CN112538438A - Recombinant yarrowia lipolytica with high oleic acid yield as well as construction method and application thereof - Google Patents
Recombinant yarrowia lipolytica with high oleic acid yield as well as construction method and application thereof Download PDFInfo
- Publication number
- CN112538438A CN112538438A CN202011464493.6A CN202011464493A CN112538438A CN 112538438 A CN112538438 A CN 112538438A CN 202011464493 A CN202011464493 A CN 202011464493A CN 112538438 A CN112538438 A CN 112538438A
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- Prior art keywords
- yarrowia lipolytica
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- recombinant
- expression cassette
- oleic acid
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Links
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- C12N15/905—Stable introduction of foreign DNA into chromosome using homologous recombination in yeast
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Abstract
The invention provides a recombinant yarrowia lipolytica strain with high oleic acid yield, a construction method and application thereof, belonging 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 coenzyme A desaturase, and experiments prove that the recombinant yarrowia lipolytica can efficiently ferment and produce the oleic acid, and realizes the efficient synthesis of the plant-derived natural product oleic acid in yarrowia lipolytica.
Description
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)18H34O2) The learning name is as follows: cis-9-octadecenoic acid, an Omega-9 type monounsaturated fatty acid, is mainly present in oilseed rape (Brassica napus), Camellia (Camellia japonica) and other oil crops. 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 further 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 invention, the promoter of each expression cassette is lipolyticPromoter P of yarrowiaTEF、Php4d、PTEFin、PYAT1、PFBA、PFBAin、PPOX2、PPOT1Or PGPDAny one of the above; the terminator is terminator T of yarrowia lipolyticaxpr2t、Tmig1t、Tlip2t、Tcyc1t、Tpex3t、Tpex10tOr Tpex20tAny 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 then 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 in any one of claims 1-6 by using 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 responsible for coding the non-homologous recombination gene ku70 and is knocked out, so that the homologous recombination capability of the Yarrowia lipolytica is enhanced, the gene integration 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, in which IntC-up represents the upstream homology arm of IntC site, IntC-dm represents the downstream homology arm of IntC site, and hp4d represents promoter Php4dMig1T denotes a terminator Tmig1tTEFin denotes the promoter PTEFinAnd lip2T denotes a terminator Tlip2tURA denotes an orotidine-5' -phosphate decarboxylase-encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P)TEFinA terminator Txpr2t) ACC1 is an acetyl-CoA carboxylase-encoding gene, and DGA1 is a diacylglycerol acyltransferase-encoding gene.
FIG. 2 is a diagram of the structure of the recombinant plasmid pUC-HUH-Fad2, in which Fad2-up represents the upstream homology arm at Fad2 site, Fad2-dm represents the downstream homology arm at Fad2 site, URA represents the orotidine-5' -phosphate decarboxylase encoding gene expression cassette (containing the promoter P endogenous to Yarrowia lipolytica)TEFinA terminator Txpr2t)。
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 Tcyc1tTEFin denotes the promoter PTEFinURA denotes an orotidine-5' -phosphate decarboxylase-encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P)TEFinA terminator Txpr2t) PgSCD is a stearoyl-CoA desaturase encoding gene.
FIG. 4 is a diagram of the structure of the recombinant plasmid pUC-HUH-lip1-PgSCD, wherein lip1-up represents the upstream homology arm at the lip1 site, lip1-dm represents the downstream homology arm at the lip1 site, cyc1T represents the terminator Tcyc1tYAT1 denotes promoter PYAT1URA denotes an orotidine-5' -phosphate decarboxylase-encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P)TEFinA terminator Txpr2t) PgSCD is a stearoyl-CoA desaturase encoding gene.
FIG. 5 is a diagram of the structure of recombinant plasmid pUC-HUH-SCP2-MaELO2-YLELO1, in which SCP2-up represents the upstream homology arm at the SCP2 site, SCP2-dm represents the downstream homology arm at the SCP2 site, and GPD represents promoter PGPDAnd pex20T denotes a terminator Tpex20tFBA represents the promoter PFBAAnd pex10T denotes a terminator Tpex10tURA denotes an orotidine-5' -phosphate decarboxylase-encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P)TEFinA terminator Txpr2t) MaELO2 is the fatty acid elongase 2 coding gene, and YLELO1 is the 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 American type culture Collection, accession number ATCC MYA-2613.
