CN112538438B

CN112538438B - Recombinant yarrowia lipolytica with high oleic acid yield as well as construction method and application thereof

Info

Publication number: CN112538438B
Application number: CN202011464493.6A
Authority: CN
Inventors: 王金鹏; 纪晓俊; 王凯峰; 陆然; 支咏妍; 朱芮莹; 胡佳颖
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2023-04-04
Anticipated expiration: 2040-12-11
Also published as: CN112538438A

Abstract

The invention provides a recombinant yarrowia lipolytica strain capable of producing oleic acid at high yield, a construction method and application thereof, and belongs to the field of bioengineering. The recombinant yarrowia lipolytica with high oleic acid yield is yarrowia lipolytica (Yarrowia lipolytica) The strain XJ-9 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2020707. Yarrowia lipolytica of the invention: (Yarrowia lipolytica) The XJ-9 strain knocks out the oleate desaturase, over-expresses endogenous acetyl coenzyme A carboxylase, diacylglycerol acyltransferase and fatty acid elongase 1, and over-expresses heterologous fatty acid elongase 2 and stearoyl-CoA desaturase, and experiments prove that the recombinant yarrowia lipolytica can efficiently ferment and produce the oleic acid, thereby realizing the efficient synthesis of the plant-derived natural product oleic acid in the yarrowia lipolytica.

Description

Recombinant yarrowia lipolytica with high oleic acid yield as well as construction method and application thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to recombinant yarrowia lipolytica with high oleic acid yield, and a construction method and application thereof.

Background

Oleic acid (Oleic acid, C) ₁₈ H ₃₄ O ₂ ) The learning name is as follows: cis-9-octadecenoic acid, an Omega-9 type monounsaturated fatty acid, is mainly found in oilseed rape (Brassica napus), camellia (Camellia japonica), etc. In the medical field, oleic acid has the function of regulating physiological functions, can selectively regulate high-density and low-density cholesterol in human blood and reduce the risk of cardiovascular diseases. In addition, oleic acid has good adjuvant therapeutic effect on cancer, autoimmune diseases and inflammatory diseases. In the energy field, oleic acid is an excellent biodiesel component due to its low temperature fluidity and oxidation stability. In the field of materials, oleic acid can be converted into fatty acid derivatives such as dihydric alcohol, dibasic acid, diamine, omega-amino fatty acid, omega-hydroxy fatty acid and the like with different chain lengths by multi-enzyme concerted catalysis, and the derivatives can be used as monomers to synthesize series polymers such as polyester, polyamide and the like and are used for producing products such as plastics, lubricating oil, surfactants, plasticizers, spices, coatings, fuels and the like.

The traditional source of oleic acid is extracted from oil crops such as Brassica napus and Camellia japonica. However, the plant extraction method requires a large area of cultivation and has a long growth cycle, and is also susceptible to the production area, climate, and the like. Compared with a plant extraction method, the microbial fermentation method has a short growth period and can produce the oleic acid in all weather, and the oleaginous microorganisms have high oil content and large biomass, so that the production of the oleic acid by the oleaginous microorganisms is an economic and efficient mode. In the prior art, microorganisms capable of efficiently producing oleic acid are lacking.

Disclosure of Invention

The invention aims to provide a recombinant yarrowia lipolytica capable of efficiently synthesizing oleic acid.

The invention also aims to provide a construction method of the recombinant yarrowia lipolytica, which is efficient and simple to operate.

Still another object of the present invention is to provide the use of said recombinant yarrowia lipolytica for the production of oleic acid.

The purpose of the invention is realized by adopting the following technical scheme:

a recombinant Yarrowia lipolytica strain for high yield of oleic acid is Yarrowia lipolytica (Yarrowia lipolytica) XJ-9 strain which is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2020707.

In the present invention, the Yarrowia lipolytica (Yarrowia lipolytica) XJ-9 strain is obtained by knocking out the oleate desaturase in the Yarrowia lipolytica genome and inserting an acetyl-CoA carboxylase expression cassette, a diacylglycerol acyltransferase expression cassette, a fatty acid elongase 1 expression cassette, a fatty acid elongase 2 expression cassette and a stearoyl-CoA desaturase expression cassette.

In the present invention, the acetyl-CoA carboxylase, diacylglycerol acyltransferase, fatty acid elongase 1-encoding gene is derived from Yarrowia lipolytica; the fatty acid elongase 2 coding gene is obtained by optimizing a fatty acid elongase 2 coding gene derived from Mortierella alpina (Mortierella alpina) by a codon; the coding gene of stearoyl-CoA desaturase is obtained by codon optimization of a coding gene of stearoyl-CoA desaturase from Puccinia graminis.

In the present invention, the promoter of each expression cassette is the promoter P of yarrowia lipolytica _TEF 、P _hp4d 、P _TEFin 、P _YAT1 、P _FBA 、P _FBAin 、P _POX2 、P _POT1 Or P _GPD Any one of the above; the terminator is terminator T of yarrowia lipolytica _xpr2t 、T _mig1t 、T _lip2t 、T _cyc1t 、T _pex3t 、T _pex10t Or T _pex20t Any one of them.

In the present invention, the integration site of the expression cassette is any one of the A08 site, 26s rDNA site, intA site, intB site, intC site, intD site, intE site, intF site, lip1 site, SCP2 site, or YLSCD site of yarrowia lipolytica.

In the invention, the sequence of the coding gene of the stearoyl-CoA desaturase is shown as SEQ ID No. 1; the sequence of the fatty acid elongase 2 coding gene is shown in SEQ ID No. 2.

The invention also provides a construction method of the recombinant yarrowia lipolytica for high oleic acid yield, which comprises the steps of introducing an oleic acid desaturase knockout box, an acetyl coenzyme A carboxylase expression box, a diacylglycerol acyltransferase expression box, a fatty acid elongase 1 expression box, a fatty acid elongase 2 expression box and a stearoyl coenzyme A desaturase expression box into the yarrowia lipolytica in a plasmid form, and integrating the yarrowia lipolytica on the genome through homologous recombination.

In the present invention, the yarrowia lipolytica knocks out the ku70 gene.

The invention also provides application of the recombinant bacterium in producing oleic acid, which comprises the step of culturing the recombinant bacterium by adopting a fermentation culture medium to obtain a fermentation product.

In the invention, the fermentation medium contains 50-70g/L of glucose, 1.6-1.8g/L of nitrogen source of aminoyeast and 1.2-1.4g/L of ammonium sulfate.

Has the advantages that: the Yarrowia lipolytica (Yarrowia lipolytica) XJ-9 strain of the invention is based on the Yarrowia lipolytica which is in charge of coding the non-homologous recombination gene ku70, so that the homologous recombination capability of the Yarrowia lipolytica is enhanced, the integration of the gene is realized through the homologous recombination function of the Yarrowia lipolytica, and the genetic stability of the introduced gene can be greatly improved. The construction method of the recombinant yarrowia lipolytica is efficient and simple to operate. The Yarrowia lipolytica (Yarrowia lipolytica) XJ-9 strain of the invention knocks out the oleate desaturase, overexpresses endogenous acetyl-CoA carboxylase, diacylglycerol acyltransferase and fatty acid elongase 1, and overexpresses heterologous fatty acid elongase 2 and stearoyl-CoA desaturase, and experiments prove that the recombinant Yarrowia lipolytica can efficiently ferment and produce oleic acid, and realizes the efficient synthesis of plant-derived natural product oleic acid in Yarrowia lipolytica.

Drawings

FIG. 1 is a diagram showing the structure of recombinant plasmid pUC-HUH-IntC-ACC1-DGA1, wherein IntC-up represents the upstream homology arm of IntC site, intC-dm represents the downstream homology arm of IntC site, hp4d represents promoter P _hp4d Mig1T represents a terminator T _mig1t TEFin denotes the promoter P _TEFin And lip2T represents a terminator T _lip2t URA denotes an orotidine-5' -phosphate decarboxylase-encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P) _TEFin A terminator T _xpr2t ) ACC1 is an acetyl-CoA carboxylase-encoding gene, and DGA1 is a diacylglycerol acyltransferase-encoding gene.

