at-80deg.C to obtain engineering strain Polf competent cells;
s3. (1) construction of yarrowia lipolytica engineering Strain Po1f-D2
Taking out yarrowia lipolytica Polf competent cells stored at-80deg.C, and placing on ice for 10min; 1 mug of recombinant plasmid pUC-Intf3-DGAT with uracil genetic marker obtained in example 2 is added into yarrowia lipolytica Po1f competent cells, gently blown and mixed, then 500 mug LFrozen-EZ Solution3 is added, mixed evenly, incubated in a shaker at 28 ℃ and 220rpm for 60min, coated on YNB solid medium for screening, and the grown transformant is verified, namely the engineering strain Po1f-D2, and the engineering strain Po1f-D2 is the genetic engineering strain for producing alpha-linolenic acid.
(2) Construction of yarrowia lipolytica engineering Strain Po1f-D3
Taking out yarrowia lipolytica Polf competent cells stored at-80deg.C, and placing on ice for 10min; 1 mug of recombinant plasmid pUC-Intf3-GPAT with uracil genetic marker obtained in example 2 is added into competent cells of yarrowia lipolytica Po1f, gently blown and mixed uniformly, then 500 mug LEZ3 is added, mixed uniformly, incubated in a shaker at 28 ℃ and 220rpm for 60min, coated on YNB solid medium for screening, and positive strains are selected as engineering strains Po1f-D3, and the engineering strains Po1f-D3 are genetic engineering strains for producing alpha-linolenic acid.
(3) Construction of yarrowia lipolytica engineering Strain Po1f-D4
Taking out yarrowia lipolytica Polf competent cells stored at-80deg.C, and placing on ice for 10min; 1 mug of recombinant plasmid pUC-Intf3-LPAAT with uracil genetic marker obtained in example 2 is added into yarrowia lipolytica Po1f competent cells, gently blown and mixed, then 500 mug LEZ3 is added, mixed evenly, incubated in a shaker at 28 ℃ and 220rpm for 60min, coated on YNB solid medium for screening, and positive strains are selected as engineering strains Po1f-D4, and the engineering strains Po1f-D4 are the genetic engineering strains for producing alpha-linolenic acid.
(4) Construction of yarrowia lipolytica engineering Strain Po1f-D5
Taking out competent cells of yarrowia lipolytica Polf-D2 stored at-80deg.C, and placing on ice for 10min; 1 μg of recombinant plasmid pUC-scp2-GPAT-LPAAT with uracil genetic marker obtained in example 2 is added into yarrowia lipolytica Polf-D2 competent cells, gently beaten and mixed uniformly, then 500 μl EZ3 is added and mixed uniformly, incubated in a shaker at 28 ℃ and 220rpm for 60min, and coated on YNB solid medium for screening, and positive strains are selected as engineering strains Po1f-D5, and the engineering strains Po1f-D5 are the genetic engineering strains for producing alpha-linolenic acid.
Wherein, the proportion of YPD-5FOA fermentation medium is shown in Table 9.
TABLE 9 proportion Table of YPD-5FOA solid Medium
Example 4 Synthesis of alpha-linolenic acid and Total grease by engineering Strain Polf-D1, engineering Strain Polf-D2, engineering Strain Polf-D3, engineering Strain Polf-D4, and engineering Strain Polf-D5 on YNB Nitrogen-Limited Medium
This example examined the effect of engineering strains Polf-D1, polf-D2, polf-D3, polf-D4 and Polf-D5 constructed in example 3 on increasing oil content during fermentation in YNB nitrogen-limited medium, with the objective of examining the effect of engineering strains overexpressing Δ15 desaturase and then three acylases and simultaneously overexpressing both acylases on increasing oil content, comprising the following steps in sequence:
S1, preparation of seed liquid
Inoculating single colonies of engineering strains Polf-D1, polf-D2, polf-D3, polf-D4 and Polf-D5 to 10mL of conventional YPD solid culture medium, and culturing for 24h to obtain first-stage seeds; inoculating the first seed into 10mL YPD culture medium, culturing again for 24 hr to obtain second seed fermentation broth, wherein the viable count of seed broth is 1.2X10% 8 ～2.4×10 8 CFU/mL。
S2, fermenting engineering strains
Respectively taking engineering strain seed fermentation liquid, inoculating the engineering strain seed fermentation liquid into YNB nitrogen-limited fermentation medium according to the inoculum size of 1-5% by volume ratio, ensuring that the number of viable bacteria inoculated into the culture medium is the same, and carrying out ventilation culture for 96 hours at 28 ℃ and 220rpm respectively to obtain a fermentation product.