Yarrowia lipolytica (Yarrowia lipolytica) Po1 f. delta. ku70 (MatA. delta. ku70:: hisG, leu2-270, ura3-302, xpr2-322, axp1-2), abbreviated as Yarrowia lipolytica Po1 f. delta. ku 70. Yarrowia lipolytica Po1 f. delta. ku70 was constructed from Yarrowia lipolytica Po1f after 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 1402bp upstream sequence (upstream homology arm) of the start codon and a 1396bp downstream sequence (downstream homology arm) of the stop codon of the IntC site on chromosome C of Yarrowia lipolytica Po1 f. delta. ku70 genome into pUC57-hisG-ura-hisG vector (construction method is shown in example 1), 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 1539bp upstream sequence (upstream homology arm) of the Fad2 site initiation codon and 1503bp downstream sequence (downstream homology arm) of the termination codon in chromosome B of Yarrowia lipolytica Po1 f. delta. ku70 genome into pUC57-hisG-ura-hisG vector (construction method is described in example 1), and two hisG tag-encoding genes were located between the Fad2 site upstream and downstream homology arms.
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 positioned between the upstream and downstream homology arms of the YLSCD site.
The lip1 site integration plasmid is obtained by inserting a sequence with the upstream size of 1458bp of the initiation codon (upstream homology arm) and a sequence with the downstream size of 1438bp of the stop codon (downstream homology arm) of the lip1 site on chromosome E of 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 positioned 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 the initiation codon of the SCP2 site being 1523bp and a sequence (downstream homology arm) with the downstream size of the termination codon being 1524bp into a pUC57-hisG-ura-hisG vector (the construction method is shown in example 1) on chromosome E of Yarrowia lipolytica Po1f delta ku70 genome, 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 (preparation of target genes)
Based on the nucleotide sequence of stearoyl-CoA desaturase encoding gene (GenBank accession No.: NW _003526568.1) from Puccinia graminis (NCBI), codon optimization was performed, and the optimized stearoyl-CoA desaturase encoding gene PgSCD (SEQ ID No.: 1) was synthesized by Kinzymenia wisdom-only Biotech, Suzhou and inserted into plasmid pUC57 to obtain plasmid pUC 57-PgSCD.
According to the nucleotide sequence of the fatty acid elongase 2 encoding gene (GenBank accession number: AB468587.1) from Mortierella alpina (Mortierella alpina) provided at NCBI, after codon optimization, Suzhou Jinzhi Biotechnology Limited 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-MaELO 2.
Based on the nucleotide sequence of orotidine-5 '-phosphate decarboxylase encoding gene ura of Yarrowia lipolytica (GenBank accession No.: AJ306421.1) and hisG tag (GenBank accession No.: AF324729.1) provided on NCBI, the synthesis of Suzhou Jinzhi Biotechnology Ltd was entrusted, two hisG tag encoding gene sequences were inserted into plasmid pUC57, and an orotidine-5' -phosphate decarboxylase encoding gene expression cassette (endogenous promoter P of Yarrowia lipolytica) was inserted between the two hisG tag encoding gene sequencesTEFinOrotidine-5' -phosphate decarboxylase-encoding gene ura and terminator Txpr2tComposition) to achieve ura tag recovery, resulting in plasmid pUC 57-hisG-ura-hisG.
acetyl-CoA carboxylase encoding gene ACC1(GenBank accession number: YALI0C11407g) was amplified using Yarrowia lipolytica Po 1F. delta. ku70 genomic DNA as a template and IntC:: ACC1-F and IntC:: ACC1-R as primers.
Diacylglycerol acyltransferase encoding gene DGA1(GenBank accession number: YALI0E3276 32769g) was amplified using Yarrowia lipolytica Po 1F. delta. ku70 genomic DNA as template and IntC:: DGA1-F and IntC:: DGA1-R as primers.