FIG. 2 is a diagram showing the structure of recombinant plasmid pUC-HUH-Fad2, in which Fad2-up represents the upstream homology arm of Fad2 site, fad2-dm represents the downstream homology arm of Fad2 site, URA represents the orotidine-5' -phosphate decarboxylase-encoding gene expression cassette (containing promoter P endogenous to Yarrowia lipolytica) _TEFin A terminator T _xpr2t )。

FIG. 3 is a diagram showing the structure of the recombinant plasmid pUC-HUH-YLSCD-PgSCD, wherein YLSCD-up represents the upstream homology arm of YLSCD site, YLSCD-dm represents the downstream homology arm of YLSCD site, cyc1T represents terminator T _cyc1t TEFin denotes the promoter P _TEFin URA denotes the orotidine-5' -phosphate decarboxylase encoding gene expression cassette (containing the endogenous promoter P of Yarrowia lipolytica) _TEFin A terminator T _xpr2t ) PgSCD is a stearoyl-CoA desaturase encoding gene.

FIG. 4 is a diagram showing the structure of recombinant plasmid pUC-HUH-lip1-PgSCD, wherein lip1-up represents the upstream homology arm of lip1 site, lip1-dm represents the downstream homology arm of lip1 site, and cyc1T represents terminator T _cyc1t YAT1 denotes the promoter P _YAT1 URA denotes the orotidine-5' -phosphate decarboxylase encoding gene expression cassette (containing the endogenous promoter P of Yarrowia lipolytica) _TEFin A terminator T _xpr2t ) PgSCD is a stearoyl-CoA desaturase encoding gene.

FIG. 5 is a diagram showing the structure of recombinant plasmid pUC-HUH-SCP2-MaELO2-YLELO1, in which SCP2-up represents the homology arm upstream of the SCP2 site, SCP2-dm represents the homology arm downstream of the SCP2 site, and GPD represents promoter P _GPD And pex20T represents a terminator T _pex20t FBA denotes the promoter P _FBA And pex10T represents a terminator T _pex10t URA denotes the orotidine-5' -phosphate decarboxylase encoding gene expression cassette (containing the endogenous promoter P of Yarrowia lipolytica) _TEFin A terminator T _xpr2t ) MaELO2 is a fatty acid elongase 2 coding gene, and YLELO1 is a fatty acid elongase 1 coding gene.

FIG. 6 shows a GC map of the production of oleic acid by Yarrowia lipolytica XJ-9 strain (recombinant strain 5).

Detailed Description

The present invention will be further illustrated by the following specific examples.

The experimental procedures used in the following examples are all conventional ones unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Yarrowia lipolytica (Yarrowia lipolytica) Po1f, available from the American type culture Collection, accession number ATCC MYA-2613.

Yarrowia lipolytica (Yarrowia lipolytica) Po1f Δ ku70 (MatA, Δ ku70:: hisG, leu2-270, ura3-302, xpr2-322, axp1-2), abbreviated to Yarrowia lipolytica Po1f Δ ku70.Yarrowia lipolytica Po1 f. DELTA.ku 70 was constructed from Yarrowia lipolytica Po1f by knocking out the coding gene ku70 responsible for non-homologous recombination (disclosed in Kretzschmar A, et al, current Genetics,2013,59 (1-2): 63-72).

The IntC site integration plasmid was obtained by inserting a sequence having an upstream region of 1402bp (upstream homology arm) from the initiation codon of the IntC site and a sequence having a downstream region of 1396bp (downstream homology arm) from the termination codon into pUC57-hisG-ura-hisG vector (the construction method is described in example 1) on chromosome C of Yarrowia lipolytica Po1 f.DELTA.ku 70 genome, and two hisG tag-encoding genes were present between the upstream and downstream homology arms of the IntC site.

The Fad2 site integration plasmid was obtained by inserting a sequence (upstream homology arm) with 1539bp upstream of the initiation codon of Fad2 site and a sequence (downstream homology arm) with 1503bp downstream of the termination codon into pUC57-hisG-ura-hisG vector (the construction method is described in example 1) on chromosome B of Yarrowia lipolytica Po1 f.DELTA.ku 70 genome, and two hisG tag-encoding genes were present between the upstream and downstream homology arms of Fad2 site.

The YLSCD site integration plasmid is obtained by inserting a sequence (upstream homology arm) with the upstream size of 1529bp of the start codon of the YLSCD site and a sequence (downstream homology arm) with the downstream size of 1496bp of the stop codon on chromosome C of the Yarrowia lipolytica Po1f delta ku70 genome into a pUC57-hisG-ura-hisG vector (the construction method is shown in example 1), and two hisG tag coding genes are arranged between the upstream and downstream homology arms of the YLSCD site.

The lip1 site integration plasmid is obtained by inserting a sequence with 1458bp upstream of the initiation codon (upstream homology arm) and a sequence with 1438bp downstream of the termination codon (downstream homology arm) of the lip1 site on chromosome E in Yarrowia lipolytica Po1f delta ku70 genome into pUC57-hisG-ura-hisG vector (the construction method is shown in example 1), and two hisG tag coding genes are arranged on the lip1 site and between the downstream homology arms.

The SCP2 site integration plasmid is obtained by inserting a sequence (upstream homology arm) with the upstream size of 1523bp of the starting codon of the SCP2 site and a sequence (downstream homology arm) with the downstream size of 1524bp of the stopping codon of chromosome E in a Yarrowia lipolytica Po1f delta ku70 genome into a pUC57-hisG-ura-hisG vector (the construction method is shown in example 1), and two hisG tag coding genes are positioned on the SCP2 site and between the downstream homology arms.

Example 1 amplification of Gene elements and preparation of target plasmids

(first) preparation of target Gene

According to the nucleotide sequence of a stearoyl-CoA desaturase coding gene (GenBank accession No.: NW _ 003526568.1) from Puccinia graminis (Puccinia graminis) provided at NCBI, after codon optimization, suzhou Jinzhi Biotech limited was committed to synthesize the optimized stearoyl-CoA desaturase coding gene PgSCD (SEQ ID No.: 1), and inserted into plasmid pUC57, to obtain plasmid pUC57-PgSCD.

According to the nucleotide sequence of the fatty acid elongase 2-encoding gene (GenBank accession No.: AB 468587.1) from Mortierella alpina (Mortierella alpina) provided at NCBI, after codon optimization, suzhou Jinwei Zhi Biotechnology Co., ltd.was entrusted to synthesize the optimized fatty acid elongase 2-encoding gene MaELO2 (SEQ ID No.: 2), and inserted into plasmid pUC57, to obtain plasmid pUC57-MaELO2.

Based on the nucleotide sequence of the orotidine-5 '-phosphate decarboxylase encoding gene ura from Yarrowia lipolytica (GenBank accession No.: AJ 306421.1) and the hisG tag (GenBank accession No.: AF 324729.1) provided on NCBI, the synthesis of Suzhou Jinzhi Biotechnology, inc. was entrusted, two hisG tag encoding gene sequences were inserted into plasmid pUC57, and an orotidine-5' -phosphate decarboxylase encoding gene expression cassette (derived from the endogenous promoter P polytica from Yarrowia lipolytica) was inserted between the two hisG tag encoding gene sequences _TEFin Orotidine-5' -phosphate decarboxylase-encoding gene ura and terminator T _xpr2t Composition) to achieve ura tag recovery, resulting in plasmid pUC57-hisG-ura-hisG.

acetyl-CoA carboxylase encoding gene ACC1 (GenBank accession number: YALI0C11407 g) was amplified using Yarrowia lipolytica Po 1F. DELTA. Ku70 genomic DNA as a template and IntC: (ACC 1-F) and IntC: (ACC 1-R) as primers.

Diacylglycerol acyltransferase encoding gene DGA1 (GenBank accession: YALI0E32769 g) was amplified using Yarrowia lipolytica Po 1F. DELTA. Ku70 genomic DNA as a template and IntC:: DGA1-F and IntC:: DGA1-R as primers.

A fatty acid elongase 1-encoding gene YLELO1 (GenBank accession No.: YALI0F06754 g) was amplified using Yarrowia lipolytica Po 1F. DELTA. Ku70 genomic DNA as a template and SCP2:: YLELO1-F and SCP2:: YLELO1-R as primers.