Wherein, the proportion of YNB nitrogen-limited fermentation medium is shown in Table 10.
TABLE 10 YNB nitrogen limiting fermentation Medium formulation Table
| Name of the name | Yeast powder | YNB | Nitrogen source |
| Content (g/L) | 2.5 | 1.7 | 6 |
S3, analysis of fermentation culture products
Respectively carrying out solid-liquid separation on the obtained fermentation products, collecting solids to obtain grease thalli, adding 0.5% (v/v) of muramidase by volume percentage, carrying out enzymolysis for 1h at 55 ℃, adding 30mL of n-hexane for extraction, standing for layering, taking upper organic phase liquid, repeatedly extracting, volatilizing a solvent, and respectively measuring total grease;
taking 30 mu L of grease obtained in the previous step, and carrying out methyl esterification on fatty acid: 500. Mu.L of 1mol/L NaOH in methanol was added and the mixture was centrifuged at 1200rpm for 30min at room temperature; then 40. Mu.L of sulfuric acid and 500. Mu.L of chromatographic grade n-hexane are added to extract fatty acid methyl ester; and finally centrifuging at 8000rpm for 2min, removing impurities from the upper layer through a microporous filter membrane, injecting into a gas chromatograph, and analyzing and calculating the content of alpha-linolenic acid.
The specific detection method of the gas chromatograph is as follows:
detection conditions: the temperature of the sample inlet is 100 ℃, the sample inlet volume is 1.0 mu L, and the split ratio is 69.8:1;
chromatographic column: DB-23 (60.0mX0.25mm X0.25μm);
chromatographic conditions: the initial temperature was 100deg.C, and 25 deg.C/min was raised to 196 deg.C. Then, heating to 220 ℃ at 2 ℃/min, and preserving heat for 6min;
detector temperature: 280 ℃.
The alpha-linolenic acid and the grease content of the engineering strain Polf-D1 after fermentation are shown in figure 4.
The alpha-linolenic acid and the grease content of the engineering strain Polf-D2 after fermentation are shown in figure 5.
The alpha-linolenic acid and the grease content of the engineering strain Polf-D3 after fermentation are shown in figure 6.
The alpha-linolenic acid and the grease content of the engineering strain Polf-D4 after fermentation are shown in figure 7.
The alpha-linolenic acid and the grease content of the engineering strain Polf-D5 after fermentation are shown in figure 8.
From FIG. 4, from the aspect of total oil content of the fermentation product, the engineering strain Po1f-D1 is obviously improved compared with the engineering strain Po1f, and particularly, the engineering strain Po1f is improved from 0.480g/L to 0.680g/L; from the aspect of the alpha-linolenic acid content of the fermentation product, the engineering strain Po1f-D1 is lifted to 0.3079924g/L;
from FIG. 5, from the aspect of total oil content of the fermentation product, the engineering strain Po1f-D2 is obviously improved compared with the engineering strain Po1f-D1, and particularly from 0.680g/L to 1.598g/L; from the aspect of the alpha-linolenic acid content of the fermentation product, the engineering strain Po1f-D2 is lifted to 0.78789290g/L;
From FIG. 6, from the aspect of total oil content of the fermentation product, the engineering strain Po1f-D3 is obviously improved compared with the engineering strain Po1f-D1, and specifically, the total oil content is improved from 0.680g/L to 1.103g/L; from the aspect of the alpha-linolenic acid content of the fermentation product, the engineering strain Po1f-D3 is lifted to 0.51102599g/L;
from FIG. 7, from the aspect of total oil content of the fermentation product, the engineering strain Po1f-D4 is obviously improved compared with the engineering strain Po1f-D1, and specifically, the total oil content is improved from 0.680g/L to 1.368g/L; from the aspect of the alpha-linolenic acid content of the fermentation product, the engineering strain Po1f-D4 is lifted to 0.64696824g/L;
from the alpha-linolenic acid content of the fermentation product, the engineering strains Po1f-D2 are obviously higher than the engineering strains Po1f-D3 and Po1f-D4, and the alpha-linolenic acid content in the fermentation liquid of the engineering strains Po1f-D2 is 0.78789290g/L which is about 2.56 times of the alpha-linolenic acid content 0.307992g/L in the fermentation liquid of the engineering strains Po1 f-D1. Thus, the engineering strain Po1f-D2 is more suitable than the engineering strains Po1f-D3 and Po1f-D4 as a strain for producing alpha-linolenic acid.
