Recombinant yarrowia lipolytica with high yield of palmitoleic acid, construction method and application thereof

Abstract

The application provides a recombinant Yarrowia lipolytica capable of producing palmitoleic acid at a high yield, a construction method and application thereof, and belongs to the technical field of bioengineering. Yarrowia lipolytica The recombinant Yarrowia lipolytica is obtained by knocking out peroxisome biogenesis factor 10 and triacylglycerol lipase 4 in the genome of Yarrowia lipolytica and inserting an acetyl-CoA carboxylase, a Δ9 desaturase, an acetyl-CoA diacylglycerol acyltransferase, a glycerol-3-phosphate acyltransferase, a lysophosphatidylcholine acyltransferase and a phosphatidylcholine:diacylglycerol acyltransferase gene expression cassette. Experiment proves that the recombinant Yarrowia lipolytica can efficiently ferment and produce palmitoleic acid, and realizes efficient synthesis of the natural product palmitoleic acid from plants in Yarrowia lipolytica.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a recombinant lipophilic yeast strain that produces high levels of palmitoleic acid, its construction method, and its application. Background Technology

[0002] Palmitoleic acid (C16:1Δ9) is a 16-carbon monounsaturated fatty acid, classified as an omega-7 fatty acid based on the position of its double bonds. As a unique fatty acid, palmitoleic acid has multiple functional applications. Since the 20th century, the increase in chronic diseases (such as obesity and cardiovascular disease) has posed a significant threat to human health. Many studies have linked these diseases to unhealthy diets, primarily referring to excessive intake of macronutrients such as sugar, protein, and fat. Recent studies have demonstrated that palmitoleic acid is a key indicator of fatty acids in the human body and may be closely linked to the development of various diseases, including obesity, cardiovascular disease, and fatty liver. In the pharmaceutical field, it is also widely used as an antithrombotic drug to prevent stroke. Due to its ability to inhibit melanin production, it is also commonly used in skin cleansers and has the potential to be an effective skin whitening agent.

[0003] Currently, palmitoleic acid synthesis mainly relies on plant synthesis and microalgae production. However, plant-based palmitoleic acid production has many disadvantages. First, these plants require specific climatic conditions, and the high cost of land and long growth cycle introduce uncontrollable factors such as climate change and human intervention. Microalgae production also faces many challenges. Gene manipulation of microalgae is difficult, metabolic modification has limited potential, and the extraction and cost of microalgae oils are obstacles to industrial-scale production. Compared to plant extraction, microbial fermentation has a shorter growth cycle, can operate around the clock, and the oil-producing microorganisms have high oil content and large biomass, making it an economical and efficient method for palmitoleic acid production. However, current technologies lack microorganisms capable of efficiently producing palmitoleic acid. Summary of the Invention

[0004] The purpose of this invention is to provide a recombinant lipophilic yeast strain capable of efficiently producing palmitic acid.

[0005] Another objective of this invention is to provide a method for constructing the recombinant Yersinia lipophila, which is efficient and simple to operate.

[0006] Another object of the present invention is to provide the application of the recombinant Yersinia lipolytica in the production of palmitic acid.

[0007] The recombinant lipophilic yeast strain that produces high levels of palmitoleic acid is derived from *Yarrowia lipophilia* (…). Yarrowia lipolyticaThe genome was obtained by knocking out peroxisome biogenesis factor 10 and triacylglycerol lipase 4, and inserting acetyl-CoA carboxylase, Δ9 desaturase, acetyl-CoA diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, lysophosphatidylcholine acyltransferase, and phosphatidylcholine:diacylglycerol acyltransferase gene expression cassettes.

[0008] In this invention, the acetyl-CoA carboxylase gene is derived from Yersinia lipolytica (Yersinia lipolytica). Yarrowia lipolytica ); Δ9 desaturase is obtained by codon optimization of a gene from Caenorhabditis elegans; glycerol-3-phosphoacyltransferase, lysophosphatidylcholine acyltransferase, acetyl-CoA diacylglycerol acyltransferase, phosphatidylcholine:diacylglycerol acyltransferase are obtained by processing sea buckthorn fruit ( Sea buckthorn The gene was obtained by codon optimization.

[0009] In this invention, the gene sequences of the Δ9 desaturase, glycerol-3-phosphoacyltransferase, lysophosphatidylcholine acyltransferase, acetyl-CoA diacylglycerol acyltransferase, and phosphatidylcholine:diacylglycerol acyltransferase are shown in SEQ ID No. 1-5, respectively.

[0010] In this invention, the gene expression cassettes of Δ9 desaturase, acetyl-CoA carboxylase, acetyl-CoA diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, lysophosphatidylcholine acyltransferase, and phosphatidylcholine:diacylglycerol acyltransferase are expressed in *Yersinia lipolytica* (Yersinia lipolytica). Yarrowia lipolytica The sites integrated into the genome are, in order: A08 site, IntA, DGAT2, GPAT, LPAAT, and PDAT.

[0011] In this invention, the promoter of the expression cassette is any one of the TEF promoter, hp4d promoter, TEFin promoter, POX2 promoter or GPDin promoter of Yersinia lipophila; the terminator is any one of the xpr2t terminator, mig1t terminator or lip2t terminator of Yersinia lipophila.

[0012] In this invention, P in the recombinant Yersinia lipophila genome ELO1 promoter sites and / or P SCD An endogenous copper ion repressor promoter P was also inserted at the promoter site. CTR1 .

[0013] In this invention, each gene expression cassette is inserted into the genome using homologous recombination, and the gene to be knocked out is knocked out using homologous recombination.

[0014] The present invention also provides the application of the recombinant Yersinia lipolytica in the preparation of palmitoleic acid.

[0015] In this invention, the recombinant Yeast lipolyticis is fermented in the following culture medium to prepare palmitoleic acid: glucose 130-170 g / L, ammonium sulfate 10-12 g / L, yeast extract 2.5-3.5 g / L, corn peptone 0.085-0.105 g / L, potassium dihydrogen phosphate 3.5-4.5 g / L, magnesium sulfate 1.8-2.2 g / L, calcium sulfate 0.78-0.82 g / L, sodium chloride 0.35-0.45 g / L, ammonium sulfate hydrochloride 10-14 mg / L, biotin 0.85-1.15 mg / L, sodium molybdate 140-180 mg / L, copper sulfate 0.18-0.22 mg / L, boric acid 38-42 mg / L, manganese sulfate 160-200 mg / L, and ferrous chloride 70-80 mg / L.

[0016] In this invention, glucose is added during the fermentation process.

[0017] Beneficial effects: The recombinant lipophilic yeast of this invention ( Yarrowia lipolytica This invention relates to *Yersinia lipolytica*, which is based on the knockout of the gene *ku70*, which encodes non-homologous recombination, thereby enhancing its homologous recombination ability. The gene is integrated through the homologous recombination function of *Yersinia lipolytica* itself, significantly improving the genetic stability of the introduced gene. This recombinant *Yersinia lipolytica* construction method is efficient and simple to operate. This invention relates to recombinant *Yersinia lipolytica* (…). Yarrowia lipolytica Peroxisome biogenesis factor 10 (PEX10 site) and triacylglycerol lipase 4 were knocked out, and Caenorhabditis elegans Δ9 desaturase, acetyl-CoA carboxylase, heterologous acetyl-CoA diacylglycerol acyltransferase, heterologous glycerol-3-phosphate acyltransferase, heterologous lysophosphatidylcholine acyltransferase, and heterologous phosphatidylcholine: diacylglycerol acyltransferase were overexpressed. The experiment proved that the recombinant Yersinia lipolytica can efficiently ferment and produce palmitoleic acid, realizing the efficient synthesis of palmitoleic acid, a natural product of plant origin, in Yersinia lipolytica. Attached Figure Description

[0018] Figure 1This diagram illustrates the palmitoleic acid synthesis strategy provided in this embodiment of the invention. ACC1: Acetyl-CoA carboxylase; DGAT2: Diacylglycerol acyltransferase; PDAT: Phosphatidylcholine: Diacylglycerol acyltransferase; ELO1: Fatty acid elongation enzyme 1; ELO2: Fatty acid elongation enzyme 2; FAD2: Δ12 oleic acid desaturase; FAS: Fatty acid elongation enzyme; TGL4: Triacylglycerol lipase 4; GPAT: 3-phosphoglycerol acyltransferase; LPAAT: Lysophosphatidyl acyltransferase; LPCAT: Lysophosphatidylcholine acyltransferase; CPT: Choline phosphotransferase.