The fatty acid elongase 1 coding gene YLELO1(GenBank accession number: YALI0F06754g) 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 lipolyticahp4dThe nucleotide sequence of (A) is shown as SEQ ID No. 3; endogenous promoter P of Yarrowia lipolyticaTEFinThe nucleotide sequence of (A) is shown as SEQ ID No. 4; endogenous promoter P of Yarrowia lipolyticaYAT1The nucleotide sequence of (A) is shown as SEQ ID No. 5; endogenous promoter P of Yarrowia lipolyticaFBAThe nucleotide sequence of (A) is shown as SEQ ID No. 6; endogenous promoter P of Yarrowia lipolyticaGPDThe nucleotide sequence of (A) is shown as SEQ ID No. 7; endogenous terminator T of Yarrowia lipolyticamig1tThe nucleotide sequence of (A) is shown as SEQ ID No. 8; endogenous terminator T of Yarrowia lipolyticalip2tThe nucleotide sequence of (A) is shown as SEQ ID No. 9; endogenous terminator T of Yarrowia lipolyticacyc1tThe nucleotide sequence of (A) is shown as SEQ ID No. 10; endogenous terminator T of Yarrowia lipolyticapex10tThe nucleotide sequence of (A) is shown as SEQ ID No. 11; endogenous terminator T of Yarrowia lipolyticapex20tThe nucleotide sequence is shown as SEQ ID No. 12; endogenous terminator T of Yarrowia lipolyticaxpr2tThe 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 is characterized in that pUC57-hisG-ura-hisG is used as a framework, an IntC-up upstream homology arm IntC-up of an IntC site initiation codon and an IntC-dm downstream homology arm of a termination codon in Yarrowia lipolytica Po1f delta ku70 are inserted, and an ACC1 gene expression cassette (P) is also inserted between the IntC-up upstream homology arm and the IntC-dm downstream homology arm (P)hp4d-ACC1-Tmig1t) And DGA1 gene expression cassette (P)TEFin-DGA1-Tlip2t) Orotidine-5' -phosphate decarboxylase encoding gene expression cassettes (containing the Yarrowia lipolytica endogenous promoter P)TEFinA terminator Txpr2t) Also between the upstream and downstream homology arms, the detailed structure is shown in figure1。
Using IntC as the reference Php4dF and IntC:: Php4d-R is a primer, and the ACC1 expression cassette promoter P is amplified by using Yarrowia lipolytica Po1f delta ku70 genome DNA as a templatehp4d. Using IntC as the basemig1t-F and IntC:: Tmig1t-R is a primer, and the ACC1 expression cassette terminator T is amplified by using Yarrowia lipolytica Po1f delta ku70 genomic DNA as a templatemig1t。
Uses Yarrowia lipolytica Po1F delta ku70 genome DNA as a template, uses IntC (ACC 1-F) and IntC (ACC 1-R) as primers, and amplifies two ends of the primer with a promoter Php4dAnd a terminator Tmig1tACC1 gene of homologous arm.
Using IntC as the reference PTEFinF and IntC:: PTEFin-R is a primer, and the DGA1 expression cassette promoter P is amplified by using Yarrowia lipolytica Po1f delta ku70 genome DNA as a templateTEFin. Using IntC as the baselip2t-F and IntC:: Tlip2t-R is a primer, and uses Yarrowia lipolytica Po1f delta ku70 genome DNA as a template to amplify DGA1 expression cassette terminator Tlip2t。
Uses Yarrowia lipolytica Po1F delta ku70 genome DNA as a template, uses IntC:: DGA1-F and IntC:: DGA1-R as primers, and amplifies the primers with promoters P at two endsTEFinAnd a terminator Tlip2tDGA1 gene of homologous arm.
The PCR amplification system is as follows:
components | Volume of |
PrimerSTAR Max Premix | 25ul |
Form | 1ul |
Primer | |
1 | |
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 ═ length of target fragment/1 kb in min), and 30 cycles of repetition.
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 examplehp4dACC1 gene and terminator Tmig1t) One-Step Cloning is realized by using the Clon express Multi S One Step Cloning Kit of Nanjing Novowed Biotechnology Limited, an 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 | x ng |
Insert fragment | y ng |
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 of the fragment or linearized vector used was [0.02 × base number of fragment or linearized vector ] ng.
The circular recombinant vector is transformed into escherichia coli DH5 alpha competent cells, and a positive recombinant plasmid pUC-HUH-IntC-ACC1 is obtained through ampicillin resistance plate screening and colony PCR and sequencing verification.