Endogenous promoter P of Yarrowia lipolytica _hp4d The nucleotide sequence of (A) is shown as SEQ ID No. 3; endogenous promoter P of Yarrowia lipolytica _TEFin The nucleotide sequence of (A) is shown as SEQ ID No. 4; endogenous promoter P of Yarrowia lipolytica _YAT1 The nucleotide sequence of (A) is shown as SEQ ID No. 5; endogenous promoter P of Yarrowia lipolytica _FBA The nucleotide sequence of (A) is shown as SEQ ID No. 6; endogenous promoter P of Yarrowia lipolytica _GPD The nucleotide sequence of (A) is shown as SEQ ID No. 7; endogenous terminator T of Yarrowia lipolytica _mig1t The nucleotide sequence of (A) is shown as SEQ ID No. 8; endogenous terminator T of Yarrowia lipolytica _lip2t The nucleotide sequence of (A) is shown as SEQ ID No. 9; endogenous terminator T of Yarrowia lipolytica _cyc1t Nucleotide sequences of e.g.SEQ ID No. 10; endogenous terminator T of Yarrowia lipolytica _pex10t The nucleotide sequence of (A) is shown as SEQ ID No. 11; endogenous terminator T of Yarrowia lipolytica _pex20t The nucleotide sequence is shown as SEQ ID No. 12; endogenous terminator T of Yarrowia lipolytica _xpr2t The nucleotide sequence is shown as SEQ ID No. 13.

(II) construction of recombinant plasmid

The structure of the recombinant plasmid is shown in Table 1 and FIGS. 1-5; the primers used to construct the recombinant plasmids are shown in Table 2.

1. Construction of recombinant plasmid pUC-HUH-IntC-ACC1-DGA1

The recombinant plasmid pUC-HUH-IntC-ACC1-DGA1 takes pUC57-hisG-ura-hisG as a framework, an intC-up homology arm IntC-up of an IntC site initiation codon and an intC-dm of a homology arm downstream of a termination codon in Yarrowia lipolytica Po1f delta ku70 are inserted, and an ACC1 gene expression cassette (P1 gene expression cassette) is inserted between the upstream homology arm and the downstream homology arm _hp4d -ACC1-T _mig1t ) And DGA1 gene expression cassette (P) _TEFin -DGA1-T _lip2t ) Orotidine-5' -phosphate decarboxylase encoding gene expression cassettes (containing the Yarrowia lipolytica endogenous promoter P) _TEFin A terminator T _xpr2t ) Also between the upstream and downstream homology arms, the specific structure is shown in FIG. 1.

Using IntC as the reference P _hp4d F and IntC:: P _hp4d -R is a primer, and the promoter P of the ACC1 expression cassette is amplified by using Yarrowia lipolytica Po1f delta ku70 genomic DNA as a template _hp4d . Using IntC as the base _mig1t -F and IntC:: T _mig1t -R is a primer, and uses Yarrowia lipolytica Po1f delta ku70 genome DNA as a template to amplify ACC1 expression cassette terminator T _mig1t 。

Uses Yarrowia lipolytica Po1F delta ku70 genome DNA as a template, uses IntC:: ACC1-F and IntC:: ACC1-R as primers, and amplifies the two ends of the primer with promoter P _hp4d And a terminator T _mig1t ACC1 gene of homologous arm.

From IntC:: P _TEFin F and IntC:: P _TEFin -R is a primer, and uses Yarrowia lipolytica Po1f delta ku70 genome DNA as a template to amplify DGA1 expression cassette promoter P _TEFin . Using IntC as the base _lip2t -F and IntC:: T _lip2t -R is a primer, and uses Yarrowia lipolytica Po1f delta ku70 genome DNA as a template to amplify DGA1 expression cassette terminator T _lip2t 。

The genomic DNA of Yarrowia lipolytica Po1F delta ku70 is taken as a template, intC:: DGA1-F and IntC:: DGA1-R are taken as primers, and the two ends of amplification are respectively provided with a promoter P _TEFin And a terminator T _lip2t DGA1 gene of homologous arm.

The PCR amplification system is as follows:

components	Volume of
		PrimerSTAR Max Premix	25ul
Form panel	1ul
		Primer
1	2ul
		Primer
2	2ul
		Distilled water	20ul

Of these, primerSTAR Max Premix was purchased from Baozi physician technology (Beijing) Inc.

The procedure for the above PCR was as follows: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ (extension time = target fragment length/1 kb in min), and 30 cycles were repeated.

Each Fragment was purified and recovered by TaKaRa MiniBEST DNA Fragment Purification Kit (purchased from Shanghai Baisai Biotechnology Ltd.).

The IntC site integration plasmid was digested with the restriction enzyme PacI from NEB, and the linearized IntC site integration plasmid was recovered by agarose gel electrophoresis.

The linearized IntC site integration plasmid and each element (promoter P) in the ACC1 gene expression cassette constructed in title 1 of this example _hp4d ACC1 gene and terminator T _mig1t ) One-Step Cloning is realized by using the Clon express Multi S One Step Cloning Kit of Nanjing Novowed Biotechnology Co., ltd, the ACC1 gene expression cassette is inserted between the upstream and downstream homology arms of the IntC locus integration plasmid, and the two hisG tag coding genes are positioned at the same side of the ACC1 gene expression cassette. The reaction system is shown in the following table. After incubating the reaction system at 37 ℃ for 30min, the circular recombinant vector is obtained.

The system for one-step cloning is as follows:

components	Volume of
		5×CE MultiS Buffer	4ul
Exnase MultiS	2ul
		Linearized vector	xng
Insert fragment	yng
		Distilled water	Make up the volume to 10ul

Wherein, the usage amount of the linearized vector (x) and the insert (y) can be calculated by the following formula: the optimum amount used per fragment or linearized vector = [0.02 × number of bases of fragment or linearized vector ] ng.

And transforming the circular recombinant vector into escherichia coli DH5 alpha competent cells, and obtaining a positive recombinant plasmid pUC-HUH-IntC-ACC1 by screening an ampicillin resistant plate and verifying colony PCR and sequencing.

After the plasmid pUC-HUH-IntC-ACC1 was digested with the restriction enzyme Kpn I of NEB, the linearized pUC-HUH-IntC-ACC1 plasmid was recovered by agarose gel electrophoresis.

The linearized pUC-HUH-IntC-ACC1 plasmid and each element (promoter P) in the DGA1 gene expression cassette constructed under the title 1 of this example _TEFin Gene DGA1 and terminator T _lip2t ) One-Step Cloning is realized by using Clonexpress Multi S One Step Cloning Kit of Nanjing Novozam Biotechnology Co., ltd, and a recombinant plasmid pUC-HUH-IntC-ACC1-DGA1 is obtained.

2. Construction of recombinant plasmid pUC-HUH-Fad2

The recombinant plasmid pUC-HUH-Fad2 takes pUC57-hisG-ura-hisG as a framework, a homologous arm (Fad 2-up) with the upstream of the initiation codon of the Fad2 site of 1539bp and a homologous arm (Fad 2-dm) with the downstream of the termination codon of 1503bp in Yarrowia lipolytica Po1f delta ku70 are inserted, and an orotidine-5' -phosphate decarboxylase encoding gene expression cassette (containing a promoter P endogenous to Yarrowia lipolytica) _TEFin A terminator T _xpr2t ) Also between the upstream and downstream homology arms, the specific structure is shown in FIG. 2.

Using Fad2-up-F and Fad2-up-R as primers and Yarrowia lipolytica Po1F delta ku70 genome DNA as a template to amplify Fad2-up of the upstream homology arm of the initiation codon of the Fad2 site.

After digesting pUC57-hisG-ura-hisG with EcoRI, a restriction enzyme of NEB, the linearized pUC57-hisG-ura-hisG plasmid was recovered by agarose gel electrophoresis.

The linearized pUC57-hisG-ura-hisG plasmid and Fad2-up (with pUC57-hisG-ura-hisG homology sequences at both ends) upstream homology arm of the start codon of Fad2 site constructed in title 2 of this example were used

II One Step Cloning was carried out by One Step Cloning Kit to obtain circular recombinant vector.

And transforming the circular recombinant vector into escherichia coli DH5 alpha competent cells, and obtaining a positive recombinant plasmid pUC-HUH-Fad2-up by screening an ampicillin resistant plate and verifying colony PCR and sequencing.

Fad2-dm-F and Fad2-dm-R are used as primers, and Yarrowia lipolytica Po1F delta ku70 genome DNA is used as a template to amplify Fad2-dm of a homologous arm at the downstream of a Fad2 site stop codon.

After the plasmid pUC-HUH-Fad2-up was digested with the restriction enzyme Hind III from NEB, the linearized pUC-HUH-Fad2-up plasmid was recovered by agarose gel electrophoresis.