[0019] Figure 2 This is the structural diagram of the recombinant plasmid pUC-Leu-A08-FAT5, where A08-up represents the upstream homologous arm A08-up of the start codon at site A08, A08-dm represents the downstream homologous arm A08-dm of the stop codon at site A08, and TEFin represents the promoter P. TEFin xpr2t indicates the termination sub-T xpr2t LEU represents the gene expression cassette encoding 3(β)-isopropylmalate dehydrogenase, and FAT5 is a Δ9 desaturase derived from Caenorhabditis elegans.

[0020] Figure 3 It is the recombinant plasmid pUC-HUH-P ELO1 -P CTR1 The structural diagram, where P ELO1 -up indicates P ELO1 upstream homologous arm of the site, P ELO1 -dm indicates P ELO1 Downstream homologous arm, URA, represents the expression cassette containing the orotate nucleoside-5'-phosphate decarboxylase gene encoding gene (containing... Yarrowia lipolytica Endogenous promoter P TEFin orotate nucleoside-5'-phosphate decarboxylase and terminator T xpr2t CTR1 is P CTR1 Gene (Yarrowia lipolyticis endogenous copper ion repressor promoter).

[0021] Figure 4 This is a structural diagram of the recombinant plasmid pUC-HUH-GPAT-SbGPAT, where GPAT-up represents the upstream homologous arm of the start codon at the GPAT site, GPAT-dm represents the downstream homologous arm of the stop codon at the SbGPAT site, and GPDin represents the promoter P. GPDin mig1t indicates the termination sub-T mig1t URA indicates that orotate nucleoside-5'-phosphate decarboxylase-encoding gene expression cassette (containing...) Yarrowia lipolytica Endogenous promoter P TEFin orotic nucleoside-5'-phosphate decarboxylase and terminator Txpr2t SbGPAT is a glycerol-3-phosphoacyltransferase from sea buckthorn fruit.

[0022] Figure 5 This is a structural diagram of the recombinant plasmid pUC-HUH-TGL4, where TGL4-up represents the upstream homologous arm of the start codon at the TGL4 site, TGL4-down represents the downstream homologous arm of the stop codon at the TGL4 site, and URA represents the expression cassette encoding the orotate nucleoside-5'-phosphate decarboxylase gene (containing...). Yarrowia lipolytica Endogenous promoter P TEFin orotic nucleoside-5'-phosphate decarboxylase and terminator T xpr2t ).

[0023] Figure 6 This is a fatty acid composition analysis diagram from Example 3 of the present invention. Detailed Implementation

[0024] The present invention will be further described below through specific embodiments.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Yarrowia lipolytica ( Yarrowia lipolytica Po1f, purchased from the American Culture Collection, catalog number [number missing].

[0028] ATCC MYA-2613.

[0029] Yarrowia lipolytica ( Yarrowia lipolytica )Po1f Δku70(MatA, Δku70::hisG,leu2-270, Ura3-302, xpr2-322, axp1-2), abbreviated as Yarrowia lipolytica Po1f Δku70. Yarrowia lipolytica Po1f Δku70 is produced by Yersinia lipophila ( Yarrowia lipolytica The Po1f gene was constructed by knocking out the ku70 gene responsible for non-homologous recombination (published in Kretzschmar A, et al., CurrentGenetics, 2013, 59(1-2): 63-72).

[0030] P SCD Site integration plasmids are plasmids that integrate P on chromosome C of the Yarrowia lipolytica Po1f Δku70 genome. SCDA 2059 bp sequence (upstream homologous arm) upstream of the start codon of the promoter at the site (GenBank accession number: XP_501496) is inserted at the EcoRI restriction site of the pUC57-hisG-Ura-hisG vector (construction method see Example 1), thereby inserting the P sequence on chromosome C of the Yarrowia lipolytica Po1f Δku70 genome into the target region. SCD The sequence (downstream homologous arm) of 2019 bp downstream of the stop codon of the promoter site is inserted into the Hind III restriction site of the pUC57-hisG-Ura-hisG vector. The two hisG tag encoding genes are located between the upstream and downstream homologous arms of the promoter site.

[0031] P ELO1 Site integration plasmids are plasmids that integrate P chromosome F from the genome of Yarrowia lipolytica Po1f Δku70. ELO1 A 1501 bp sequence (upstream homologous arm) upstream of the start codon in the pUC57-hisG-Ura-hisG vector was inserted at the EcoRI restriction site, thus inserting the P sequence on chromosome F of the Yarrowia lipolytica Po1f Δku70 genome into the pUC57-hisG-Ura-hisG vector. ELO1 The sequence (downstream homologous arm) of 1501 bp downstream of the stop codon is inserted into the Hind III restriction site of the pUC57-hisG-Ura-hisG vector. The two hisG tag encoding genes are located between the upstream and downstream homologous arms at the promoter site.

[0032] The GPAT site integration plasmid was obtained by inserting a 1500 bp sequence upstream of the start codon of the GPAT site on chromosome C of the Yarrowia lipolytica Po1f Δku70 genome (GenBank accession number: XM_501275.1) (upstream homologous arm GPAT-up) into the Hind III restriction site of the pUC57-hisG-Ura-hisG vector, and a 1500 bp sequence downstream of the stop codon of the GPAT site on chromosome C of the Yarrowia lipolytica Po1f Δku70 genome (downstream homologous arm GPAT-dm) into the EcoRI restriction site of the pUC57-hisG-Ura-hisG vector. The two hisG tag encoding genes are located between the upstream and downstream homologous arms at the site.

[0033] The LPAAT site integration plasmid is created by inserting a 1500 bp sequence upstream of the start codon (upstream homologous arm LPAAT-up) of the LPAAT site on chromosome E of the Yarrowia lipolytica Po1f Δku70 genome (GenBank accession number: XM_504127.1) into the KpnⅠ restriction site of the pUC57-hisG-Ura-hisG vector, and a 1500 bp sequence downstream of the stop codon (downstream homologous arm LPAAT-dm) of the LPAAT site on chromosome E of the Yarrowia lipolytica Po1f Δku70 genome into the HindⅢ restriction site of the pUC57-hisG-Ura-hisG vector. The two hisG tag encoding genes are located between the upstream and downstream homologous arms at the site.

[0034] The PDAT site integration plasmid was obtained by inserting a 1666 bp sequence (upstream homologous arm) upstream of the start codon of the PDAT site on chromosome E of the Yarrowia lipolytica Po1f Δku70 genome (GenBank accession number: XM_504038.1) into the EcoRI restriction site of the pUC57-hisG-Ura-hisG vector, and a 1666 bp sequence (downstream homologous arm) downstream of the stop codon of the PDAT site on chromosome E of the Yarrowia lipolytica Po1f Δku70 genome into the PacRI restriction site of the pUC57-hisG-Ura-hisG vector. The two hisG tag encoding genes are located between the upstream and downstream homologous arms at the site.

[0035] The DGAT2 site integration plasmid was obtained by inserting a 1500 bp sequence (upstream homologous arm) upstream of the start codon of the DGAT2 site on chromosome E of the Yarrowia lipolytica Po1f Δku70 genome (GenBank accession number: XM_504700.1) into the KpnⅠ restriction site of the pUC57-hisG-Ura-hisG vector, and a 1486 bp sequence (downstream homologous arm) downstream of the stop codon of the DGAT2 site on chromosome E of the Yarrowia lipolytica Po1f Δku70 genome into the EcoRI restriction site of the pUC57-hisG-Ura-hisG vector. The two hisG tag encoding genes are located between the upstream and downstream homologous arms at the site.

[0036] The PEX10 site integration plasmid is used to... Yarrowia lipolyticaA 2250 bp sequence (upstream homologous arm) upstream of the start codon at the PEX10 site on chromosome C (GenBank accession number: XM_501311.1) in the Po1f Δku70 genome was inserted into the Hind III restriction site of the pUC57-hisG-Ura-hisG vector. Yarrowia lipolytica The Po1f Δku70 genome was obtained by inserting a 1500bp sequence downstream of the stop codon at the PEX10 site on chromosome C (downstream homologous arm) into the EcoRI restriction site of the pUC57-hisG-Ura-hisG vector. The two hisG tag encoding genes are located between the upstream and downstream homologous arms at the PEX10 site.

