Construction method and application of recombinant yarrowia lipolytica with high ricinoleic acid yield

Abstract

The invention provides a construction method and application of recombinant yarrowia lipolytica with high ricinoleic acid yield, and belongs to the field of bioengineering. The recombinant yarrowia lipolytica with high ricinoleic acid yield can produce 4.23 g / L of ricinoleic acid after fed-batch fermentation, no organic solvent needs to be added in the fermentation process, and efficient synthesis of the plant source natural product ricinoleic acid in yarrowia lipolytica cells is achieved. The construction method of the recombinant yarrowia lipolytica is easy to operate, and efficient synthesis of ricinoleic acid can be achieved.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and particularly to a method for constructing and applying recombinant Yarrowia lipolytica with high ricinoleic acid production. Background Art

[0002] Ricinoleic Acid, 12-hydroxy-9-cis-octadecenoic acid, with the molecular formula C 18 H 34 O 3 , is a colorless to light yellow oily liquid with a special odor. As a major component of fatty acids, ricinoleic acid has a high degree of unsaturation and stability. Due to its unique chemical structure and properties, it has received increasing attention in the fields of chemistry, industry, medicine, etc. in recent years. Currently, industrially, due to its low viscosity and high lubricity, ricinoleic acid can be used to prepare surfactants, plasticizers, lubricating oil additives, etc., and can also be used in the production of sebacic acid and undecylenic acid. In medicine, ricinoleic acid can specifically activate the EP3 prostaglandin receptor of prostaglandin E2 due to its analgesic and anti-inflammatory effects. In addition, ricinoleic acid can further produce a variety of products with special properties through chemical reactions, such as methyl epoxyacetylricinoleate. These derivatives also have important application values in the fields of coating industry, plastic industry, metal processing, etc.

[0003] Traditionally, ricinoleic acid was mainly obtained from the seed oil extracted from the seeds of mature castor plants (Ricinus communis, Euphorbiaceae) or the sclerotia of ergot. Industrially, ricinoleic acid can be prepared through the saponification or fractionation process after hydrolyzing castor oil. However, this method faces problems such as large planting area, long plant growth cycle, toxicity of the raw material castor, and influence of climate conditions. In comparison, the microbial fermentation method has a short growth cycle and can be produced all-weather, so using microorganisms to ferment and produce ricinoleic acid is an economical and efficient way. In the prior art, it is necessary to further transform microorganisms through synthetic biology to improve the yield of heterologous synthesis of ricinoleic acid.

[0004] When ricinoleic acid is synthesized through metabolic engineering in Yarrowia lipolytica, its intracellular secretion and extracellular secretion strategies each have significant advantages. Intracellular secretion relies on the host's natural lipid droplet storage mechanism, which can effectively isolate the potential toxicity of the product to the cell membrane, and at the same time use the stable microenvironment of lipid droplets to avoid the oxidative degradation of ricinoleic acid, especially suitable for product accumulation under high-density culture. In addition, the concentrated intracellular storage can be efficiently recovered through cell lysis and solvent extraction, reducing the separation complexity. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide a method for constructing and applying recombinant Yarrowia lipolytica with high ricinoleic acid production in view of the deficiencies of the prior art.

[0006] To solve the above technical problems, the present invention discloses a method for constructing and applying a recombinant Yarrowia lipolytica with high ricinoleic acid production. It is realized by the following technical solutions:

[0007] A method for constructing a recombinant Yarrowia lipolytica with high ricinoleic acid production, wherein the recombinant Yarrowia lipolytica expresses the Δ-12 hydroxylase gene FAH12, and also expresses any one or a combination of multiple kinds of lysophosphatidylcholine acyltransferase gene LPCAT, phosphatidyl-diacylglycerol acyltransferase gene PDAT, and choline phosphotransferase CPT; the recombinant Yarrowia lipolytica simultaneously knocks out any one or a combination of multiple kinds of acyl-CoA diacylglycerol acyltransferase 1 gene DGA1, acyl-CoA diacylglycerol acyltransferase 2 gene DGA2, peroxisome biogenesis factor 10 gene PEX10, and triacylglycerol lipase 4 gene TGL4.