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 in title 1 of this exampleTEFinGene DGA1 and terminator Tlip2t) One-Step Cloning is realized by using the Clonexpress Multi S One Step Cloning Kit of Nanjing Novozam Biotechnology Co., LtdTo the recombinant plasmid pUC-HUH-IntC-ACC1-DGA 1.
2. Construction of recombinant plasmid pUC-HUH-Fad2
The recombinant plasmid pUC-HUH-Fad2 is characterized in that pUC57-hisG-ura-hisG is used as a framework, a homologous arm (Fad2-up) with the upstream of the initiation codon of Fad2 site of Yarrowia lipolytica Po1f delta ku70 being 1539bp and a homologous arm (Fad2-dm) with the downstream of the termination codon being 1503bp are inserted, and an orotidine-5' -phosphate decarboxylase encoding gene expression cassette (containing a promoter P endogenous to Yarrowia lipolytica)TEFinA terminator Txpr2t) Also between the upstream and downstream homology arms, the specific structure is shown in FIG. 2.
Fad2-up-F and Fad2-up-R are used as primers, Yarrowia lipolytica Po1F delta ku70 genome DNA is used as a template, and Fad2-up which is the upstream homology arm of the initiation codon of Fad2 site is amplified.
After the plasmid pUC57-hisG-ura-hisG was digested 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 site initiation codon upstream homology Fad2-up (with pUC57-hisG-ura-hisG homology sequences at both ends) constructed in title 2 of this example were used from Nanjing Nozao Biotech LtdII One Step Cloning was carried out by One Step Cloning Kit to obtain circular recombinant vector.
The circular recombinant vector is transformed into escherichia coli DH5 alpha competent cells, and positive recombinant plasmid pUC-HUH-Fad2-up is obtained through ampicillin resistance plate screening and colony PCR and sequencing verification.
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 which is a homologous arm at the downstream of a termination codon at the Fad2 site.
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.
Linearizing pUC-HUH-Fad2The Fad2 site termination codon downstream of the homology arm Fad2-dm (pUC-HUH-Fad 2-up homology arm sequence at both ends) constructed in title 2 of this example was cloned using Nanjing Nodezaki Biotech LtdII One Step Cloning was performed with the One Step Cloning Kit to obtain recombinant plasmid pUC-HUH-Fad 2.
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 start codon of YLSCD site and a downstream homology arm YLSCD-dm of the stop codon in Yarrowia lipolytica Po1f delta ku70 are inserted, and a PgSCD gene expression cassette (P-HUH-YLSCD-PgSCD) is also inserted between the upstream and downstream homology armsTEFin-PgSCD-Tcyc1t) Orotidine-5' -phosphate decarboxylase encoding gene expression cassettes (containing the Yarrowia lipolytica endogenous promoter P)TEFinA terminator Txpr2t) Also between the upstream and downstream homology arms, the specific structure is shown in FIG. 3.
P is expressed by YLSCDTEFinF and YLSCD PTEFin-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 PTEFin. T is defined by YLSCDcyc1tT and YLSCDcyc1t-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 cassettecyc1t。
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 amplificationTEFinAnd a terminator Tcyc1tPgSCD 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 exampleTEFinThe gene PgSCD and the terminator Tcyc1t) Utilizing Nanjing Nuozan biotechnologyThe Clon express Multi S One Step Cloning Kit of Limited company realizes One-Step Cloning to obtain a 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 lip1 site initiation codon and a downstream homology arm lip1-dm of 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 armsYAT1-PgSCD-Tcyc1t) Orotidine-5' -phosphate decarboxylase encoding gene expression cassettes (containing the Yarrowia lipolytica endogenous promoter P)TEFinA terminator Txpr2t) Also between the upstream and downstream homology arms, the specific structure is shown in FIG. 4.
P is expressed by lip1YAT1F and lip1:: PYAT1-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 PYAT1. T is defined as lip1cyc1tF and lip1:: Tcyc1t-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 cassettecyc1t。
Plasmid pUC57-PgSCD is used as a template, lip1, PgSCD-F and lip1 are used as primers, and promoter P is respectively arranged at two amplified endsYAT1And a terminator Tcyc1tPgSCD gene of homologous arm.