[0037] The TGL4 site integration plasmid is used to... Yarrowia lipolytica A 1500 bp sequence (upstream homologous arm) upstream of the start codon at the TGL4 site on chromosome F (GenBank accession number: XM_505230.1) in the Po1f Δku70 genome was inserted into the PacⅠ restriction site of the pUC57-hisG-Ura-hisG vector. Yarrowia lipolytica A 1500bp sequence downstream of the stop codon at the TGL4 site on chromosome F in the Po1fΔku70 genome (downstream homologous arm) is inserted into the EcoRI restriction site of the pUC57-hisG-Ura-hisG vector, resulting in two hisG tag encoding genes located between the upstream and downstream homologous arms at the TGL4 site.

[0038] IntA site integration plasmid is used to... Yarrowia lipolytica A 1633 bp sequence (upstream homologous arm) upstream of the start codon at the IntA site (GenBank accession number: CR382127.1) on chromosome A in the Po1f Δku70 genome was inserted into the EcoRI restriction site of the pUC57-hisG-Ura-hisG vector. Yarrowia lipolytica The Po1fΔku70 genome was obtained by inserting a 1621 bp sequence (downstream homologous arm) downstream of the stop codon at the IntA site on chromosome A into the pUC57-hisG-Ura-hisG vector at the Hind III restriction site. The two hisG tag encoding genes are located between the upstream and downstream homologous arms of the IntA site.

[0039] The A08 site integration plasmid is used to... Yarrowia lipolytica A 2521 bp sequence (upstream homologous arm) upstream of the start codon at the A08 site (GenBank accession number: CP096871.1) on the chromosome of the Po1f Δku70 genome was inserted into the EcoRI restriction site of the pUC57-Leu vector (construction method see Example 1).Yarrowia lipolytica The Leu gene was obtained by inserting a 2031 bp sequence downstream of the stop codon at the A08 site on the chromosome of the Po1f Δku70 genome (downstream homologous arm) into the Hind III restriction site of the pUC57-Leu vector (construction method is described in Example 1). The Leu gene is located between the upstream and downstream homologous arms at the A08 site.

[0040] Example 1: Amplification of Gene Elements and Preparation of Target Plasmids

[0041] (I) Preparation of target gene

[0042] According to information from *C. elegans* provided by NCBI (… Caenorhabditis elegans Δ9 desaturase ( FAT5 The nucleotide sequence of the gene (GenBank No: AAF97548.1) was optimized by codons, and the optimized Δ9 desaturase gene was synthesized by GenScript Biotech Co., Ltd. FAT5 (SEQ ID No: 1), and inserted between the HindⅠ and EcoRI restriction sites of plasmid pUC57 (purchased from GenScript), to obtain plasmid pUC57-FAT5.

[0043] According to the literature, it comes from sea buckthorn fruit ( Sea buckthorn The nucleotide sequence of the glycerol-3-phosphoacyltransferase gene (Ding J, et al. BMC plant biology, 2019, 19(1): 1-18) was obtained. After codon optimization, the optimized glycerol-3-phosphoacyltransferase gene SbGPAT (SEQ ID No: 2) was synthesized by GenScript Biotech Co., Ltd. and inserted between the HindⅠ and EcoRI restriction sites of plasmid pUC57 to obtain plasmid pUC57-SbGPAT.

[0044] According to the literature, it comes from sea buckthorn fruit ( Sea buckthorn The nucleotide sequence of the lysophosphatidylcholine acyltransferase gene (Ding J, et al. BMC plant biology, 2019, 19(1): 1-18) was obtained. After codon optimization, the optimized lysophosphatidylcholine acyltransferase encoding gene SbLPAAT (SEQ ID No: 3) was synthesized by GenScript Biotech Co., Ltd. and inserted between the HindⅠ and EcoRI restriction sites of plasmid pUC57 to obtain plasmid pUC57-SbLPAAT.

[0045] According to the literature, it comes from sea buckthorn fruit ( Sea buckthornThe nucleotide sequence of acetyl-CoA diacylglycerol acyltransferase (Ding J, et al. BMC plant biology, 2019, 19(1): 1-18) was obtained. After codon optimization, the optimized acetyl-CoA diacylglycerol acyltransferase encoding gene SbDGAT2 (SEQ ID No: 4) was synthesized by GenScript Biotech Co., Ltd. and inserted between the HindIII and PacI restriction sites of plasmid pUC57 to obtain plasmid pUC57-SbDGAT2.

[0046] According to the literature, it comes from sea buckthorn fruit ( Sea buckthorn The nucleotide sequence of phosphatidylcholine: diacylglycerol acyltransferase (Ding J, et al. BMC plant biology, 2019, 19(1): 1-18) was obtained. After codon optimization, the optimized phosphatidylcholine: diacylglycerol acyltransferase encoding gene SbPDAT (SEQ ID No: 5) was synthesized by GenScript Biotech Co., Ltd. and inserted between the Hind III and Pac I restriction sites of plasmid pUC57 to obtain plasmid pUC57-SbPDAT.

[0047] According to information provided by NCBI Yarrowia lipolytica The nucleotide sequence of the orotate nucleoside-5'-phosphate decarboxylase encoding gene Ura (GenBank accession number: AJ306421.1) and the hisG tag (GenBank accession number: AF324729.1) were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. The two hisG tag encoding gene sequences were inserted between the Hind III and Pac I restriction sites of plasmid pUC57 (from Genewiz). An orotate nucleoside-5'-phosphate decarboxylase encoding gene expression cassette (made by...) was inserted between the two hisG tag encoding gene sequences. Yarrowia lipolytica Endogenous promoter P TEFin The Ura gene encoding orotic nucleoside-5'-phosphate decarboxylase and the terminator T xpr2t Composition, abbreviated as P TEFin -URA-T xpr2t In order to achieve Ura tag recovery, plasmid pUC57-hisG-Ura-hisG is obtained.

[0048] According to information provided by NCBI Yarrowia lipolytica The nucleotide sequence of the 3(β)-isopropylmalate dehydrogenase encoding gene Leu (GenBank accession number: CP061014.1) was synthesized by Suzhou Genewise Biotech Co., Ltd. A cassette for the 3(β)-isopropylmalate dehydrogenase encoding gene (created by [unclear]) was inserted between the KpnⅠ and PacⅠ restriction sites on plasmid pUC57.Yarrowia lipolytica Endogenous promoter P TEFin The gene encoding 3(β)-isopropylmalate dehydrogenase, Leu, and the terminator T xpr2t Composition), resulting in plasmid pUC57-Leu.

[0049] by Yarrowia lipolytica Po1f Using Δku70 genomic DNA as a template, the ACC1 gene encoding acetyl-CoA carboxylase was amplified using IntA::ACC1-F and IntA::ACC1-R primers (GenBank accession number: YALI0C11407g).

[0050] by Yarrowia lipolytica Po1f Using Δku70 genomic DNA as a template, with P ELO1 :: P CTR1 -F and P ELO1 :: P CTR1 -R is a primer used to amplify the copper ion repressed promoter CTR1 (GenBank accession number: YALI0B10153g).

[0051] Yarrowia lipolytica Endogenous promoter P hp4d P TEFin P GPDin and endogenous terminator T xpr2t The nucleotide sequence can be found in the literature "Wang, Kaifeng, et al." ACS Synthetic Biology 11.4 (2022): 1542-1554.

[0052] Construction of recombinant plasmids

[0053] The structures of the recombinant plasmids are shown in Table 1 and Figures 2 - 5 The primers used to construct the recombinant plasmid are shown in Table 2.

[0054] 1. Construction of recombinant plasmid pUC-leu-A08-FAT5

[0055] The recombinant plasmid pUC-leu-A08-FAT5 uses pUC57-Leu as its backbone and inserts... Yarrowia lipolytica Po1f In Δku70, the upstream homologous arm A08-up of the start codon and the downstream homologous arm A08-dm of the stop codon are located at site A08. A FAT5 expression cassette (P) is also inserted between these upstream and downstream homologous arms. TEFin -FAT5-T xpr2t ), LEU expression box (including Yarrowia lipolytica Endogenous promoter P TEFinThe gene encoding 3(β)-isopropylmalate dehydrogenase, Leu, and the terminator T xpr2t It is also located between the upstream and downstream homologous arms; see the specific structure below. Figure 2 FAT5 is a Δ9 desaturase derived from *C. elegans*. In this invention, the expression cassette is represented as promoter-target gene-terminator, for example, expression cassette P. TEFin -FAT5-T xpr2t P TEFin It is the promoter, FAT5 is the target gene, and T... xpr2t It is a terminator.