[0008] Preferably, the recombinant Yarrowia lipolytica expresses FAH12, LPCAT, PDAT, and CPT, and simultaneously knocks out DGA1, DGA2, PEX10, and TGL4.

[0009] Among them, the FAH12 is derived from Claviceps purpurea (CpFah12), and its nucleotide sequence is as shown in SEQ ID No.1, which is obtained by codon optimization of the sequence of GenBank HG326650.1.

[0010] The LPCAT is derived from Ricinus communis (RcLPCAT), and its nucleotide sequence is as shown in SEQ ID No.2, which is obtained by codon optimization of the sequence of GenBank AGO14581.1.

[0011] The PDAT is derived from Ricinus communis and / or Yarrowia lipolytica; among them, the nucleotide sequence of PDAT (RcPDAT1) derived from Ricinus communis is as shown in SEQ ID No.3, which is obtained by codon optimization of the sequence of GenBank NM_001323733.1, and the GenBank accession number of PDAT (also known as LRO1) derived from Yarrowia lipolytica is YALI0E16797g. Preferably, the recombinant Yarrowia lipolytica simultaneously expresses PDAT derived from Ricinus communis and PDAT derived from Yarrowia lipolytica.

[0012] Among them, the CPT is derived from Yarrowia lipolytica, and its GenBank accession number is YALI0C10989g.

[0013] The GenBank accession number of the DGA1 is YALI0E32769g; the GenBank accession number of the DGA2 is YALI0D07986g; the GenBank accession number of the PEX10 is YALI0C01023g; the GenBank accession number of the TGL4 is YALI0F10010g.

[0014] Among them, the recombinant Yarrowia lipolytica uses the Yarrowia lipolytica Po1f with the KU70 gene knocked out as the starting strain. Po1f was purchased from the American Type Culture Collection, and the number is ATCC MYA-2613.

[0015] Preferably, the recombinant Yarrowia lipolytica also expresses one or more marker genes; the marker genes are selected from the 3(β)-isopropylmalate dehydrogenase-encoding gene expression cassette or the orotidine-5'-phosphate decarboxylase-encoding gene expression cassette.

[0016] Preferably, the recombinant Yarrowia lipolytica is constructed according to the following method:

[0017] (1) Construct a gene expression cassette: Amplify the promoter, target gene, and terminator to obtain the promoter gene fragment, target gene fragment, and terminator gene fragment, and simultaneously ligate them into a plasmid vector containing an integration site to obtain a recombinant expression plasmid vector containing the gene expression cassette;

[0018] (2) Construct a gene knockout cassette: Amplify the upstream homologous arm and downstream homologous arm of the knockout gene, and ligate them into a plasmid vector to obtain a recombinant knockout plasmid vector containing the upstream and downstream homologous arms of the knockout gene;

[0019] (3) Introduce the recombinant expression plasmid vector described in step (1) and the recombinant knockout plasmid vector described in step (2) into Yarrowia lipolytica, so that the gene expression cassette and the gene knockout cassette are integrated into the Yarrowia lipolytica genome.

[0020] More preferably, in step (1), the target gene includes FAH12 and any one or more combinations of LPCAT, PDAT, or CPT; the promoter is the promoter P of Yarrowia lipolytica TEF 、P hp4d 、P TEFin 、P FBA 、P FBAin 、P POX2 、PPOT1 or P GPD any one of them; the terminator is the terminator T of Yarrowia lipolytica xpr2t , T mig1t , T lip2t , T cyc1t , T pex3t , T pex10t or T pex20t any one of them; the integration site is any one or more 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.

[0021] In step (2), the knocked-out gene is any one or a combination of more than one of DGA1, DGA2, PEX10, and TGL4.

[0022] In the second aspect, the present invention provides a recombinant Yarrowia lipolytica constructed by the construction method described in the first aspect.

[0023] In the third aspect, the present invention provides the application of the recombinant Yarrowia lipolytica described in the second aspect in the fermentation production of ricinoleic acid.

[0024] Preferably, the fermentation includes the following steps: culturing the recombinant Yarrowia lipolytica described in the second aspect in a fermentation medium without adding an organic solvent, and that's it.