The linearized pUC-HUH-Fad2-up plasmid and the Fad2 site termination codon downstream of the homology arm Fad2-dm (with pUC-HUH-Fad2-up homology arm sequence at both ends) constructed in the title 2 of this example were used from Nanjing Novozam Biotech Ltd

II One Step Cloning Kit to obtain recombinant plasmid pUC-HUH-Fad2.

3. Construction of recombinant plasmid pUC-HUH-YLSCD-PgSCD

The recombinant plasmid pUC-HUH-YLSCD-PgSCD takes pUC57-hisG-ura-hisG as a framework, an upstream homology arm YLSCD-up of the initiation codon of YLSCD site and a downstream homology arm YLSCD-dm of a termination codon in Yarrowia lipolytica Po1f delta ku70 are inserted, and a PgSCD gene expression cassette (P gSCD gene expression cassette) is also inserted between the upstream and downstream homology arms _TEFin -PgSCD-T _cyc1t ) Orotidine-5' -phosphate decarboxylase encoding gene expression cassette (Package)Containing Yarrowia lipolytica endogenous promoter P _TEFin A terminator T _xpr2t ) Also between the upstream and downstream homology arms, the specific structure is shown in FIG. 3.

P is represented by YLSCD _TEFin F and YLSCD P _TEFin -R is a primer, and the genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify the PgSCD expression cassette promoter P _TEFin . T is defined by YLSCD _cyc1t T and YLSCD _cyc1t -R is a primer, and the genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify the terminator T of the PgSCD expression cassette _cyc1t 。

Plasmid pUC57-PgSCD is used as template, ylSCD, pgSCD-F and PgSCD-R are used as primers, and promoter P is respectively arranged at two ends of amplification _TEFin And a terminator T _cyc1t PgSCD gene of homologous arm.

The integrated plasmid of YLSCD site was digested with HindIII, a restriction enzyme from NEB, and the linearized integrated plasmid of YLSCD site was recovered by agarose gel electrophoresis.

The linearized YLSCD site integration plasmid and each element (promoter P) in the PgSCD gene expression cassette constructed in title 3 of this example _TEFin The gene PgSCD and the terminator T _cyc1t ) One-Step Cloning is realized by using Clonexpress Multi S One Step Cloning Kit of Nanjing Novowen Biotechnology Co., ltd to obtain recombinant plasmid pUC-HUH-YLSCD-PgSCD.

4. Construction of recombinant plasmid pUC-HUH-lip1-PgSCD

The recombinant plasmid pUC-HUH-lip1-PgSCD takes pUC57-hisG-ura-hisG as a framework, an upstream homology arm lip1-up of a lip1 site initiation codon and a downstream homology arm lip1-dm of a termination codon in Yarrowia lipolytica Po1f delta ku70 are inserted, and a PgSCD gene expression cassette (P gSCD gene) is also inserted between the upstream and downstream homology arms _YAT1 -PgSCD-T _cyc1t ) Orotidine-5' -phosphate decarboxylase encoding gene expression cassettes (containing the Yarrowia lipolytica endogenous promoter P) _TEFin A terminator T _xpr2t ) Also between the upstream and downstream homology arms, the specific structure is shown in FIG. 4.

Using lip1 as the reference P _YAT1 F and lip1:: P _YAT1 -R is a primer, and uses Yarrowia lipolytica Po1f delta ku70 geneGroup DNA as template, amplifying PgSCD expression box promoter P _YAT1 . T is defined as lip1 _cyc1t -F and lip1:: T _cyc1t -R is a primer, and the genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify the terminator T of the PgSCD expression cassette _cyc1t 。

Plasmid pUC57-PgSCD is used as a template, lip1, pgSCD-F and lip1, pgSCD-R are used as primers, and promoters P are respectively arranged at two amplification ends _YAT1 And a terminator T _cyc1t PgSCD gene of homologous arm.

The plasmid integrated at the lip1 site was digested with the restriction enzyme Pac I from NEB, and the linearized plasmid integrated at the lip1 site was recovered from the gel by agarose gel electrophoresis.

The linearized lip1 site integration plasmid and each element (promoter P) in the PgSCD gene expression cassette constructed in title 4 of this example _YAT1 The gene PgSCD and the terminator T _cyc1t ) One-Step Cloning is realized by using Clonexpress Multi S One Step Cloning Kit of Nanjing Novowed Biotechnology Co., ltd to obtain a recombinant plasmid pUC-HUH-lip1-PgSCD.

5. Construction of recombinant plasmid pUC-HUH-SCP2-MaELO2-YLELO1

The recombinant plasmid pUC-HUH-SCP2-MaELO2-YLELO1 takes pUC57-hisG-ura-hisG as a framework, an upstream homology arm SCP2-up and a downstream homology arm SCP2-dm of SCP2 site initiation codon and termination codon in Yarrowia lipolytica Po1f delta ku70 are inserted, and a MaELO2 gene expression cassette (P-HUH-SCP 2-YLELO 1) is inserted between the upstream and downstream homology arms _FBA -MaELO2-T _pex10t ) And YLELO1 Gene expression cassette (P) _GPD -YlELO1-T _pex20t ) Orotidine-5' -phosphate decarboxylase encoding gene expression cassettes (containing the Yarrowia lipolytica endogenous promoter P) _TEFin A terminator T _xpr2t ) Also between the upstream and downstream homology arms, the specific structure is shown in FIG. 5.

Using SCP2 as _FBA F and SCP2:: P _FBA -R is a primer, and the Yarrowia lipolytica Po1f delta ku70 genome DNA is used as a template to amplify the MaELO2 expression cassette promoter P _FBA . Using SCP2 as follows _pex10t -F and SCP2:: T _pex10t -R is a primer, and the Yarrowia lipolytica Po1f delta ku70 genome DNA is used as a template to amplify the MaELO2 expression cassetteStopper T _pex10t 。

Plasmid pUC57-MaELO2 is used as template, and SCP2:: maELO2-F and SCP2:: maELO2-R are used as primers, and two amplified ends are respectively provided with promoter P _FBA And a terminator T _pex10t MaELO2 gene of homologous arm.

Using SCP2 as follows _GPD F and SCP2:: P _GPD -R is a primer, and the genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify the YLELO1 expression cassette promoter P _GPD . Using SCP2 as follows _pex20t -F and SCP2:: T _pex20t -R is a primer, and the YLELO1 expression cassette terminator T is amplified by using Yarrowia lipolytica Po1f delta ku70 genomic DNA as a template _pex20t 。

Uses Yarrowia lipolytica Po1F delta ku70 genome DNA as a template, uses SCP2:: YLELO1-F and SCP2:: YLELO1-R as primers, and has promoters P at two ends of amplification _GPD And a terminator T _pex20t The YLELO1 gene of the homologous arm.

The SCP2 site integration plasmid was digested with HindIII, a restriction enzyme from NEB, and the linearized SCP2 site integration plasmid was recovered from the agarose gel electrophoresis.

The linearized SCP2 site integration plasmid and each element (promoter P) in the MaELO2 gene expression cassette constructed in title 5 of this example _FBA Gene MaELO2 and terminator T _pex10t ) One-Step Cloning was performed using the Clonexpress Multi S One Step Cloning Kit of Biotechnology Ltd of Nanjing Novodka to obtain recombinant plasmid pUC-HUH-SCP2-MaELO2.

The recombinant plasmid pUC-HUH-SCP2-MaELO2 was digested with the restriction enzyme HindIII from NEB, and the linearized pUC-HUH-SCP2-MaELO2 recombinant plasmid was recovered from the gel by agarose gel electrophoresis.

The linearized pUC-HUH-SCP2-MaELO2 recombinant plasmid and each element (promoter P) in the YLELO1 gene expression cassette constructed under the title 5 of this example _GPD Gene YLELO1 and terminator T _pex20t ) One-Step Cloning is realized by using Clonexpress MultiS One Step Cloning Kit of Nanjing Novowed Biotechnology Co., ltd to obtain a recombinant plasmid pUC-HUH-SCP2-MaELO2-YLELO1.

TABLE 1 insertion sequence in each recombinant plasmid

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TABLE 2 primer sequences

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Example 2 construction of recombinant yarrowia lipolytica (construction of recombinant bacterium 1)

Plasmid pUC-HUH-intC-ACC1-DGA1 containing ACC1 and DGA1 gene expression cassettes are introduced into Yarrowia lipolytica Po1f delta ku70, ACC1 and DGA1 expression cassettes are integrated at the intC locus of a genome, and then a hisG tag and a Ura selection tag are lost under the 5-fluoroorotic acid selection pressure, so that recombinant bacteria 1 are obtained.