[0056] With A08::P TEFin -F and A08::P TEFin -R is the primer, using Yarrowia lipolytica Po1f Δku70 genomic DNA as a template to amplify the FAT5 expression cassette promoter P. TEFin With A08::T xpr2t -F and A08::T xpr2t -R is the primer, using Yarrowia lipolytica Po1f Δku70 genomic DNA as a template, to amplify the FAT5 expression cassette terminator T. xpr2t .

[0057] Using plasmid pUC57-FAT5 as a template and primers A08::FAT5-F and A08::FAT5-R as primers, amplification was performed using promoters P at both ends. TEFin and Termination T xpr2t The FAT5 gene of the homologous arm.

[0058] The PCR amplification system described above is as follows:

[0059] Component Volume PrimerSTAR Max Premix 25µl Template 1µl Primer 1 2µl Primer 2 2µl Distilled water 20µl

[0060] The PrimerSTAR Max Premix was purchased from Baori Biotechnology (Beijing) Co., Ltd.

[0061] The PCR procedure described above is as follows: denaturation at 98℃ for 10 seconds, annealing at 55℃ for 5 seconds, extension at 72℃ (extension time = target fragment length / 1kb, unit: min), repeated for 30 cycles.

[0062] The fragments were purified and recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit (purchased from Shanghai Bioscientific and Technological Co., Ltd.).

[0063] The A08 site integration plasmid was processed using a restriction endonuclease from NEB. SnaBI. After enzyme digestion, the linearized A08 site integrated plasmid was recovered by agarose gel electrophoresis.

[0064] The linearized A08 site integration plasmid and the various elements (promoter P) in the FAT5 gene expression cassette constructed in Title 1 of this embodiment were integrated. TEFin FAT5 gene and terminator T xpr2t One-step cloning was achieved using the ClonExpressMultiS One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd., inserting the FAT5 gene expression cassette between the upstream and downstream homologous arms of the integration plasmid at the A08 site. The reaction system is shown in the table below. After incubating the reaction system at 37°C for 30 min, a circular recombinant vector was obtained.

[0065] The one-step cloning system is shown in the table below:

[0066] Component Volume 5×CE MultiS Buffer 4 µl Exnase MultiS 2 µl Linearized vector x ng Insert fragment y ng Distilled water Make up the volume to 10µl

[0067] The amount of linearized vector (x) and insert fragments (y = the sum of the optimal amounts of all insert fragments) used can be calculated by the following formula: Optimal amount of each fragment or linearized vector = [0.02 × number of base pairs of the fragment or linearized vector] ng.

[0068] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid pUC-leu-A08-FAT5 was obtained by ampicillin-resistant plate screening and colony PCR and sequencing verification.

[0069] Using restriction endonucleases from NEB Not I. After digesting the plasmid pUC-leu-A08-FAT5 with enzymes, the linearized pUC-leu-A08-FAT5 plasmid was recovered by agarose gel electrophoresis.

[0070] 2. Recombinant plasmid pUC-HUH-P ELO1 -P CTR1 Construction

[0071] Recombinant plasmid pUC-HUH-P ELO1 -P CTR1 It uses pUC57-hisG-Ura-hisG as the skeleton and inserts Yarrowia lipolytica Po1fΔku70 in P ELO1 upstream homology arm P of the site ELO1 -up and downstream homologous arm P ELO1 -dm, and P was also inserted between this downstream homologous arm and the hisG tag encoding gene. CTR1The gene, along with the expression cassettes of two hisG tag-encoding gene sequences and the orotate nucleoside-5'-phosphate decarboxylase-encoding gene, is also located on the upstream homology arm P. ELO1 -up and downstream homologous arm P ELO1 Between -dm, see the specific structure. Figure 3 .

[0072] by Yarrowia lipolytica Po1f Using Δku70 genomic DNA as a template, with P ELO1 :: P CTR1 -F and P ELO1 :: P CTR1 -R is a primer, with P at both ends of the amplification. ELO1 -dm and hisG tags encode the P gene homologous arm. CTR1 Gene.

[0073] P ELO1 Site-integrating plasmids were developed using restriction endonucleases from NEB. HindⅢ After enzyme digestion, the linearized pUC-HUH integrative plasmid was recovered by agarose gel electrophoresis.

[0074] Linearize P ELO1 The site-integrating plasmid and the plasmid constructed in Title 2 of this embodiment each have P at both ends. ELO1 -dm and hisG tags encode the P gene homologous arm. CTR1 Gene cloning was achieved in one step using the ClonExpress OneStep Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd., which cloned P... CTR1 Gene insertion into P ELO1 By integrating the upstream and downstream homologous arms of the site into the plasmid, a circular recombinant vector is obtained.

[0075] The circular recombinant vector was transformed into E. coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-P was obtained by ampicillin-resistant plate selection and colony PCR and sequencing verification. ELO1 -P CTR1 .

[0076] Using restriction endonucleases from NEB BstAPI For plasmid pUC-HUH-P ELO1 -P CTR1 After enzyme digestion, linearized pUC-HUH-P was recovered by agarose gel electrophoresis. ELO1 -P CTR1 Plasmid.

[0077] 3. Recombinant plasmid pUC-HUH-P SCD -P CTR1 Construction

[0078] Recombinant plasmid pUC-HUH-P SCD -P CTR1 It uses pUC57-hisG-Ura-hisG as the skeleton and inserts Yarrowia lipolytica Po1fΔku70 in P SCD upstream homology arm P of the site SCD -up and downstream homologous arm P SCD -dm, inserting P between the downstream homologous arm and the hisG tag coding gene sequence. CTR1 The gene, the two hisG tag encoding gene sequences, and the orotate nucleoside-5'-phosphate decarboxylase encoding gene expression cassette are also located between the upstream and downstream homologous arms. The recombinant plasmid pUC-HUH-P SCD -P CTR1 The structural diagram is the same as Figure 3 The only difference is that P SCD Site homology arm substitution P ELO1 Homologous arm of the site.

[0079] by Yarrowia lipolytica Po1f Using Δku70 genomic DNA as a template, with P SCD :: P CTR1 -F and P SCD :: P CTR1 -R is a primer, with P at both ends of the amplification. ELO1 -dm and hisG tags encode the P gene homologous arm. CTR1 Gene.

[0080] P SCD Site integration plasmids were prepared using restriction endonucleases from NEB. HindⅢ After enzyme digestion, linearized P was recovered by agarose gel electrophoresis. SCD Site-integrated plasmid.

[0081] linearized P SCD Site integration plasmid and P-type plasmid with homologous arms at both ends constructed in Title 3 of this embodiment. CTR1 Gene cloning was achieved in one step using the ClonExpress One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd., which cloned P... CTR1 Gene insertion into P SCD A circular recombinant vector was obtained by integrating the downstream homologous arm of the site-integrated plasmid with the hisG tag-encoding gene.

[0082] The circular recombinant vector was transformed into E. coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-P was obtained by ampicillin-resistant plate selection and colony PCR and sequencing verification. SCD -P CTR1 .

[0083] Using restriction endonucleases from NEB BstAPI For plasmid pUC-HUH-P SCD -P CTR1 After enzyme digestion, linearized pUC-HUH-P was recovered by agarose gel electrophoresis. SCD -P CTR1 Plasmid.

[0084] 4. Construction of recombinant plasmid pUC-HUH-GPAT-SbGPAT

[0085] pUC-HUH-GPAT-SbGPAT uses pUC57-hisG-Ura-hisG as its framework and inserts... Yarrowia lipolytica In Po1fΔku70, the GPAT site contains an upstream homologous arm GPAT-up from the start codon and a downstream homologous arm GPAT-dm from the stop codon. An SbGPAT gene expression cassette (P) is also inserted between this downstream homologous arm and the hisG tag coding gene. GPDin -SbGPAT-T mig1t ), URA expression box (including Yarrowia lipolytica Endogenous promoter P TEFin orotic nucleoside-5'-phosphate decarboxylase and terminator T xpr2t It also lies between the upstream and downstream homologous arms. The structural diagram of the recombinant plasmid pUC-HUH-GPAT-SbGPAT is shown below. Figure 4 .

[0086] With GPAT::P GPDin -F and GPAT::P GPDin -R is a primer, with Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the SbGPAT expression cassette promoter P was amplified. GPDin . Using GPAT::T mig1t -F and GPAT::T mig1t -R is a primer, with Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the SbGPAT expression cassette terminator T was amplified. mig1t .