The plasmid integrated at the lip1 site was digested with the restriction enzyme Pac I from NEB, and the linearized lip1 site integrated plasmid was recovered from the agarose gel electrophoresis gel.
The linearized lip1 site integration plasmid and each element (promoter P) in the PgSCD gene expression cassette constructed in title 4 of this exampleYAT1The gene PgSCD and the terminator Tcyc1t) One-Step Cloning was carried out using the Clonexpress MultiS One Step Cloning Kit of Nanjing Novowed Biotechnology Ltd to obtain recombinant plasmid pUC-HUH-lip 1-PgSCD.
5. Construction of recombinant plasmid pUC-HUH-SCP2-MaELO2-YLELO1
The recombinant plasmid pUC-HUH-SCP2-MaELO2-YLELO1 is pUC57-hisG-ura-hisG is a skeleton, an upstream homology arm SCP2-up of an initiation codon of an SCP2 site and a downstream homology arm SCP2-dm of a termination codon in Yarrowia lipolytica Po1f delta ku70 are inserted, and a MaELO2 gene expression cassette (P2 gene expression cassette is inserted between the upstream and downstream homology armsFBA-MaELO2-Tpex10t) And the YLELO1 Gene expression cassette (P)GPD-YlELO1-Tpex20t) Orotidine-5' -phosphate decarboxylase encoding gene expression cassettes (containing the Yarrowia lipolytica endogenous promoter P)TEFinA terminator Txpr2t) Also between the upstream and downstream homology arms, the specific structure is shown in FIG. 5.
Using SCP2 as the raw material PFBAF and SCP2:: PFBA-R is a primer, and the genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify the promoter P of the MaELO2 expression cassetteFBA. T is prepared from SCP2pex10tF and SCP2:: Tpex10t-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 MaELO2 expression cassettepex10t。
Plasmid pUC57-MaELO2 is used as template, SCP2, MaELO2-F and SCP2, MaELO2-R are used as primer, and two ends of amplification are respectively provided with promoter PFBAAnd a terminator Tpex10tThe MaELO2 gene of homologous arm.
Using SCP2 as the raw material PGPDF and SCP2:: PGPD-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 PGPD. T is prepared from SCP2pex20tF and SCP2:: Tpex20t-R is a primer, genomic DNA of Yarrowia lipolytica Po1f delta ku70 is taken as a template, and the terminator T of the YLELO1 expression cassette is amplifiedpex20t。
The genomic DNA of Yarrowia lipolytica Po1F delta ku70 is taken as a template, SCP2, YLELO1-F and SCP2, YLELO1-R are taken as primers, and promoters P are respectively arranged at two ends of amplificationGPDAnd a terminator Tpex20tThe YLELO1 gene of the homologous arm.
The SCP2 site integration plasmid was digested with HindIII restriction enzyme from NEB, and the linearized SCP2 site integration plasmid was recovered from agarose gel electrophoresis.
The linearized SCP2 site integration plasmid and the MaELO2 gene constructed in title 5 of this example were expressedElements in the cassette (promoter P)FBAGene MaELO2 and terminator Tpex10t) One-Step Cloning was carried out using the Clonexpress MultiS One Step Cloning Kit of Nanjing Novowed Biotechnology Ltd to obtain recombinant plasmid pUC-HUH-SCP2-MaELO 2.
The recombinant plasmid pUC-HUH-SCP2-MaELO2 was digested with HindIII, a restriction enzyme from NEB, and the linearized pUC-HUH-SCP2-MaELO2 recombinant plasmid was recovered 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 exampleGPDGene YLELO1 and terminator Tpex20t) One-Step Cloning was carried out using the Clonexpress MultiS One Step Cloning Kit of Nanjing Novowed Biotechnology Ltd to obtain recombinant plasmid pUC-HUH-SCP2-MaELO2-YLELO 1.
TABLE 1 insertion sequence in each recombinant plasmid
TABLE 2 primer sequences
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 site of genome, and then a hisG tag and a Ura selection tag are lost under 5-fluoroorotic acid selection pressure, so that recombinant bacterium 1 is obtained.
The specific method comprises the following steps:
after overnight culture of Yarrowia lipolytica Po1 f. delta. ku70 in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose), competent cells were prepared.