[0025] Beneficial effects:

[0026] The recombinant Yarrowia lipolytica constructed by the present invention is based on Yarrowia lipolytica with the non-homologous recombination gene KU70 knocked out, which enhances its homologous recombination ability. Gene integration is achieved through the homologous recombination function of Yarrowia lipolytica itself, which can greatly improve the genetic stability of the imported gene. The method for constructing this recombinant Yarrowia lipolytica is efficient and simple to operate.

[0027] Compared with the prior art, the recombinant Yarrowia lipolytica of the present invention expresses the Δ-12 hydroxylase gene FAH12, lysophosphatidylcholine acyltransferase gene LPCAT, phosphatidyl-diacylglycerol acyltransferase gene PDAT, and choline phosphotransferase CPT, while knocking out the acyl-CoA diacylglycerol acyltransferase 1 gene DGA1, acyl-CoA diacylglycerol acyltransferase 2 gene DGA2, peroxisome biogenesis factor 10 gene PEX10, and triacylglycerol lipase 4 gene TGL4. Experiments have shown that this recombinant Yarrowia lipolytica can produce 4.23 g / L of ricinoleic acid after fed-batch fermentation, and no organic solvents need to be added during the fermentation process, realizing the efficient synthesis of the natural product ricinoleic acid of plant origin in the cells of Yarrowia lipolytica. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further specifically described below in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0029] Figure 1 It is a synthetic metabolic map of ricinoleic acid provided by an embodiment of the present invention.

[0030] Figure 2 It is a plasmid map of the recombinant plasmid pUC-HUH-LIP1-CpFAH12, where LIP1-up represents the upstream homologous arm of the LIP1 locus, LIP1-dm represents the downstream homologous arm of the LIP1 locus, TEFin represents the promoter P TEFin , xpr2t represents the terminator T xpr2t , URA represents the orotidine-5'-phosphate decarboxylase-encoding gene expression cassette (including the promoter P endogenous to Yarrowia lipolytica TEFin , terminator T xpr2t ), and CpFAH12 is the gene encoding the Δ-12 hydroxylase of Claviceps.

[0031] Figure 3 It is a plasmid map of the recombinant plasmid pUC-HUH-PEX10, where PEX10-up represents the upstream homologous arm of the PEX10 locus, PEX10-dm represents the downstream homologous arm of the PEX10 locus, and URA represents the orotidine-5'-phosphate decarboxylase-encoding gene expression cassette (including the promoter P endogenous to Yarrowia lipolytica TEFin , terminator T xpr2t ).

[0032] Figure 4 It is a graph of the production of ricinoleic acid by the recombinant bacteria 1-8 in fermentation. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The present invention will be further described below through specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0034] In the following embodiments, the formula of the YPD liquid medium is: 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose.

[0035] The formula of the YPD plate containing 5-fluoroorotic acid is as follows: Dissolve 1 g of 5-fluoroorotic acid powder in 10 mL of dimethyl sulfoxide (DMSO) to obtain a 5-fluoroorotic acid solution. Add 1 volume part of the 5-fluoroorotic acid solution to 99 volume parts of the YPD solid medium (23 g / L agar powder is added to the YPD liquid medium) to obtain the YPD plate containing 5-fluoroorotic acid.

[0036] The formula of the screening medium SD-Leu is: 20 g / L glucose, 6.7 g / L YNB (yeast nitrogen base without amino acids, purchased from BBI Life Sciences), 0.67 g / L CSM-Leu (complete supplement mixture without leucine, purchased from MP Biomedicals), 23 g / L agar powder, and the solvent is water.

[0037] The formula of the screening medium SD-Ura is: 20 g / L glucose, 6.7 g / L YNB (yeast nitrogen base without amino acids, purchased from BBI Life Sciences), 0.67 g / L CSM-Ura (complete supplement mixture without uracil, purchased from MP Biomedicals), 23 g / L agar powder, and the solvent is water.

[0038] Yarrowia lipolytica Po1f was purchased from the American Type Culture Collection with the accession number ATCC MYA - 2613. Yarrowia lipolytica Po1fΔku70 (MatA, Δku70::HisG, leu2 - 270, ura3 - 302, xpr2 - 322, axp1 - 2), abbreviated as Yarrowia lipolytica Po1fΔku70, was constructed by knocking out the coding gene KU70 responsible for non - homologous recombination from Yarrowia lipolytica Po1f (published in Kretzschmar A, et al., Current Genetics, 2013, 59(1 - 2):63 - 72).