From the total oil content of the fermentation product, the engineering strains Po1f-D2 are obviously higher than the engineering strains Po1f-D3 and Po1f-D4, and the total oil content 1.598g/L in the fermentation liquid of the engineering strains Po1f-D2 is about 2.35 times of the total oil content 0.680g/L in the fermentation liquid of the engineering strains Po1 f-D1. Thus, the engineering strain Po1f-D2 is more suitable than the engineering strains Po1f-D3 and Po1f-D4 as a strain for producing an increased total lipid content.
Thus, the engineering strain Polf-D5 was constructed by simultaneously overexpressing glycerol-3-phosphate acyl-transferase (GPAT) derived from Phaeodactylum tricornutum and lysophosphatidic acid acyl-transferase (lysophosphatidate acyl-transferase, LPAAT) derived from Phaeodactylum tricornutum on the basis of the engineering strain Po1 f-D2.
From FIG. 8, from the aspect of total oil content of the fermentation product, the engineering strain Po1f-D5 is obviously improved compared with the engineering strain Po1f-D2, and specifically, the total oil content is improved from 1.368g/L to 2.3893g/L; from the point of the alpha-linolenic acid content of the fermentation product, the engineering strain Po1f-D4 is improved to 1.24948g/L.
The engineering strain Po1f-D5 finally obtained by optimization is calculated, the oil content in 1L fermentation liquor reaches 2.3893g, and the alpha-linolenic acid content reaches 1.24948g.
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Claims (10)
1. An engineered strain of yarrowia lipolytica for high-level production of alpha-linolenic acid, characterized in that: the strain is obtained by taking yarrowia lipolytica (Yarrowia lipolytica Po f) as an initial strain, expressing delta 15 desaturase genes and then cooperatively and over-expressing acyltransferase.
2. The engineered strain of yarrowia lipolytica for high-level production of alpha-linolenic acid according to claim 1, wherein:
the delta 15 desaturase gene is derived from orange Lin Youzhi yeast Lipomyces kononenkoaeLKFAD, and the sequence of the delta 15 desaturase gene is shown as SEQ ID No. 1;
the acyltransferase is glycerol-3-phosphate linear transferase GPAT and/or lysophosphatidic acid acyltransferase LPAAT and/or diacylglycerol acyltransferase DGAT;
the diacylglycerol acyltransferase is derived from schizochytrium (Sp) ScDGAT, and the ScDGAT gene sequence is shown as SEQ ID NO. 2;
the glycerol-3-phosphate linear transferase is derived from Phaeodactylum tricornutum (Phaeodactylum tricornutum) PtGPAT, and the PtGPAT gene sequence is shown in SEQ ID NO. 3;
the lysophosphatidic acid acyltransferase is derived from PtLPAAT of Phaeodactylum tricornutum (Phaeodactylum tricornutum), and the PtLPAAT gene sequence is shown in SEQ ID NO. 4.
3. The method for constructing a yarrowia lipolytica engineering strain for high-level production of alpha-linolenic acid according to claim 1 or 2, comprising the steps of: the method comprises the following steps:
(1) Construction of recombinant plasmid PUC-Intc-Delta15 DEs
Taking pUC-Intc plasmid as a framework, and inserting an LKFAD15 gene expression cassette into the plasmid framework to obtain a recombinant plasmid PUC-Intc-delta 15DEs;
(2) Construction of genetically engineered strain Polf-D1 for producing alpha-linolenic acid
Transferring the recombinant plasmid pUC-Intc-delta 15DEs into uracil auxotroph yarrowia lipolytica Polf to obtain a yarrowia lipolytica engineering strain Polf-D1;
(3) Preparation of yarrowia lipolytica engineering strain Polf-D1 (URA-)
Inoculating the yarrowia lipolytica engineering strain Polf-D1 into a YPD-5FOA solid culture medium, and after single colony grows out, carrying out streak activation again in the YPD solid culture medium to obtain a yarrowia lipolytica engineering strain Polf-D1 (URA-);
(4) Construction of recombinant plasmid pUC-Intf3-DGAT
Taking pUC-Intf3 plasmid as a framework, and inserting a ScDGAT gene expression cassette into the plasmid framework to obtain a recombinant plasmid pUC-Intf3-DGAT;
(5) Preparation of yarrowia lipolytica engineering strain Polf-D2
Transferring the recombinant plasmid pUC-Intf3-DGAT into a yarrowia lipolytica engineering strain Polf-D1 (URA-) to obtain a yarrowia lipolytica engineering strain Polf-D2;
(6) Construction of recombinant plasmid pUC-Intf3-GPAT
Taking pUC-Intf3 plasmid as a framework, and inserting a PtGPAT gene expression cassette into the plasmid framework to obtain a recombinant plasmid pUC-Intf3-GPAT;