② homologous recombination was carried out by transforming Yarrowia lipolytica Po1 f. delta. ku70 competent cells with pUC-HUH-IntC-ACC1-DGA1 using Zymogen FROzen EZ Yeast Transformation Kit II from Zymo Research Corporation.
And thirdly, screening positive clones by adopting 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 23 g/L.
Fourthly, the positive clones which are correctly identified by PCR are spread 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 are placed 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
The recombinant plasmid pUC-HUH-Fad2 is introduced into the recombinant bacterium 1, a hisG-Ura-hisG fragment is integrated to a genome Fad2 site through homologous recombination, so that an oleate desaturase gene is knocked out, and then a hisG label and a Ura selection marker are lost under the 5-fluoroorotic acid selection pressure, so that the recombinant bacterium 2 is obtained.
The specific method comprises the following steps:
the recombinant bacterium 1 was cultured overnight in YPD liquid medium (containing 2% peptone, 1% Yeast extract, 2% glucose) to prepare competent cells, the recombinant plasmid pUC-HUH-Fad2 was transformed into the recombinant bacterium 1 using Zymogen FROZEN EZ Yeast Transformation Kit II from 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.
Secondly, coating the positive clones which are correctly identified by PCR on a YPD plate containing 5-fluoroorotic acid (in the process of constructing the recombinant bacterium 1), and simultaneously drawing lines on the YPD plate containing 5-fluoroorotic acid and an SD-Ura plate by taking a single colony. 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
And (2) introducing the recombinant plasmid pUC-HUH-YLSCD-PgSCD into the recombinant strain 2, integrating the PgSCD expression cassette into a genome YLSCD locus through homologous recombination, and then losing a hisG label and a Ura screening label under the 5-fluoroorotic acid screening pressure to obtain a recombinant strain 3.
The specific method comprises the following steps:
the recombinant bacterium 2 was cultured overnight in YPD liquid medium (containing 2% peptone, 1% Yeast extract, 2% glucose) to prepare competent cells, the recombinant plasmid pUC-HUH-YLSCD-PgSCD was transformed into the 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.
Secondly, coating positive clones which are correctly identified by PCR on YPD plates containing 5-fluoroorotic acid, and taking single colonies to streak on the 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 3.
(IV) construction of recombinant bacterium 4
And (3) introducing the recombinant plasmid pUC-HUH-lip1-PgSCD into the recombinant bacterium 3, integrating the PgSCD expression cassette into a lip1 locus of a genome, and then losing a hisG label and a Ura screening label under the 5-fluoroorotic acid screening pressure to obtain a recombinant bacterium 4.
The specific method comprises the following steps:
first, recombinant bacterium 3 was cultured overnight in YPD liquid medium (containing 2% peptone, 1% Yeast extract, 2% glucose) to prepare competent cells, and recombinant plasmid pUC-HUH-lip1-PgSCD was transformed into recombinant bacterium 3 using Zymogen FROzen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination, and positive clones were selected using screening 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.
Secondly, coating positive clones which are correctly identified by PCR on YPD plates containing 5-fluoroorotic acid, and taking single colonies to streak on the 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 4.
(V) construction of recombinant bacterium 5
The recombinant plasmid pUC-HUH-SCP2-MaELO2-YLELO1 is introduced into the recombinant bacterium 4, an expression cassette of MaELO2 and YLELO1 is integrated to a genome SCP2 site, and then a hisG tag and a Ura screening marker are lost under the 5-fluoroorotic acid screening pressure, so that the recombinant bacterium 5 is obtained.
The specific method comprises the following steps:
recombinant bacterium 4 was cultured overnight in YPD liquid medium (containing 2% peptone, 1% Yeast extract, 2% glucose) to prepare competent cells, 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.