[0039] The LIP1 - site integration plasmid described in the following examples was obtained by inserting a 1458 - bp sequence (upstream homologous arm) upstream of the start codon and a 1438 - bp sequence (downstream homologous arm) downstream of the stop codon of the LIP1 site on the chromosome of the Yarrowia lipolytica Po1fΔku70 genome into the pUC57 - hisG - ura - hisG vector (the construction method is shown in Example 1). The two hisG tag - encoding genes are between the upstream and downstream homologous arms of the LIP1 site.

[0040] The 7H - site integration plasmid described in the following examples was obtained by inserting a 2035 - bp sequence (upstream homologous arm) upstream of the start codon and a 2244 - bp sequence (downstream homologous arm) downstream of the stop codon of the 7H site on the chromosome of the Yarrowia lipolytica Po1fΔku70 genome into the pUC57 - hisG - ura - hisG vector (the construction method is shown in Example 1). The two hisG tag - encoding genes are between the upstream and downstream homologous arms of the 7H site.

[0041] The IntC - site integration plasmid described in the following examples was obtained by inserting a 1402 - bp sequence (upstream homologous arm) upstream of the start codon and a 1396 - bp sequence (downstream homologous arm) downstream of the stop codon of the IntC site on the chromosome of the Yarrowia lipolytica Po1fΔku70 genome into the pUC57 - hisG - ura - hisG vector (the construction method is shown in Example 1). The two hisG tag - encoding genes are between the upstream and downstream homologous arms of the IntC site.

[0042] The promoter P used in the following examplesTEFin and P FBAin , terminator T mig1t and T xpr2t The specific nucleotide sequences of and are shown in Chinese Patent CN202410521402X.

[0043] The synthesis strategy diagram of ricinoleic acid provided by the embodiments of the present invention is shown in Figure 1 .

[0044] Example 1 Amplification of Gene Elements and Preparation of Target Plasmids

[0045] (1) Preparation of Target Genes

[0046] According to the nucleotide sequence of the coding gene of ergot Δ-12 hydroxylase from Claviceps purpurea provided on NCBI (GenBank accession number: HG326650.1), after codon optimization, the optimized coding gene CpFAH12 (SEQ ID No.1) of Δ-12 hydroxylase was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. and inserted into plasmid pUC57 (purchased from GenScript) to obtain plasmid pUC57-CpFAH12.

[0047] According to the nucleotide sequence of the coding gene of lysophosphatidylcholine acyltransferase from Ricinus communis provided on NCBI (GenBank accession number: AGO14581.1), after codon optimization, the optimized coding gene RcLPCAT (SEQ ID No.2) of lysophosphatidylcholine acyltransferase was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. and inserted into plasmid pUC57 (purchased from GenScript) to obtain plasmid pUC57-RcLPCAT.

[0048] According to the nucleotide sequence of the coding gene of phosphatidyl-diacylglycerol acyltransferase 1 from Ricinus communis provided on NCBI (GenBank accession number: NM_001323733.1), after codon optimization, the optimized coding gene RcPDAT1 (SEQ ID No.3) of phosphatidyl-diacylglycerol acyltransferase 1 was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. and inserted into plasmid pUC57 (purchased from GenScript) to obtain plasmid pUC57-RcPDAT1.

[0049] According to the nucleotide sequence of the orotidine-5'-phosphate decarboxylase-encoding gene URA of Yarrowia lipolytica (GenBank accession number: AJ306421.1) and the HisG tag (GenBank accession number: AF324729.1) provided on NCBI, it was commissioned to Suzhou Genewiz Biotechnology Co., Ltd. for synthesis. The two HisG tag-encoding gene sequences were inserted into plasmid pUC57, and the orotidine-5'-phosphate decarboxylase-encoding gene expression cassette (consisting of the promoter P TEFin from Yarrowia lipolytica endogenous, the orotidine-5'-phosphate decarboxylase-encoding gene URA and the terminator T xpr2t ) was inserted between the two HisG tag-encoding gene sequences to enable Ura marker recycling, obtaining plasmid pUC57-HisG-Ura-HisG (pUC57-HUH).