(7) Preparation of yarrowia lipolytica engineering strain Polf-D3
Transferring the recombinant plasmid pUC-Intf3-GPAT into a yarrowia lipolytica engineering strain Polf-D1 (URA-) to obtain a yarrowia lipolytica engineering strain Polf-D3;
(8) Construction of recombinant plasmid pUC-Intf3-LPAAT
Taking pUC-Intf3 plasmid as a framework, and inserting PtLPAAT gene expression cassettes into the plasmid framework to obtain recombinant plasmid pUC-Intf3-LPAAT;
(9) Preparation of yarrowia lipolytica engineering strain Polf-D4
Transferring the recombinant plasmid pUC-Intf3-LPAAT into a yarrowia lipolytica engineering strain Polf-D1 (URA-) to obtain a yarrowia lipolytica engineering strain Polf-D4;
(10) Construction of recombinant plasmid pUC-scp2-GPAT-LPAAT
Taking pUC-scp2 plasmid as a framework, and inserting a PtGPAT gene expression cassette and a ScDGAT gene expression cassette into the plasmid framework to obtain a recombinant plasmid pUC-scp2-GPAT-LPAAT;
(11) Preparation of yarrowia lipolytica engineering strain Polf-D5
Transferring the recombinant plasmid pUC-scp2-GPAT-LPAAT into yarrowia lipolytica engineering strain Polf-D2 (URA-) to obtain yarrowia lipolytica engineering strain Polf-D5.
4. A method of construction according to claim 3, wherein: the construction method of uracil auxotroph yarrowia lipolytica Polf in the step (2) comprises the following steps: knocking out uracil-encoding genes in yarrowia lipolytica Po1f by CRISPR/Cas9 technology;
specific construction methods are described in journal Journal of Agricultural and Food Chemistry, 2021, volume 69, 46, pages 13831-13837, under the paper designation "Harnessing Yarrowia lipolytica Peroxisomes as a Subcellular Factory for. Alpha. -Humulene Overproduction".
5. A method of construction according to claim 3, wherein: the construction method of the recombinant plasmid PUC-Intc-delta 15DEs in the step (1) comprises the following steps: the PUC-Intc plasmid with uracil genetic marker is used as a skeleton, and a delta 15 desaturase gene expression cassette is inserted into the plasmid skeleton.
6. A method of construction according to claim 3, wherein: in the step (1), the LKFAD15 gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoters or P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (4), the ScDGAT gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (6), the PtGPAT gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (8), the PtLPAAT gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in step (10), the PtGPAT gene expression cassette and the ScDGAT gene expression cassette promoterIs P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators.
7. Use of an engineered strain of yarrowia lipolytica according to claim 1 or 2 for the production of alpha-linolenic acid.
8. A method for the fermentative production of α -linolenic acid using the engineered strain of yarrowia lipolytica according to claim 1 or 2, characterized in that: the yarrowia lipolytica engineering strain is used for producing alpha-linolenic acid by fermenting on YNB nitrogen-limited culture medium;
the formula of the YNB nitrogen limiting medium is as follows: yeast powder 2.5g/L, YNB medium 1.7g/L, nitrogen source 6g/L,115 degrees centigrade sterilization 30 minutes.
9. The method according to claim 8, wherein: the method comprises the following steps:
(1) Preparation of seed liquid
Activating yarrowia lipolytica engineering strains, inoculating the yarrowia lipolytica engineering strains into YPD liquid culture medium, and culturing the yarrowia lipolytica engineering strains to logarithmic growth phase to obtain seed liquid;
(2) Fermentation production of alpha-linolenic acid
Inoculating the seed solution into YNB nitrogen-limiting culture medium according to the volume ratio of 1% -5%, fermenting at 28-30 ℃ and 200-240 rpm, and carrying out ventilation culture for 96-120 h to obtain a fermentation product alpha-linolenic acid;
wherein, the formula of YNB nitrogen limiting medium is: 2.5g/L yeast powder, 1.0-2.5 g/L YNB culture medium, 5-7 g/L nitrogen source, and sterilizing at 115 ℃ for 30 minutes.
10. The method according to claim 8, wherein: in the step (1), the number of viable bacteria in the seed liquid is 1.2X10 8 ～2.4×10 8 CFU/mL；
In the step (2), the nitrogen source addition amount in the fermentation culture is 6g/L, and the YNB addition amount is 1.7g/L.
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