Secondly, coating positive clones which are correctly identified by PCR on YPD plates containing 5-fluoroorotic acid, and taking single colonies to streak on the 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 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) Po1f Δ ku70, 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
Weighing about 1.5g of dry thallus, adding 10ml of 4M hydrochloric acid into the thallus, and reacting for 20 min; transferring to boiling water bath and heating for 10 min; freezing at-80 deg.C for 15 min; adding 10mL of chloroform and 5mL of methanol, and shaking at 200rpm for 30 min; taking the lower lipid-soluble layer, putting the lower lipid-soluble layer into a 10ml centrifugal tube, and drying the lower lipid-soluble layer in a fume hood by using nitrogen; vacuum drying for 2h to obtain the grease. 0.1g of oil is weighed in a 2ml centrifuge tube, 1ml of n-hexane and 0.1ml of potassium hydroxide/methanol solution are added, and the mixture is shaken quickly for 1min to be mixed completely. The reaction solution was left standing at room temperature for 15min to perform methyl esterification. And (4) after standing, setting the centrifugation condition to be 5000rpm for 5min, taking 200 mu l of supernatant, placing the supernatant into a 2ml centrifuge 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: 50:1, chromatographic 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 fatty acid mix standard 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, 25.85g of oleic acid is produced per liter of fermentation liquor, which is obviously higher than that of the initial bacteria and the recombinant bacteria 2.
TABLE 3 oleic acid yields of initial, recombinant 2 and recombinant 5 bacteria
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
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<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 (10)
1. A recombinant yarrowia lipolytica strain 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.
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 promoter of each expression cassette is the promoter P of yarrowia lipolyticaTEF、Php4d、PTEFin、PYAT1、PFBA、PFBAin、PPOX2、PPOT1Or PGPDAny one of the above; the terminator is terminator T of yarrowia lipolyticaxpr2t、Tmig1t、Tlip2t、Tcyc1t、Tpex3t、Tpex10tOr Tpex20tAny one of them.
5. The recombinant yarrowia lipolytica for high oleic acid production of claim 4, wherein said integration site of said 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.
6. The recombinant yarrowia lipolytica for high oleic acid production of claim 5, wherein said stearoyl-CoA desaturase encoding gene sequence is set forth in SEQ ID number 1; the sequence of the fatty acid elongase 2 coding gene is shown in SEQ ID No. 2.
7. The method of 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 said yarrowia lipolytica in the form of a plasmid, followed by integration by homologous recombination into the genome of said yarrowia lipolytica.
8. The method of claim 7, wherein said yarrowia lipolytica knocks out the ku70 gene.
9. Use of the recombinant bacterium of any one of claims 1 to 7 for producing oleic acid, comprising the step of culturing the recombinant bacterium of any one of claims 1 to 6 in a fermentation medium to obtain a fermentation product.
10. The use of the recombinant bacterium of claim 9 in the production of oleic acid, wherein the fermentation medium comprises 50-70g/L glucose, 1.6-1.8g/L nitrogen source without amino yeast, and 1.2-1.4g/L ammonium sulfate.
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CN114606150A (en) * | 2022-04-18 | 2022-06-10 | 南京师范大学 | Genetic engineering strain for producing gamma-linolenic acid, construction method and application thereof |
CN114891653A (en) * | 2022-05-17 | 2022-08-12 | 南京工业大学 | Recombinant yarrowia lipolytica strain capable of producing nervonic acid at high yield as well as construction method and application thereof |
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CN106544284A (en) * | 2016-11-01 | 2017-03-29 | 临沂大学 | A kind of restructuring Yarrowia lipolytica engineered strain and its construction method and application |
CN109913380A (en) * | 2019-03-25 | 2019-06-21 | 南京工业大学 | Produce (-)-α-bisabolol recombination Yarrowia lipolytica and its construction method and application |
CN110846239A (en) * | 2019-11-29 | 2020-02-28 | 南京工业大学 | Recombinant yarrowia lipolytica with high homologous recombination efficiency as well as construction method and application thereof |
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CN106544284A (en) * | 2016-11-01 | 2017-03-29 | 临沂大学 | A kind of restructuring Yarrowia lipolytica engineered strain and its construction method and application |
CN109913380A (en) * | 2019-03-25 | 2019-06-21 | 南京工业大学 | Produce (-)-α-bisabolol recombination Yarrowia lipolytica and its construction method and application |
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CN114606150B (en) * | 2022-04-18 | 2023-10-27 | 南京师范大学 | Gene engineering strain for producing gamma-linolenic acid, construction method and application thereof |
CN114891653A (en) * | 2022-05-17 | 2022-08-12 | 南京工业大学 | Recombinant yarrowia lipolytica strain capable of producing nervonic acid at high yield as well as construction method and application thereof |
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