The specific method comprises the following steps:

(1) competent cells were prepared after overnight culture of Yarrowia lipolytica Po1 f. DELTA.ku 70 in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose).

(2) pUC-HUH-IntC-ACC1-DGA1 was transformed into Yarrowia lipolytica Po1 f. DELTA.ku 70 competent cells using Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination.

(3) Screening positive clones by using a screening culture medium SD-Ura, and carrying out PCR identification. Wherein the screening medium SD-Ura contains: glucose 20g/L, YNB (Aminoyeast nitrogen source free from BBI Life Sciences) 6.7g/L, CSM-Ura (complete supplement mixture for uracil removal, from MP Biomedicals) 0.67g/L, agar powder 23g/L.

(4) Positive clones identified by PCR as correct were plated on YPD plates containing 5-fluoroorotic acid (containing 1g/L of 5-fluoroorotic acid, 20g/L of peptone, 10g/L of yeast extract, 20g/L of glucose and 23g/L of agar powder), and cultured in an incubator at 30 ℃ for 3 days. Taking single bacterial colony to streak on YPD plate and SD-Ura plate containing 5-fluoroorotic acid at the same time, observing growth condition of bacterial colony. A single colony that grew on YPD plates containing 5-fluoroorotic acid but did not grow on SD-Ura plates was selected and designated as recombinant bacterium 1.

(II) construction of recombinant bacterium 2

And (3) introducing a recombinant plasmid pUC-HUH-Fad2 into the recombinant bacterium 1, a hisG-Ura-hisG fragment, and integrating the fragment to a gene Fad2 site through homologous recombination, so as to knock out an oleate desaturase gene, and then losing a hisG label and a Ura screening marker under the 5-fluoroorotic acid screening pressure, so as to obtain a recombinant bacterium 2.

The specific method comprises the following steps:

(1) after culturing recombinant bacterium 1 in YPD liquid medium (containing 2% peptone, 1% Yeast extract, 2% glucose) overnight, competent cells were prepared, recombinant plasmid pUC-HUH-Fad2 was transformed into recombinant bacterium 1 using Zymogen FROzen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination, positive clones were selected using selection medium SD-Ura, and PCR was performed. Wherein the screening culture medium SD-Ura contains 20g/L glucose, 6.7g/L YNB, 0.67g/L CSM-Ura, 23g/L agar powder and water as solvent.

(2) The positive clones identified correctly by PCR were spread on YPD plates containing 5-fluoroorotic acid (in the same recombinant bacterium 1 construction process), and single colonies were streaked on YPD plates containing 5-fluoroorotic acid and SD-Ura plates at the same time. A single colony that grew on YPD plates containing 5-fluoroorotic acid but failed to grow on SD-Ura plates was selected and designated as recombinant bacterium 2.

(III) construction of recombinant bacterium 3

The recombinant plasmid pUC-HUH-YLSCD-PgSCD is introduced into a recombinant bacterium 2, a PgSCD expression box is integrated to a genome YLSCD locus through homologous recombination, and then a hisG label and a Ura screening label are lost under the 5-fluoroorotic acid screening pressure to obtain a recombinant bacterium 3.

The specific method comprises the following steps:

(1) after culturing recombinant bacterium 2 overnight in YPD liquid medium (containing 2% peptone, 1% Yeast extract, 2% glucose), competent cells were prepared, recombinant plasmid pUC-HUH-YLSCD-PgSCD was transformed into recombinant bacterium 2 using Zymogen FROzen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination, positive clones were selected using selection medium SD-Ura, and then identified by PCR. Wherein the screening culture medium SD-Ura contains 20g/L glucose, 6.7g/L YNB, 0.67g/L CSM-Ura, 23g/L agar powder and water as solvent.

(2) The positive clones identified by PCR were spread on YPD plates containing 5-fluoroorotic acid, and single colonies were streaked on both YPD and SD-Ura plates containing 5-fluoroorotic acid. A single colony that grew on YPD plates containing 5-fluoroorotic acid but failed to grow on SD-Ura plates was selected and designated as recombinant bacterium 3.

(IV) construction of recombinant bacterium 4

The recombinant plasmid pUC-HUH-lip1-PgSCD is introduced into a recombinant bacterium 3, a PgSCD expression cassette is integrated to a genome lip1 locus, and then a hisG label and a Ura screening label are lost under the 5-fluoroorotic acid screening pressure to obtain a recombinant bacterium 4.

The specific method comprises the following steps:

(1) recombinant strain 3 was cultured overnight in YPD liquid medium (containing 2% peptone, 1% Yeast extract, 2% glucose) to prepare competent cells, recombinant plasmid pUC-HUH-lip1-PgSCD was transformed into recombinant strain 3 using Zymogen Frozen EZ Yeast Transformation Kit II of Zymo Research Corporation for homologous recombination, and positive clones were selected using selection medium SD-Ura for PCR identification. Wherein the screening culture medium SD-Ura contains 20g/L glucose, 6.7g/L YNB, 0.67g/L CSM-Ura, 23g/L agar powder and water as solvent.

(2) The positive clones identified by PCR were spread on YPD plates containing 5-fluoroorotic acid, and single colonies were streaked on both YPD and SD-Ura plates containing 5-fluoroorotic acid. A single colony that grew on YPD plates containing 5-fluoroorotic acid but failed to grow on SD-Ura plates was selected and designated as recombinant bacterium 4.

(V) construction of recombinant bacterium 5

Introducing a recombinant plasmid pUC-HUH-SCP2-MaELO2-YLELO1 into a recombinant bacterium 4, integrating an expression cassette of MaELO2 and YLELO1 into a site of a genome SCP2, and then losing a hisG tag and a Ura screening marker under the 5-fluoroorotic acid screening pressure to obtain a recombinant bacterium 5.

The specific method comprises the following steps:

(1) after culturing recombinant bacterium 4 overnight in YPD liquid medium (containing 2% peptone, 1% Yeast extract, 2% glucose), competent cells were prepared, recombinant plasmid pUC-HUH-SCP2-MaELO2-YLELO1 was transformed into recombinant bacterium 4 using Zymogen FROzen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination, positive clones were selected using selection medium SD-Ura, and PCR was performed. Wherein the screening culture medium SD-Ura contains 20g/L glucose, 6.7g/L YNB, 0.67g/L CSM-Ura, 23g/L agar powder and water as solvent.

(2) Positive clones identified by PCR as correct were spread on YPD plates containing 5-fluoroorotic acid, and single colonies were streaked on both YPD plates containing 5-fluoroorotic acid and SD-Ura plates. A single colony that grew on YPD plates containing 5-fluoroorotic acid but failed to grow on SD-Ura plates was selected to obtain recombinant bacterium 5. Recombinant bacterium 5 was named Yarrowia lipolytica (Yarrowia lipolytica) strain XJ-9.

The above 5 recombinant strains were constructed, wherein Yarrowia lipolytica (Yarrowia lipolytica) XJ-9 strain was deposited in China Center for Type Culture Collection (CCTCC) at 11/9 of 2020 at address: wuhan university, classified and named as: yarrowia lipolytica XJ-9 or Yarrowia lipolytica XJ-9, accession number: CCTCC NO: M2020707.

Example 3 application of recombinant yarrowia lipolytica in production of oleic acid (culture of engineering bacteria) and extraction of products

Oleic acid was produced using the initial strain Yarrowia lipolytica (Yarrowia lipolytica) Po1 f. DELTA. Ku70, and the recombinant strains 2 and 5 of example 2, respectively. The specific method comprises the following steps: the activated strain was cultured in YPD liquid medium (containing 2% peptone, 1% yeast extract, 2% glucose, solvent water) at 30 ℃ and 220rpm for 16 hours to obtain a seed solution. The seed solution was inoculated in 50ml of a fermentation medium at an inoculum size of 1%, and shake-cultured at 220rpm at 30 ℃ for 5 days. After the fermentation is finished, transferring the fermentation liquor to a 50ml centrifuge tube, centrifuging for 15min at 5000rpm, removing supernatant, and drying in an oven (75 ℃) to constant weight.