[0087] Using plasmid pUC57-SbGPAT as a template and GPDin::SbGPAT-F and GPDin::SbGPAT-R as primers, amplification was performed using promoters P at both ends. GPDin and Termination T mig1t The SbGPAT gene of the homologous arm.

[0088] Using restriction endonucleases from NEB Pac Ⅰ After enzyme digestion of the GPAT site integration plasmid, the linearized GPAT plasmid was recovered by agarose gel electrophoresis.

[0089] The linearized GPAT site integration plasmid and the elements (P) in the SbGPAT gene expression cassette constructed in Title 4 of this embodiment were integrated. GPDin Gene SbGPAT and terminator T mig1t One-step cloning was achieved using the ClonExpress®II One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd., to obtain a circular recombinant vector.

[0090] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-GPAT-SbGPAT was obtained by ampicillin-resistant plate screening and colony PCR and sequencing verification.

[0091] Using restriction endonucleases from NEB Not I. After digesting the plasmid pUC-HUH-GPAT-SbGPAT with enzymes, the linearized pUC-HUH-GPAT-SbGPAT plasmid was recovered by agarose gel electrophoresis.

[0092] 5. Construction of recombinant plasmid pUC-HUH-LPAAT-SbLPAAT

[0093] The recombinant plasmid pUC-HUH-LPAAT-SbLPAAT uses pUC57-hisG-Ura-hisG as its backbone and inserts... Yarrowia lipolytica In Po1fΔku70, the LPAAT site has an upstream homologous arm LPAAT-up from the start codon and a downstream homologous arm LPAAT-dm from the stop codon. An SbLPAAT gene expression cassette (P) is also inserted between this downstream homologous arm and the hisG tag-encoding gene. TEFin -SbLPAAT-T xpr2t ), orotate nucleoside-5'-phosphate decarboxylase encoding gene expression cassette (containing Yarrowia lipolytica Endogenous promoter P TEFin orotic nucleoside-5'-phosphate decarboxylase and terminator T xpr2tIt is also located between upstream and downstream homologous arms. The structural diagram of the recombinant plasmid pUC-HUH-LPAAT-Sb LPAAT is the same. Figure 4 The only difference is that LPAAT-up and LPAAT-dm replace GPAT-up and GPAT-dm respectively, and SbLPAAT gene expression cassette replaces SbGPAT gene expression cassette.

[0094] LPAAT-up::P TEFin -F and LPAAT-up::P TEFin -R is a primer, with Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the SbLPAAT gene expression cassette promoter P was amplified. TEFin LPAAT-dm::T xpr2t -F and LPAAT-dm::T xpr2t -R is a primer, with Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the SbLPAAT expression cassette terminator T was amplified. xpr2t .

[0095] by Yarrowia lipolytica Po1f Using Δku70 genomic DNA as a template, with P TEFin ::SbLPAAT-F and T xpr2t ::SbLPAAT-R is the primer, with promoter P at both ends for amplification. TEFin and Termination T xpr2t The SbLPAAT gene of the homologous arm.

[0096] Using restriction endonucleases from NEB Pac Ⅰ After digesting the LPAAT site-integrating plasmid with enzymes, the linearized LPAAT plasmid was recovered by agarose gel electrophoresis.

[0097] The linearized LPAAT site integration plasmid and the elements (P) in the SbLPAAT gene expression cassette constructed in Title 5 of this embodiment were integrated. TEFin SbLPAAT gene and terminator T xpr2t One-step cloning was achieved using the ClonExpress® II One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd., to obtain a circular recombinant vector.

[0098] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-LPAAT-SbLPAAT was obtained by ampicillin-resistant plate screening and colony PCR and sequencing verification.

[0099] Using restriction endonucleases from NEB Not I. After digesting the plasmid pUC-HUH-LPAAT-SbLPAAT with enzymes, the linearized pUC-HUH-LPAAT-SbLPAAT plasmid was recovered by agarose gel electrophoresis.

[0100] 6. Construction of recombinant plasmid pUC-HUH-DGAT2-SbDGAT2

[0101] The recombinant plasmid pUC-HUH-DGAT2-SbDGAT2 uses pUC57-hisG-Ura-hisG as its backbone and inserts... Yarrowia lipolytica Po1f In Δku70, the DGAT2 site contains an upstream homologous arm (DGAT2-up) of the start codon and a downstream homologous arm (DGAT2-dm) of the stop codon. An SbDGAT2 gene expression cassette (P) is inserted between this downstream homologous arm and the hisG tag-encoding gene. TEFin -SbDGAT2-T xpr2t ), orotate nucleoside-5'-phosphate decarboxylase encoding gene expression cassette (containing Yarrowia lipolytica Endogenous promoter P TEFin orotic nucleoside-5'-phosphate decarboxylase and terminator T xpr2t It is also located between upstream and downstream homologous arms. The structural diagram of the recombinant plasmid pUC-HUH-DGAT2-SbDGAT2 is the same as that of the upstream and downstream homologous arms. Figure 4 The only difference is that DGAT2-up and DGAT2-dm replace GPAT-up and GPAT-dm respectively, and SbDGAT2 gene expression cassette replaces SbGPAT gene expression cassette.

[0102] With DGAT2::P TEFin -F and DGAT2::P TEFin -R is a primer, with Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the SbDGAT2 gene expression cassette promoter P was amplified. TEFin Using DGAT2::T xpr2t -F and DGAT2::T xpr2t -R is a primer, with Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the SbDGAT2 gene expression cassette terminator T was amplified. xpr2t .

[0103] by Yarrowia lipolytica Po1f Using Δku70 genomic DNA as a template, with P TEFin :: SbDGAT2-F and T xpr2t:: SbDGAT2-R is the primer, with promoter P at both ends for amplification. TEFin and Termination T xpr2t The SbDGAT2 gene of the homologous arm.

[0104] Using restriction endonucleases from NEB Hind III. The DGAT2 site integration plasmid was digested with enzymes, and the linearized DGAT2 site integration plasmid was recovered by agarose gel electrophoresis.

[0105] The linearized DGAT2 site integration plasmid and the various elements (promoter P) in the SbDGAT2 gene expression cassette constructed in Title 6 of this embodiment were integrated. TEFin SbDGAT2 gene and terminator T xpr2t One-step cloning was achieved using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd., to obtain a circular recombinant vector.

[0106] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-DGAT2-SbDGAT2 was obtained by ampicillin-resistant plate screening and colony PCR and sequencing verification.

[0107] Using restriction endonucleases from NEB EcoR I. After digesting the plasmid pUC-HUH-DGAT2-SbDGAT2 with enzymes, the linearized pUC-HUH-DGAT2-SbDGAT2 plasmid was recovered by agarose gel electrophoresis.

[0108] 7. Construction of recombinant plasmid pUC-HUH-IntA-ACC1

[0109] The recombinant plasmid pUC-HUH-IntA-ACC1 uses pUC57-hisG-Ura-hisG as its backbone and inserts... Yarrowia lipolytica In Po1fΔku70, the IntA site contains an upstream homologous arm (IntA-up) of the start codon and a downstream homologous arm (IntA-dm) of the stop codon. An ACC1 gene expression cassette (P) is also inserted between this downstream homologous arm and the hisG tag coding gene. hp4d -ACC1-T mig1t ), orotate nucleoside-5'-phosphate decarboxylase encoding gene expression cassette (containing Yarrowia lipolytica Endogenous promoter P TEFin orotic nucleoside-5'-phosphate decarboxylase and terminator T xpr2t It is also located between upstream and downstream homologous arms. The structural diagram of the recombinant plasmid pUC-HUH-IntA-ACC1 is the same as that of the upstream and downstream homologous arms. Figure 4The only difference is that IntA-up and IntA-dm replace GPAT-up and GPAT-dm respectively, and the ACC1 gene expression cassette replaces the GPAT gene expression cassette.

[0110] IntA::P hp4d -F and IntA::P hp4d -R is a primer, with Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the ACC1 gene expression cassette promoter P was amplified. hp4d . Using IntA::T mig1t -F and IntA::T mig1t -R is a primer, with Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the ACC1 expression cassette terminator T was amplified. mig1t .

[0111] by Yarrowia lipolytica Po1f Using Δku70 genomic DNA as a template and IntA::ACC1-F and IntA::ACC1-R as primers, amplification was performed using promoters P at both ends. hp4d and Termination T mig1t The ACC1 gene of the homologous arm.

[0112] The IntA site integration plasmid was digested with NEB restriction endonuclease PacⅠ, and the linearized IntA site integration plasmid was recovered by agarose gel electrophoresis.