[0050] (II) Construction of recombinant plasmids

[0051] The structure of the recombinant plasmid is shown in Table 1; the primers used for constructing the recombinant plasmid are shown in Table 2.

[0052] 1. Construction of recombinant integration plasmids

[0053] The recombinant integration plasmids pUC-HUH-LIP1-CpFAH12, pUC-HUH-IntC-RcLPCAT, pUC-HUH-IntC-RcLPCAT-CPT, pUC-HUH-7H-RcPDAT1 and pUC-HUH-PEX10-LRO1 were based on pUC57-HisG-Ura-HisG, and inserted with the upstream homologous arm of the start codon and the downstream homologous arm of the stop codon at the LIP1 site, IntC site, 7H site and PEX10 site in Yarrowia lipolytica Po1fΔku70, and the target gene expression cassette was inserted between the upstream and downstream homologous arms. The target gene expression cassette consists of a promoter, a target gene and a terminator. The specific structure of the recombinant plasmid is shown in Table 1.

[0054] Among them, the CpFAH12 expression cassette (P TEFin -CpFAH12-T xpr2t ), the RcLPCAT expression cassette (P FBAin -RcLPCAT-T mig1t ), the CPT expression cassette (P TEFin -CPT-T xpr2t ), the RcPDAT1 expression cassette (P TEFin -RcPDAT1-T mig1t ), the LRO1 expression cassette (P TEFin-LRO1-T xpr2t ) PEX10-up, PEX10-dm amplify the promoter, coding gene, and terminator fragments using the primers described in Table 2 respectively.

[0055] Taking the recombinant plasmid pUC-HUH-LIP1-CpFAH12 as an example for the specific construction method, the recombinant plasmid pUC-HUH-LIP1-CpFAH12 uses pUC57-HisG-Ura-HisG as the backbone, inserts the upstream homologous arm LIP1-up of the start codon and the downstream homologous arm LIP1-dm of the stop codon at the LIP1 locus in Yarrowia lipolytica Po1fΔku70, and also inserts the CpFAH12 expression cassette (P TEFin -CpFAH12-T xpr2t ) between the upstream and downstream homologous arms, and the orotidine-5'-phosphate decarboxylase coding gene URA expression cassette (including the promoter P TEFin and terminator T xpr2t ) of Yarrowia lipolytica endogenous is also between the upstream and downstream homologous arms.

[0056] Using the genomic DNA of Yarrowia lipolytica Po1fΔku70 as the template, amplify the promoter P TEFin and terminator T xpr2t of the CpFAH12 expression cassette using the primers in Table 2 respectively. Using the plasmid pUC57-CpFAH12 as the template, amplify the CpFAH12 gene using the primers in Table 2. The above PCR amplification system is: Primer STAR Max Premix 25 μL, template 1 μL, primer F 2 μL, primer R 2 μL, distilled water 20 μL.

[0057] Among them, Primer STAR Max Premix is purchased from Takara Biotechnology (Beijing) Co., Ltd.

[0058] The procedure of the above PCR is as follows: denaturation at 98 °C for 10 s, annealing at 55 °C for 5 s, extension at 72 °C (extension time = target fragment length / 1 kb, unit min), repeat 30 cycles.

[0059] Purify and recover each fragment using TaKaRa MiniBEST DNA Fragment Purification Kit (purchased from Shanghai Biosciences Co., Ltd.).

[0060] After digesting the LIP1 locus integration plasmid with the restriction endonuclease HindⅢ from NEB, recover the linearized LIP1 locus integration plasmid by agarose gel electrophoresis gel recovery.

[0061] Integrate the linearized LIP1-site integration plasmid and each element (promoter P TEFin , gene CpFAH12, and terminator T xpr2t ) in the CpFAH12 gene expression cassette constructed in this example using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novoprotein Science and Technology Co., Ltd. to achieve one-step cloning. Insert the CpFAH12 gene expression cassette between the upstream and downstream homologous arms of the LIP1-site integration plasmid, and the two hisG tag-encoding genes are on the same side of the CpFAH12 gene expression cassette to obtain a circular recombinant vector.