Wherein the formula of the fermentation medium is as follows: 60g/L glucose, 1.7g/L nitrogen source (YNB) without amino yeast, 1.3g/L ammonium sulfate and water as solvent.

Qualitative and quantitative analysis of (di) oleic acid

1. Methyl esterification of fatty acids

About 1.5g of dry thallus is weighed, and 10ml of 4M hydrochloric acid is added into the thallus for reaction for 20min; transferring to boiling water bath, and heating for 10min; freezing at-80 deg.C for 15min; adding 10mL of chloroform and 5mL of methanol, and shaking at 200rpm for 30min; taking down the lipid-soluble layer, putting the lipid-soluble layer into a 10ml centrifuge tube, and drying the tube in a fume hood by using nitrogen; vacuum drying for 2h to obtain the grease. 0.1g of grease is weighed in a 2ml centrifuge tube, added with 1ml of normal hexane and 0.1ml of potassium hydroxide/methanol solution and shaken quickly for 1min to be mixed completely. The reaction solution was allowed to stand at room temperature for 15min to conduct the methyl esterification reaction. And (4) after standing, setting the centrifugal condition to be 5000rpm for centrifugation for 5min, taking 200 mu l of supernatant, placing the supernatant into a 2ml centrifugal tube, and waiting for gas phase analysis.

2. Oleic acid detection

Detection conditions are as follows: FID detector, injection port temperature 250 ℃, injection volume 1 μ l, split ratio: 50, column: DB-23 (60m 0.25 m 0.15 m). Chromatographic conditions are as follows: the initial temperature is 100 deg.C, and the temperature is increased to 196 deg.C at a rate of 25 deg.C/min, then increased to 220 deg.C at a rate of 2 deg.C/min, and maintained for about 2 min. Qualitative and quantitative determinations were performed using a mixed standard of fatty acids from Sigma-Aldrich.

After 5 days of fermentation, the yield of oleic acid of the recombinant bacteria 5 is the highest (figure 6 and table 3), and reaches 25.85g/L, namely, each liter of fermentation liquor produces 25.85g of oleic acid, which is obviously higher than that of the initial bacteria and the recombinant bacteria 2.

TABLE 3 oleic acid yields of original, recombinant 2 and recombinant 5 bacteria

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SEQUENCE LISTING

<110> Nanjing university of industry

<120> recombinant yarrowia lipolytica strain with high oleic acid yield, and construction method and application thereof

<130> 20201211

<160> 13

<170> PatentIn version 3.3

<210> 1

<211> 1524

<212> DNA

<213> artificial

<220>

<223> optimized stearoyl-CoA desaturase-encoding gene PgSCD

<400> 1

atgtccaagc cctctccctc cactcccgcc actgcccctc atttaagaca gcgacaacga 60

aaaaacctcc ccgattacga ccccgactcc gatttatctg aatctgaagg tctcggtggt 120

ctccgatccc aagttggcaa cacttgggag gacgacgagg agaccgccgt ggacgacgac 180

tcctacgtgc aacgaactct gcgaaaggag aagcccctcc cccccatcac ttggtgcaac 240

ttctaccgag agatcaacat gatttccacc ctcgctttaa ccgtggtccc catcctcgct 300

atttacggtg ccttcaccac ccctttatac agatctaccc tcgcttggtc cattctctac 360

tactacttta ctggcctcgg catcactgcc ggctaccacc gactctgggc ccatcgatct 420

tacaacgctt ctttacctct gcagtacttt ctcgctttag gcggttctgg cgccgtggag 480

ggttctatcc gatggtgggc ccgaggccac cgagcccacc acagatatac cgacaccgat 540

ctcgacccct actccgccca taaaggcctc ctctggtccc atgtgggttg gatgattgtg 600

aagccccgaa gaaagcccgg tgtggccgat gtgtctgatt tatcccgaaa ccaagttgtc 660

cgatggcagc accgatggta tttacctctg atcttcggca tgggtttctt tttccccacc 720

ctcgtggctg gtctcggttg gggtgattgg cgaggcggtt ttttctacgc tggtgctgct 780

cgactcctct ttgtgcacca ctccaccttc tgtgtgaact ctctcgctca ctggctcggt 840

gaggctccct tcgacgacaa gcataccccc cgagaccaca ttattaccgc tttcgtcacc 900

atcggtgagg gttaccataa cttccaccac gagttccccc aagatttcag aaacgccatc 960

cgatggtacc agtatgaccc cactaagtgg tttattgccg tggctgcctt cctcggttta 1020

gcttccgagc tcaagacctt ccccgataac gaggtgcgaa agggccagta ctccatgaaa 1080

ctcaaggagc tgcagcgaga tttccgagac gtcaagtggc ccaagtcctc caacgatctg 1140

cccattgtca cttgggagca attcgtggag gaagccgaca agaagaacgg ccgagacctc 1200

attgtcgtcg gtggtttcat tcacgacgtg accgagttta tcgacgagca ccccggtggt 1260

cgagctttaa tcaagaccag actgggcaag gatgctacca ccgcctttca cggtggcgtg 1320

tacgaccact ctaacgctgc tcacaattta ctcgctatgc tgcgagtcgg tgtcatcgag 1380

ggcggttacg aggtcgagca tttaaagaag aaggtgggcg tcttcagaaa ggagcagcag 1440

atccctatct gcggccctaa gtctctgggc accatctcta cccccgaatc tcccgtggtc 1500

gaggtcaagc ccatctacac ttaa 1524

<210> 2

<211> 828

<212> DNA

<213> artificial

<220>

<223> optimized fatty acid elongase 2 encoding gene MaELO2

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atggagtctg gccccatgcc cgccggaatt cccttccccg agtactacga cttcttcatg 60

gactggaaga cccctttagc cattgccgcc acttacaccg ctgccgtcgg tttattcaac 120

cctaaggtgg gcaaggtctc ccgagtggtg gccaagtctg ccaacgccaa gcccgccgag 180

agaactcagt ctggcgctgc catgaccgcc ttcgtgtttg tgcacaattt aatcctctgc 240

gtctactccg gcatcacctt ctactacatg ttccccgcca tggtgaagaa cttccgaacc 300

cacactttac acgaggccta ctgcgacacc gaccagtctt tatggaacaa cgctttaggc 360

tactggggtt atttattcta tttatccaag ttctacgagg tcatcgatac catcatcatt 420

attctgaagg gacgacgatc ctctttactg cagacctacc accacgctgg cgccatgatc 480

accatgtggt ctggcatcaa ctaccaagct acccccatct ggattttcgt ggtctttaac 540

tccttcattc acaccattat gtactgctac tatgccttca cctccatcgg cttccatccc 600

cccggtaaga agtatttaac ctctatgcag attacccagt ttttagtcgg catcaccatt 660

gccgtgtctt atttattcgt ccccggatgc atcagaaccc ccggcgctca gatggccgtg 720

tggatcaacg tgggctatct gttccctctg acctacttat tcgtggactt cgccaagcga 780

acctattcca agcgatccgc catcgccgcc cagaagaagg cccagtaa 828

<210> 3

<211> 545

<212> DNA

<213> yarrowia lipolytica

<400> 3

gtgcatgctg aggtgtctca caagtgccgt gcagtcccgc ccccacttgc ttctctttgt 60

gtgtagtgta cgtacattat cgagaccgtt gttcccgccc acctcgatcc ggcatgctga 120

ggtgtctcac aagtgccgtg cagtcccgcc cccacttgct tctctttgtg tgtagtgtac 180

gtacattatc gagaccgttg ttcccgccca cctcgatccg gcatgctgag gtgtctcaca 240

agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt gtagtgtacg tacattatcg 300