[0113] The linearized IntA site integration plasmid and the various elements (promoter P) in the ACC1 gene expression cassette constructed in Title 7 of this embodiment were integrated. hp4d ACC1 gene and terminator T mig1t Using the ClonExpressMultiS One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd., one-step cloning was achieved by inserting the ACC1 gene expression cassette between the upstream and downstream homologous arms of the IntA site integration plasmid to obtain a circular recombinant vector.

[0114] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid pUC-HUH-IntA-ACC1 was obtained by ampicillin-resistant plate screening and colony PCR and sequencing verification.

[0115] Using restriction endonucleases from NEB EcoR I. After digesting the plasmid pUC-HUH-IntA-ACC1 with enzymes, the linearized pUC-HUH-IntA-ACC1 plasmid was recovered by agarose gel electrophoresis.

[0116] 8. Construction of recombinant plasmid pUC-HUH-PDAT-SbPDAT

[0117] The recombinant plasmid pUC-HUH-PDAT-SbPDAT uses pUC57-hisG-Ura-hisG as its backbone and inserts... Yarrowia lipolytica In Po1fΔku70, the PDAT site contains an upstream homologous arm PDAT-up from the start codon and a downstream homologous arm PDAT-dm from the stop codon. An SbPDAT gene expression cassette (P) is also inserted between this downstream homologous arm and the hisG tag-encoding gene. TEFin -SbPDAT-T xpr2t ), orotate nucleoside-5'-phosphate decarboxylase encoding gene expression cassette (containing Yarrowia lipolytica Endogenous promoter P TEFin orotic nucleoside-5'-phosphate decarboxylase and terminator T xpr2t It is also located between the upstream and downstream homologous arms. The structural diagram of the recombinant plasmid pUC-HUH-PDAT-SbPDAT is the same. Figure 4 The only difference is that PDAT-up and PDAT-dm replace GPAT-up and GPAT-dm respectively, and SbPDAT gene expression cassette replaces SbGPAT gene expression cassette.

[0118] With PDAT::P TEFin -F and PDAT::P TEFin -R is a primer, with Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the SbPDAT expression cassette promoter P was amplified. TEFin . with PDAT::T xpr2t -F and PDAT::T xpr2t -R is a primer, with Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the SbPDAT expression cassette terminator T was amplified. xpr2t .

[0119] Using pUC57-SbPDAT as a template, and P TEFin :: SbPDAT-F and T xpr2t :: SbPDAT-R is the primer, with promoter P at both ends for amplification. TEFin and Termination T xpr2t The SbPDAT gene of the homologous arm.

[0120] The PDAT site integrative plasmid was processed using a restriction endonuclease from NEB. Pac I. After enzyme digestion, the linearized PDAT site integrated plasmid was recovered by agarose gel electrophoresis.

[0121] The linearized PDAT site integration plasmid and the various elements (promoter P) in the SbPDAT gene expression cassette constructed in Title 8 of this embodiment were integrated. TEFin SbPDAT gene and terminator T xpr2t Using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd., one-step cloning was achieved by inserting the SbPDAT gene expression cassette between the downstream homologous arm of the PDAT site integration plasmid and the hisG tag encoding gene, and simultaneously between the upstream and downstream homologous arms, to obtain a circular recombinant vector.

[0122] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-PDAT-SbPDAT was obtained by ampicillin-resistant plate screening and colony PCR and sequencing verification.

[0123] Using restriction endonucleases from NEB Not I. After digesting the plasmid pUC-HUH-PDAT-SbPDAT with enzymes, the linearized pUC-HUH-PDAT-SbPDAT plasmid was recovered by agarose gel electrophoresis.

[0124] 9. Construction of the recombinant knockout plasmid pUC-HUH-PEX10

[0125] The recombinant plasmid pUC-HUH-PEX10 uses pUC57-hisG-Ura-hisG as its backbone and inserts... Yarrowia lipolytica In Po1fΔku70, the 1500bp homologous arm PEX10-up upstream of the start codon at the PEX10 site and the 1500bp homologous arm PEX10-dm downstream of the stop codon are contained in the orotate nucleoside-5'-phosphate decarboxylase gene expression cassette (containing...). Yarrowia lipolytica Endogenous promoter P TEFin orotic nucleoside-5'-phosphate decarboxylase and terminator T xpr2t It is also located between the upstream and downstream homologous arms; see the specific structure below. Figure 5 The only difference is that PEX10-up and PEX10-dm replace TGL4-up and TGL4-down respectively.

[0126] Using PEX10-up-F and PEX10-up-R as primers, Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the upstream homologous arm PEX10-up of the start codon at the TGL4 site was amplified.

[0127] Using restriction endonucleases from NEB EcoRI. After digesting the plasmid pUC57-hisG-Ura-hisG with enzymes, the linearized pUC57-hisG-Ura-hisG plasmid was recovered by agarose gel electrophoresis.

[0128] The linearized pUC57-hisG-Ura-hisG plasmid and the upstream homologous arm PEX10-up (with pUC57-hisG-Ura-hisG homologous arm sequences at both ends) of the PEX10 site start codon constructed in Title 9 of this embodiment were cloned in one step using the ClonExpress® II One Step Cloning Kit of Nanjing Novizan Biotechnology Co., Ltd., to obtain a circular recombinant vector.

[0129] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid pUC-HUH-PEX10-up was obtained by ampicillin-resistant plate screening and colony PCR and sequencing verification.

[0130] Using PEX10-dm-F and PEX10-dm-R as primers, Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the downstream homologous arm PEX10-dm of the PEX10 stop codon was amplified.

[0131] Using restriction endonucleases from NEB Hind III. After digesting the plasmid pUC-HUH-PEX10-up with enzymes, the linearized pUC-HUH-PEX10-up plasmid was recovered by agarose gel electrophoresis.

[0132] The linearized pUC-HUH-PEX10-up plasmid and the downstream homologous arm PEX10-dm (with pUC-HUH-PEX10-up homologous arm sequences at both ends) of the PEX10 site stop codon constructed in Title 9 of this embodiment were cloned in one step using the ClonExpress® II One Step Cloning Kit of Nanjing Novizan Biotechnology Co., Ltd. to obtain the recombinant plasmid pUC-HUH-PEX10.

[0133] Using restriction endonucleases from NEB Hind III. After digesting the plasmid pUC-HUH-PEX10 with enzymes, the linearized pUC-HUH-PEX10 plasmid was recovered by agarose gel electrophoresis.

[0134] 10. Construction of the recombinant knockout plasmid pUC-HUH-TGL4

[0135] The recombinant plasmid pUC-HUH-TGL4 uses pUC57-hisG-Ura-hisG as its backbone and inserts...Yarrowia lipolytica In Po1fΔku70, the TGL4 site contains a 1500bp homologous arm upstream of the start codon (TGL4-up) and a 1500bp homologous arm downstream of the stop codon (TGL4-down), and an orotate nucleoside-5'-phosphate decarboxylase-encoding gene expression cassette (containing...). Yarrowia lipolytica Endogenous promoter P TEFin orotic nucleoside-5'-phosphate decarboxylase and terminator T xpr2t Between the upstream and downstream homologous arms, see the specific structure. Figure 5 .

[0136] Using TGL4-up-F and TGL4-up-R as primers, Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the upstream homologous arm TGL4-up of the start codon at the TGL4 site was amplified.

[0137] Using restriction endonucleases from NEB EcoR I. After digesting the plasmid pUC57-hisG-Ura-hisG with enzymes, the linearized pUC57-hisG-Ura-hisG plasmid was recovered by agarose gel electrophoresis.

[0138] The linearized pUC57-hisG-Ura-hisG plasmid and the upstream homologous arm TGL4-up (with pUC57-hisG-Ura-hisG homologous arm sequences at both ends) of the TGL4 site start codon constructed in Title 9 of this embodiment were cloned in one step using the ClonExpress® II One Step Cloning Kit of Nanjing Novizan Biotechnology Co., Ltd., to obtain a circular recombinant vector.

[0139] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-TGL4-up was obtained by ampicillin-resistant plate screening and colony PCR and sequencing verification.

[0140] Using TGL4-dm-F and TGL4-dm-R as primers, Yarrowia lipolytica Using Po1fΔku70 genomic DNA as a template, the downstream homologous arm TGL4-down of the TGL4 stop codon was amplified.

[0141] Using restriction endonucleases from NEB Hind III. After digesting the plasmid pUC-HUH-TGL4-up with enzymes, the linearized pUC-HUH-TGL4-up plasmid was recovered by agarose gel electrophoresis.

[0142] The linearized pUC-HUH-TGL4-up plasmid and the downstream homologous arm TGL4-down (with pUC-HUH-TGL4-up homologous arm sequences at both ends) of the TGL4 site stop codon constructed in Title 9 of this embodiment were cloned in one step using the ClonExpress® II One Step Cloning Kit of Nanjing Novizan Biotechnology Co., Ltd. to obtain the recombinant plasmid pUC-HUH-TGL4.

[0143] Using restriction endonucleases from NEB Hind III. After digesting the plasmid pUC-HUH-TGL4 with enzymes, the linearized pUC-HUH-TGL4 plasmid was recovered by agarose gel electrophoresis.

[0144] Table 1

[0145]

[0146] Table 2 Primer sequences

[0147]

[0148] Example 2: Construction of recombinant Yersinia lipolyticis

[0149] (I) Construction of recombinant strain 1

[0150] Linearized recombinant plasmid pUC-HUH-P ELO1 -P CTR1 Importing Yarrowia lipolytica Po1f Δku70, P CTR1 Gene integration into the genome P ELO1 At the site, and then under 5-fluoroorotic acid selection pressure, a hisG tag and an Ura selection marker were lost, resulting in recombinant strain 1.

[0151] The specific method is as follows:

[0152] ① Linearized recombinant plasmid pUC-HUH-P ELO1 -P CTR1 Introduction and screening and identification of positive recombinant bacteria: Yarrowia lipolytica Po1f Δku70 was cultured overnight in YPD liquid medium (containing 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose) to prepare competent cells. The linearized recombinant plasmid pUC-HUH-P was then transfected using the ZymogenFrozen EZ Yeast Transformation Kit II from Zymo Research Corporation. ELO1 -P CTR1 ConversionYarrowia lipolytica Homologous recombination was performed in Po1f Δku70, and positive clones were screened using SD-Ura plates, followed by PCR identification. The SD-Ura plates contained 20 g / L glucose, 6.7 g / L YNB (amino-free yeast nitrogen source, purchased from BBI LifeSciences), 0.67 g / L CSM-Ura (completely supplemented mixture to remove uracil, purchased from MP Biomedicals), 23 g / L agar powder, and water as the solvent.

[0153] ② Loss of one hisG tag and Ura selection marker: Positive clones identified by PCR were plated on YPD plates containing 5-fluoroorotic acid (containing 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, 5-fluoroorotic acid (2.5 g / L), and 23 g / L agar powder). Single colonies were then streaked simultaneously on both YPD and SD-Ura plates containing 5-fluoroorotic acid. Single colonies that grew on the YPD plate containing 5-fluoroorotic acid but not on the SD-Ura plate were selected to obtain recombinant strain 1.

[0154] (II) Construction of recombinant strain 2

[0155] Linearized recombinant plasmid pUC-HUH-P SCD -P CTR1 Imported into recombinant bacteria 1, P CTR1 Gene integration into the genome P SCD At the site, and then under 5-fluoroorotic acid selection pressure, a hisG tag and an Ura selection marker were lost, resulting in recombinant strain 2.

[0156] The specific method is as follows:

[0157] ① Linearized recombinant plasmid pUC-HUH-P SCD -P CTR1 Introduction and screening and identification of positive recombinant bacteria: Recombinant bacteria 1 was cultured overnight in YPD liquid medium to prepare competent cells. The linearized recombinant plasmid pUC-HUH-P was introduced into the cells using the Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation. SCD -P CTR1 The cells were transformed into recombinant bacteria 1 for homologous recombination. Positive clones were screened using SD-Ura plates and then identified by PCR.

[0158] ② Loss of one hisG tag and Ura selection marker: Positive clones identified correctly by PCR were plated on YPD plates containing 5-fluoroorotic acid. Single colonies were then streaked simultaneously on both YPD and SD-Ura plates containing 5-fluoroorotic acid. Single colonies that grew on the YPD plate containing 5-fluoroorotic acid but not on the SD-Ura plate were selected to obtain recombinant strain 2.

[0159] (III) Construction of recombinant strain 3

[0160] The linearized recombinant plasmid pUC-leu-A08-FAT5 was introduced into recombinant bacteria 2, and the FAT5 gene expression cassette was integrated into the A08 site of the genome, resulting in recombinant bacteria 3. The specific method is as follows:

[0161] ① Introduction of linearized recombinant plasmid pUC-leu-A08-FAT5 and screening and identification of positive recombinant bacteria: Competent cells were prepared by overnight culture of recombinant bacteria 2 in YPD liquid medium. The linearized recombinant plasmid pUC-leu-A08-FAT5 was transformed into recombinant bacteria 2 using the Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination. Positive clones were screened using SD-Leu screening medium, followed by PCR identification. The SD-Leu screening medium contained 20 g / L glucose, 6.7 g / L YNB, 0.67 g / L CSM-Ura, 23 g / L agar powder, and water as the solvent.

[0162] ② Loss of one hisG tag and Ura selection marker: Positive clones identified by PCR were plated on YPD plates containing 5-fluoroorotic acid and cultured. Then, single colonies were streaked simultaneously on both YPD plates and SD-Leu plates containing 5-fluoroorotic acid. Single colonies that grew on YPD plates containing 5-fluoroorotic acid but failed to grow on SD-Leu plates were selected to obtain recombinant bacteria 3.

[0163] (iv) Construction of recombinant strain 4

[0164] In recombinant bacteria 3, linearized plasmids pUC-HUH-TGL4, pUC-HUH-Pex10, and the linearized plasmid pUC-HUH-IntA-ACC1 carrying the ACC1 gene expression cassette were sequentially introduced. The Pex10 and TGL4 genes were knocked out by homologous recombination, and the ACC1 expression cassette was integrated into the IntA site to obtain recombinant bacteria 4.

[0165] The specific method is as follows: After overnight culture of recombinant bacteria 3 in YPD liquid medium, competent cells were prepared. Using the Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation, the linearized recombinant plasmid pUC-HUH-TGL4 was introduced into recombinant bacteria 3 for homologous recombination to knock out the TGL4 gene. Positive recombinant bacteria with the TGL4 gene knocked out were screened and identified, following the same method as described in the title (I) of this embodiment ① linearized recombinant plasmid pUC-HUH-P. ELO1 -P CTR1 The introduction of the linearized plasmids pUC-HUH-Pex10 and pUC-HUH-IntA-ACC1 and the screening and identification of positive recombinant bacteria were then performed. Using the same method, the linearized plasmids pUC-HUH-Pex10 and pUC-HUH-IntA-ACC1 were sequentially introduced into the positive recombinant bacteria obtained in the previous step. After each plasmid introduction, positive recombinant bacteria were screened and identified. One hisG tag and one Ura selection marker were lost. Positive clones identified by PCR were plated on YPD plates containing 5-fluoroorotic acid. Single colonies were then streaked simultaneously on both YPD plates and SD-Ura plates containing 5-fluoroorotic acid. Single colonies that grew on YPD plates containing 5-fluoroorotic acid but not on SD-Ura plates were selected to obtain recombinant bacteria 4.

[0166] (v) Construction of recombinant strain 5

[0167] Linearized recombinant plasmids pUC-HUH-GPAT-SbGPAT, pUC-HUH-LPAAT-SbLPAAT, pUC-HUH-DGAT2-SbDGAT2, and pUC-HUH-PDAT-SbPDAT were sequentially introduced into recombinant bacteria 4. SbGPAT, SbLPAAT, SbDGAT2, and SbPDAT were integrated into the GPAT, LPAAT, DGAT2, and PDAT sites, respectively, via homologous recombination, resulting in recombinant bacteria 5. Positive recombinant bacteria were screened after each plasmid introduction and used as host bacteria for the next plasmid introduction. The introduction and screening methods for each plasmid were the same as those described in the title (I) of this embodiment, specifically ① linearized recombinant plasmid pUC-HUH-P ELO1 -P CTR1 The introduction of the plasmid and the screening and identification of positive recombinant bacteria were carried out. After introducing the last linearized plasmid pUC-HUH-PDAT-SbPDAT, the obtained positive recombinant bacteria were screened and identified. After losing one hisG tag and Ura selection marker, recombinant bacteria 5 were obtained. The specific method for losing one hisG tag and Ura selection marker is the same as in the title (I) of this embodiment.