agaccgttgt tcccgcccac ctcgatccgg catgctgagg tgtctcacaa gtgccgtgca 360

gtcccgcccc cacttgcttc tctttgtgtg tagtgtacgt acattatcga gaccgttgtt 420

cccgcccacc tcgatccggc atgcactgat cacgggcaaa agtgcgtata tatacaagag 480

cgtttgccag ccacagattt tcactccaca caccacatca cacatacaac cacacacatc 540

cacgt 545

<210> 4

<211> 531

<212> DNA

<213> yarrowia lipolytica

<400> 4

agagaccggg ttggcggcgc atttgtgtcc caaaaaacag ccccaattgc cccaattgac 60

cccaaattga cccagtagcg ggcccaaccc cggcgagagc ccccttcacc ccacatatca 120

aacctccccc ggttcccaca cttgccgtta agggcgtagg gtactgcagt ctggaatcta 180

cgcttgttca gactttgtac tagtttcttt gtctggccat ccgggtaacc catgccggac 240

gcaaaataga ctactgaaaa tttttttgct ttgtggttgg gactttagcc aagggtataa 300

aagaccaccg tccccgaatt acctttcctc ttcttttctc tctctccttg tcaactcaca 360

cccgaaatcg ttaagcattt ccttctgagt ataagaatca ttcaaaatgg tgagtttcag 420

aggcagcagc aattgccacg ggctttgagc acacggccgg gtgtggtccc attcccatcg 480

acacaagacg ccacgtcatc cgaccagcac tttttgcagt actaaccgca g 531

<210> 5

<211> 1136

<212> DNA

<213> yarrowia lipolytica

<400> 5

acatggtttt tagggggtac tgtacatata tatctgtggt ggtcctgatt ttcgccaaac 60

catgttcttc gtgttccttt tcaccctcac tcacatgtcg tccacttgtt agcgtcatct 120

ttcttggcaa tagctactat tcaacattga aggaacagcc gtcccgaaag tcacttgtcg 180

gagtactccg tcccgcgacg catgcaaccg ctatgaacta acccgtgtca gtgaagtcgg 240

gggatagtct tctgccattg ttggtgaaac tgttctgctt attcacgtga atttcaacat 300

tcacacatcg atcgacaagc gaggctatta tttaaaaact gtcttagtga gttaccctgc 360

tgacgaagca atgataatct gattcgagga gagatgatga acacgactga agtgagtggt 420

tattccccca ttaacgataa tatccgcatt aattattatg ctgcacggaa ctgacatcaa 480

tatcgcccac caccactcgt ttcaacacgt tgacaggctg atgacaccag ctagacgcca 540

aattttagtg atctaaacat cctcttttgg taagggtagg tttcagagcg gggctagcgg 600

gaatgccaag cgttagctgg ggtatgtgag cattgcgcca aaaaacgact gtacgagatg 660

gaccaagacg gggccaagct gtgcagaaag tgaagaacaa gcagcagccg attttagaaa 720

ggagcgacag aacgcttaag agagactcgc gtgactaaac agccttctac tccgctttca 780

tcacccaagt aagtatgttg acagccactc accttcaccg gtttgcgttt gacaatacag 840

ttggacccct gcagagatac tacccacgtg ggtggtatcg agctgtaatt ggcatccttc 900

agtaataaac ctgaccagcc gtcagtcgcc gagaaccaag aacacgggct agccaatcac 960

gcagatccaa ttagccatca aagtctttgt tattgagtct ccacaacgat taaccagtct 1020

aaacaagcat acgaatacga gctttctctt gaaaagaatg gaaagaaaac atatatagac 1080

tgtgggacag acgaggcagg agaaaaggaa ccttacccca agtcgctcgt tcaaca 1136

<210> 6

<211> 841

<212> DNA

<213> yarrowia lipolytica

<400> 6

gtacgtagca acaacagtgt acgcagtact atagaggaac aattgccccg gagaagacgg 60

ccaggccgcc tagatgacaa attcaacaac tcacagctga ctttctgcca ttgccactag 120

gggggggcct ttttatatgg ccaagccaag ctctccacgt cggttgggct gcacccaaca 180

ataaatgggt agggttgcac caacaaaggg atgggatggg gggtagaaga tacgaggata 240

acggggctca atggcacaaa taagaacgaa tactgccatt aagactcgtg atccagcgac 300

tgacaccatt gcatcatcta agggcctcaa aactacctcg gaactgctgc gctgatctgg 360

acaccacaga ggttccgagc actttaggtt gcaccaaatg tcccaccagg tgcaggcaga 420

aaacgctgga acagcgtgta cagtttgtct tagcaaaaag tgaaggcgct gaggtcgagc 480

agggtggtgt gacttgttat agcctttaga gctgcgaaag cgcgtatgga tttggctcat 540

caggccagat tgagggtctg tggacacatg tcatgttagt gtacttcaat cgccccctgg 600

atatagcccc gacaataggc cgtggcctca tttttttgcc ttccgcacat ttccattgct 660

cggtacccac accttgcttc tcctgcactt gccaacctta atactggttt acattgacca 720

acatcttaca agcggggggc ttgtctaggg tatatataaa cagtggctct cccaatcggt 780

tgccagtctc ttttttcctt tctttcccca cagattcgaa atctaaacta cacatcacac 840

a 841

<210> 7

<211> 1088

<212> DNA

<213> yarrowia lipolytica

<400> 7

gtgaagagtc cgtgtagcct ccacaccaca cacactgtcc ataacatata ccttccataa 60

acttccatta tacaatgtac gaggtacaat atgtactcaa agttgtggaa ttcgcgtttt 120

gcagtttttt gatttccgag atttgcagct ttggcgggac agccgcactc gtgcacccca 180

agcttcctat gcgcctttct ctctgcctct ctctggcgct cgttaggccg acaccgctgc 240

gtccttatct ccgtctctct ggactctgat acgccatctg cacatccaga ggtaagaagt 300

gtgtgtctac ccgtgggtat acagataccg agccgaggta gggagagtag tctatgaggc 360

agattggggg acgctattgc tgcacctgta gctggttcaa ttggaccagg cacatctttc 420

tgcacccata tacccctcct gtcagctgtg ccgccggctg ctctcctcct tagtaaaagt 480

cgtcaatagt tgtttcttct cctcgcttac cttcacatgc gtcttccata ttcacactgg 540

tagatgcaac caagagttaa aggttcatcc caacgttggg gacggtggag tttattgaga 600

cagtagcaat ataacatggg agagcagctg gtggcgagct atgggggctt ttatagggaa 660

acaaggaaga accacagaac caagatgatg gatgacgcga aaaaaagttg cttccaaaaa 720

tcgcattgcc gctcttcatc ttacgctctc atattgccac tctcctgcac cccactctac 780

ctccgctaaa gtcctcggct ccagcacctt attcaccatt agcttgtatg atattcgtcg 840

tgttggtgga gttgtgaaga cgttgtaggg cgatattggg tctggtgaag agacggtaga 900

ccagcatcga cggcagtggc aacagaacac cgtcttctgg ctcatctcca cctcccctcc 960

cccaccccaa ctcggcccaa cgaagtgttt ttccgaacac gctttcagac gctgggctaa 1020

accctattct ctggcgggcg cctctgaaaa ttactcaacc aagatataag agatcgcatc 1080

cccataac 1088

<210> 8

<211> 502

<212> DNA

<213> yarrowia lipolytica

<400> 8

cactggccgg tcgataattt aacgtgctga gctcagcaca cgcattgccc attggctgta 60

tatagatgaa tgtaatgata ccgtaagaga atgagagcac ggtattgtat tacaggggat 120

taagtacaca tttacttgga gttctgtacc agaagacact actatacatg gtattactta 180

cattagagtc ggtgaccgta ttcgtctcgt atagacataa tattttccta ccccacattg 240

ttcctgggcc ttcggagcac atctacagtg agtgactgtt tcagttgagc ttgaggggtt 300

aagtaagtgg gggaagggtt tgcgattctg aaaaagagca tgactaatct ctctgtggag 360

gagcaatgaa gtcacgtgat gcaatcatac cggtgtatcg gatctgcctg ggtgtctgat 420

tactaatcat ttactcacct gttttcccca gctatctcat ccatctcaga gcctcggccc 480

agccttcggc ccttttgggt tt 502

<210> 9

<211> 563

<212> DNA

<213> yarrowia lipolytica

<400> 9

gctatttatc actctttaca acttctacct caactatcta ctttaataaa tgaatatcgt 60

ttattctcta tgattactgt atatgcgttc ctctaagaca aatcgaaacc agcatgcgat 120

cgaatggcat acaaaagttt cttccgaagt tgatcaatgt cctgatagtc aggcagcttg 180

agaagattga cacaggtgga ggccgtaggg aaccgatcaa cctgtctacc agcgttacga 240

atggcaaatg acgggttcaa agccttgaat ccttgcaatg gtgccttgga tactgatgtc 300

acaaacttaa gaagcagccg cttgtcctct tcctcgaaac tctcaaacac agtccagaag 360

tcctttatag tttgatctgt atccagatag cctccgtaat tggtgtgtgt cttcaaatcc 420

cagacgtcca cattggcatg tcctccactg ataagcattt gaagttcatc tgcgttgaac 480

attgagaccc acgaagggtc aatgagctgg tatagaccgc ccaagaatgc atctgtctgt 540

gttctgatac tggtgttaag ctt 563

<210> 10

<211> 248

<212> DNA

<213> yarrowia lipolytica

<400> 10

tcatgtaatt agttatgtca cgcttacatt cacgccctcc ctccacatcc gctctaaccg 60

aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt 120

tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt 180

acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt 240

taatttgc 248

<210> 11

<211> 1010

<212> DNA

<213> yarrowia lipolytica

<400> 11

tgacgaggtc tggatggaag gactagtcag cgagacacag agcatcaggg accagacacg 60

accaattcaa tcgacaacac tgtgctgcat agcagtgcac agaggtcctg ggcatgaata 120

tattttagca ttggagatat gagtggtaga gcgtatacag tattaattgt ggaggtatct 180

cgtcgcattg atagagcaat acagttactg ctgaagggaa tgataccgag tatttcggcc 240

cgattcagtt cttgatatcg tcattttgtc tctattgtct acttttcaga taacctcaac 300

aaatcttcaa caaatctccc agtaaacagt cagagatcat atccgagatc atatcagata 360

tgtcacgatc cgagtacaat aatggatatt aatctgcttg attttgaatt ctgttgcgat 420

tatgatttct ttgatttcga tatgaacaca tacggcgact cccagacctt tagaagctcc 480

agtttggatt cttagcaatg gttacactca actatatccc aagtaatact tggtaacaat 540

atgccaagtt agtcattcat tcgttatagg agttagcaag tgtttgtcag ctaaaaatgg 600

ttagtcggtc gattaccact tagatctttt cagcgtggaa cttgatggta cgcttgaacc 660

gacacttgga gtagtcgggg ctgttgatga cgtagatgac gtttcgctca gggtgaggag 720

tgcaatagta gtactccttg gggccgtctc tcagctcaaa ggttccatcg gcggcaatgt 780

caaagaccga gccctggagc ttgtagccgt agtcgccggt ccagaacaaa gcctgcagct 840

ccagataggc gatgggcatg tcgttaacag agaaggtgtt gccctcgccc tcggtgatgg 900

tgatgggttc gccgtcggtg gaggcggtga tcaggtcatc ttggtaggtg acgggcagag 960

attcgaccga ttgggcgtct gatctggtat aggtcagctt gtacttgtct 1010

<210> 12

<211> 856

<212> DNA

<213> yarrowia lipolytica

<400> 12

ggaagtgtgg atggggaagt gagtgcccgg ttctgtgtgc acaattggca atccaagatg 60

gatggattca acacagggat atagcgagct acgtggtggt gcgaggatat agcaacggat 120

atttatgttt gacacttgag aatgtacgat acaagcactg tccaagtaca atactaaaca 180

tactgtacat actcatactc gtacccgggc aacggtttca cttgagtgca gtggctagtg 240

ctcttactcg tacagtgtgc aatactgcgt atcatagtct ttgatgtata tcgtattcat 300

tcatgttagt tgcgtcaatc gtcaaatata gctatgcgtg agtttgtatc tctatcgcac 360

ctgtgcacaa ctactgtaac atgcaatctg cttatgttgg catggtctgc tcatgcatct 420

ggttagaatc ttgttctttc ctgttcagca ggaacctcat aggtatttat ctttggatac 480

ttctccatct cgtagtctgt atcgtccact gacctgttca cataatagtc aggagcaaag 540

ttgttattgg actggtttcc gtaaaccggc tgtggctgat aattgtatgc ctggtaagga 600

tttgaagctg ggtacatggc tgggttattt gctgctgcgt agttaccttg gttggcctct 660

ctgttgatct tatcgttaca gcagcaggca cagcaagagc acagcgcaca acaggcgctg 720

aaagcagata ctccaaagaa gcagcatctg aaaaaggctg tgagtaccca cagaaccagc 780

attcctccca gaacaattcc cacaatcgcc acaatcttgc aaaacgtgtt atccatgcat 840

gtatcccagc tcttga 856

<210> 13

<211> 519

<212> DNA

<213> yarrowia lipolytica

<400> 13

gatccaacta cggaacttgt gttgatgtct ttgcccccgg ctccgatatc atctctgcct 60

cttaccagtc cgactctggt actttggtct actccggtac ctccatggcc tgtccccacg 120

ttgccggtct tgcctcctac tacctgtcca tcaatgacga ggttctcacc cctgcccagg 180

tcgaggctct tattactgag tccaacaccg gtgttcttcc caccaccaac ctcaagggct 240

ctcccaacgc tgttgcctac aacggtgttg gcatttaggc aattaacaga tagtttgccg 300

gtgataattc tcttaacctc ccacactcct ttgacataac gatttatgta acgaaactga 360

aatttgacca gatattgttg taaatagaaa atctggcttg taggtggcaa aatgcggcgt 420

ctttgttcat caattccctc tgtgactact cgtcatccct ttatgttcga ctgtcgtatt 480

tcttattttc catacatatg caagtgagat gcccgtgtc 519

Claims

1. A recombinant yarrowia lipolytica strain for high oleic acid production, wherein said recombinant yarrowia lipolytica strain is yarrowia lipolytica (S) (R)Yarrowia lipolytica) The strain XJ-9 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2020707.

2. The recombinant yarrowia lipolytica for high oleic acid production of claim 1, wherein said yarrowia lipolytica (R) (R: (R)) (R)Yarrowia lipolytica) The XJ-9 strain is obtained by knocking out the oleate desaturase in the yarrowia lipolytica genome and inserting an acetyl-CoA carboxylase expression cassette, a diacylglycerol acyltransferase expression cassette, a fatty acid elongase 1 expression cassette, a fatty acid elongase 2 expression cassette and a stearoyl-CoA desaturase expression cassette.

3. The recombinant yarrowia lipolytica for high oleic acid production of claim 2, wherein said acetyl-coa carboxylase, diacylglycerol acyltransferase, fatty acid elongase 1 encoding genes are from yarrowia lipolytica (yarrowia lipolytica) ((R))Yarrowia lipolytica) (ii) a The fatty acid elongase 2 coding gene is derived from Mortierella alpina (Mortierella alpina) The fatty acid elongase 2 coding gene is obtained after codon optimization; the coding gene of stearoyl-CoA desaturase is derived from rust (C.Puccinia graminis) The coding gene of stearoyl-CoA desaturase is obtained by codon optimization.

4. The recombinant yarrowia lipolytica for high oleic acid production of claim 3, wherein said integration site of acetyl-CoA carboxylase 1 and diacylglycerol acyltransferase 1 expression cassette is IntC site, the integration site of selection marker hisG-ura-hisG is Fad2 site, the integration site of stearoyl-CoA desaturase expression cassette is lip1 site or YLSCD site, and the integration site of fatty acid elongase 1 and fatty acid elongase 2 expression cassette is SCP2 site.

5. The recombinant yarrowia lipolytica for high oleic acid production of claim 4, wherein said stearoyl-CoA desaturase encoding gene sequence is set forth in SEQ ID No. 1; the sequence of the fatty acid elongase 2 coding gene is shown in SEQ ID No. 2.

6. The method for constructing the recombinant yarrowia lipolytica bacterium capable of producing oleic acid in high yield according to claim 1, comprising the step of introducing an oleic acid desaturase knockout cassette, an acetyl-CoA carboxylase expression cassette, a diacylglycerol acyltransferase expression cassette, a fatty acid elongase 1 expression cassette, a fatty acid elongase 2 expression cassette and a stearoyl-CoA desaturase expression cassette into a yarrowia lipolytica bacterium (yarrowia lipolytica bacterium) having a ku70 gene knocked out, in the form of a plasmidYarrowia lipolytica) Po1f Δ ku70, and then integrating by homologous recombination on the genome of yarrowia lipolytica.

7. Use of the recombinant yarrowia lipolytica strain of any one of claims 1-5 for the production of oleic acid, comprising the step of culturing the recombinant strain of any one of claims 1-5 in a fermentation medium to obtain a fermentation product.

8. The use of the recombinant yarrowia lipolytica of claim 7 in the production of oleic acid, wherein said fermentation medium comprises glucose 50-70g/L, amino-free yeast nitrogen source 1.6-1.8g/L, ammonium sulfate 1.2-1.4g/L.