[0168] Example 3: Application of recombinant Yersinia lipolytica in the production of palmitic acid

[0169] (a) Cultivation of engineered bacteria and extraction of products

[0170] The initial strain was Yersinia lipophila (Yersinia lipophila). Yarrowia lipolytica Po1f Δku70 and recombinant strains 1-5 from Example 2 were used to produce palmitoleic acid. The specific method is as follows: Each strain was streaked on YPD plates (containing 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, and 10 g / L agar) and cultured overnight to activate the strains. The activated strains were then inoculated into YPD liquid medium (containing 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose, with water as the solvent) and cultured at 30°C and 220 rpm for 16 hours to obtain the seed culture. The seed culture was inoculated at a rate of 1% into 50 ml of fermentation medium and cultured with shaking at 30°C and 220 rpm for 5 days. At the 48th hour of shaking culture, 4 g / L of copper sulfate aqueous solution was added to bring the final concentration of copper sulfate in the fermentation broth to 40 μM. After fermentation, the fermentation broth was transferred to a 50ml centrifuge tube, centrifuged at 5000rpm for 15min, the supernatant was discarded, and the bacterial precipitate was placed in an oven (75℃) and dried to constant weight to obtain dried bacterial cells.

[0171] The fermentation medium formula is as follows: glucose 60g / L, amino-free yeast nitrogen source (YNB) 1.7g / L, ammonium sulfate 1.3g / L, and water as the solvent.

[0172] (II) Qualitative and quantitative analysis of palmitoleic acid

[0173] 1. Fatty acid methyl esterification

[0174] Weigh approximately 0.05 g of dried bacterial cells and add 100 μL of a 0.2408 g / L methyl tridecanoate solution in hexane as an internal standard, along with 500 μL of a 1M NaOH solution in methanol. Vortex the sample at 1200 rpm for 1 hour at room temperature (25℃), then neutralize with 40 μL of 98% sulfuric acid. Add 400 μL of hexane and vortex at 1200 rpm for 10 minutes at room temperature (25℃), at which point the fatty acid methyl esters are extracted into the hexane. Centrifuge at 8000 rpm for 2 minutes, collect the supernatant, filter it through an organic nylon membrane (0.22 μm), and transfer it to a glass bottle for GC analysis.

[0175] 2. Palmitoleic acid detection

[0176] GC detection conditions: FID detector, injection port temperature 250℃, injection volume 1 μl, split ratio: 50:1, column: DB-23 column (60 m * 0.25 μm * 0.15 μm). Chromatographic conditions: initial temperature 100℃, increased to 196℃ at a rate of 25℃ / min, then increased to 220℃ at a rate of 2℃ / min, held for 2 min. Qualitative and quantitative analysis was performed using a mixed fatty acid standard from Sigma-Aldrich.

[0177] Under the above conditions, the elution time of methyl palmitate detected by GC was approximately 6.2 min. Figure 6 ).

[0178] After 5 days of fermentation, palmitoleic acid accumulation was detected in all strains. The percentage of palmitoleic acid in total fatty acids in recombinant strains 1, 2, 3, 4, and 5 were 7.62%, 15.67%, 20.14%, 24.15%, and 41.57%, respectively. Recombinant strain 5 had the highest palmitoleic acid yield (Table 3), reaching 2.84 g / L, meaning it produced 2.84 g of palmitoleic acid per liter of fermentation broth, significantly higher than the initial strain and other recombinant strains.

[0179] Table 3. Palmitoleic acid yield of the initial strain, recombinant strain 3, and recombinant strain 5

[0180]

[0181] Recombinant strain 5 was inoculated into 50 ml of seed culture (YPD medium) and cultured for 24 h. Then, the seed culture was inoculated into a 5 L fermenter at an inoculation rate of 5%. 2.5 L of fermentation medium was added to the 5 L fermenter. Fermentation was carried out for 0-48 h, with dissolved oxygen controlled at 20%; after 48 h, dissolved oxygen was controlled at 5%. The pH was maintained constant at 5.5 throughout fermentation until completion. Feed was added twice, at 40 h and 96 h, with 800 ml of 800 g / L glucose solution added each time. The fermentation temperature was 28℃, and the stirring speed was adjusted according to dissolved oxygen levels. The culture time was 6 days. The fermentation medium contained: glucose 150 g / L, ammonium sulfate 11 g / L, yeast extract 3 g / L, corn peptone 0.1 g / L, potassium dihydrogen phosphate 4 g / L, magnesium sulfate 2 g / L, calcium sulfate 0.8 g / L, sodium chloride 0.4 g / L, ammonium sulfate hydrochloride 12 mg / L, biotin 1 mg / L, sodium molybdate 160 mg / L, copper sulfate 0.2 mg / L, boric acid 40 mg / L, manganese sulfate 180 mg / L, and ferrous chloride 75 mg / L. After 5 days of fermentation, recombinant strain 5 produced the highest palmitoleic acid yield, reaching 10.56 g / L, or 10.56 g of palmitoleic acid per liter of fermentation broth (Table 3), significantly higher than the initial strain and other recombinant strains.

Claims

1. A recombinant lipophilic yeast strain that produces high levels of palmitoleic acid, characterized in that... In Yersinia lipophila ( Yarrowia lipolytica The genome of *Yersinia lipolytica* was obtained by knocking out peroxisome biogenesis factor 10 and triacylglycerol lipase 4, and inserting acetyl-CoA carboxylase, Δ9 desaturase, acetyl-CoA diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, lysophosphatidylcholine acyltransferase, and phosphatidylcholine:diacylglycerol acyltransferase gene expression cassettes; the acetyl-CoA carboxylase gene was derived from *Yersinia lipolytica*. Yarrowia lipolytica ); Δ9 desaturase is obtained by codon optimization of a gene from Caenorhabditis elegans; glycerol-3-phosphoacyltransferase, lysophosphatidylcholine acyltransferase, acetyl-CoA diacylglycerol acyltransferase, phosphatidylcholine:diacylglycerol acyltransferase are obtained by processing sea buckthorn fruit ( Sea buckthorn The gene was obtained by codon optimization from the source; the P gene in the recombinant Yersinia lipophila genome ELO1 promoter sites and P SCD An endogenous copper ion repressor promoter P was also inserted at the promoter site. CTR1 The gene sequences of the Δ9 desaturase, glycerol-3-phosphoacyltransferase, lysophosphatidylcholine acyltransferase, acetyl-CoA diacylglycerol acyltransferase, and phosphatidylcholine:diacylglycerol acyltransferase are shown in SEQ ID No: 1-5, respectively. The gene expression cassettes of the Δ9 desaturase, acetyl-CoA carboxylase, acetyl-CoA diacylglycerol acyltransferase, glycerol-3-phosphoacyltransferase, lysophosphatidylcholine acyltransferase, and phosphatidylcholine:diacylglycerol acyltransferase are presented in *Yersinia lipolytica* (Yersinia lipolytica). Yarrowia lipolytica The sites integrated into the genome are, in order: A08 site, IntA, DGAT2, GPAT, LPAAT, and PDAT.

2. The recombinant Yersinia lipolyticis according to claim 1, characterized in that... The promoter of the expression cassette is any one of the TEF promoter, hp4d promoter, TEFin promoter, POX2 promoter or GPDin promoter of Yersinia lipophila; the terminator is any one of the xpr2t terminator, mig1t terminator or lip2t terminator of Yersinia lipophila.

3. The method for preparing the recombinant Yersinia lipolyticis according to claim 2, characterized in that... Gene expression cassettes are inserted into the genome using homologous recombination, and genes to be knocked out are knocked out using homologous recombination.

4. The application of the recombinant Yersinia lipolytica according to claim 1 in the preparation of palmitoleic acid.

5. The application according to claim 4, characterized in that... Palmitoleic acid was prepared by fermentation of the recombinant Yersinia lipolytica using the following fermentation medium: glucose 130-170 g / L, ammonium sulfate 10-12 g / L, yeast extract 2.5-3.5 g / L, corn peptone 0.085-0.105 g / L, potassium dihydrogen phosphate 3.5-4.5 g / L, magnesium sulfate 1.8-2.2 g / L, calcium sulfate 0.78-0.82 g / L, sodium chloride 0.35-0.45 g / L, thiamine hydrochloride 10-14 mg / L, biotin 0.85-1.15 mg / L, sodium molybdate 140-180 mg / L, copper sulfate 0.18-0.22 mg / L, boric acid 38-42 mg / L, manganese sulfate 160-200 mg / L, and ferrous chloride 70-80 mg / L.

6. The application according to claim 5, characterized in that... Glucose is added during the fermentation process.

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