[0062] Transform the circular recombinant vector into Escherichia coli DH5α competent cells, screen through an LB plate with ampicillin resistance, and verify by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-HUH-LIP1-CpFAH12. The plasmid map of this recombinant plasmid is as Figure 2 shown.

[0063] After digesting the plasmid pUC-HUH-LIP1-CpFAH12 with the restriction endonuclease BamHⅠ from NEB, recover the linearized pUC-HUH-LIP1-CpFAH12 plasmid by agarose gel electrophoresis.

[0064] The construction methods of the recombinant plasmids pUC-HUH-IntC-RcLPCAT, pUC-HUH-IntC-RcLPCAT-CPT, pUC-HUH-7H-RcPDAT1, and pUC-HUH-PEX10-LRO1 are the same as that of the plasmid pUC-HUH-LIP1-CpFAH12, except that the insertion sites and gene expression cassettes are different. Specifically:

[0065] Recombinant plasmid pUC-HUH-IntC-RcLPCAT: Using the Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, amplify the promoter P FBAin and terminator T mig1t of the RcLPCAT expression cassette using the primers in Table 2 respectively. Using the plasmid pUC57-RcLPCAT as a template, amplify the RcLPCAT gene using the primers in Table 2. Digest the IntC-site integration plasmid, and recover the linearized IntC-site integration plasmid by agarose gel electrophoresis. Integrate the linearized IntC-site integration plasmid and each element (promoter P FBAin , gene RcLPCAT, and terminator T mig1t) Through one-step cloning, the RcLPCAT gene expression cassette was inserted between the upstream and downstream homologous arms of the IntC site integration plasmid, and the two hisG tag-encoding genes were on the same side of the RcLPCAT gene expression cassette, resulting in the circular recombinant plasmid pUC-HUH-IntC-RcLPCAT.

[0066] Recombinant plasmid pUC-HUH-IntC-RcLPCAT-CPT: Using the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template, the promoter P of the CPT expression cassette, TEFin the CPT gene (choline phosphotransferase gene, GenBank accession number YALI0C10989g), and the terminator T xpr2t . were amplified respectively using the primers in Table 2. The pUC-HUH-IntC-RcLPCAT recombinant plasmid was digested with enzymes, and the linearized plasmid was recovered by agarose gel electrophoresis. The linearized plasmid and each element in the CPT expression cassette (P TEFin , the CPT gene and the terminator T xpr2t ) were obtained through one-step cloning.

[0067] Recombinant plasmid pUC-HUH-7H-RcPDAT1: Using the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template, the promoter P of the RcPDAT1 expression cassette TEFin and the terminator T mig1t were amplified respectively using the primers in Table 2. Using the plasmid pUC57-RcPDAT1 as a template, the RcPDAT1 gene was amplified using the primers in Table 2. The pUC-HUH-7H site integration plasmid was digested with enzymes, and the linearized plasmid was recovered by agarose gel electrophoresis. The linearized plasmid and each element in the RcPDAT1 expression cassette (P TEFin , the RcPDAT1 gene and the terminator T mig1t ) were obtained through one-step cloning.

[0068] Recombinant plasmid pUC-HUH-PEX10-LRO1: Using the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template, the promoter P of the LRO1 expression cassette, TEFin the LRO1 gene (the endogenous phosphatidylglycerol acyltransferase gene of Yarrowia lipolytica, GenBank accession number YALI0E16797g), and the terminator T xpr2t were amplified respectively using the primers in Table 2. The pUC-HUH-PEX10 plasmid was digested with enzymes, and the linearized plasmid was recovered by agarose gel electrophoresis. The linearized plasmid and each element in the LRO1 expression cassette (P TEFin , the LRO1 gene and the terminator T xpr2t) It can be obtained by one-step cloning. For the construction method of the pUC-HUH-PEX10 plasmid, please refer to "2. Construction of the recombinant knockout plasmid" in this example.

[0069] 2. Construction of the recombinant knockout plasmid

[0070] The recombinant knockout plasmids pUC-HUH-PEX10, pUC-HUH-TGL4, pUC-HUH-DGA1, and pUC-HUH-DGA2 use pUC57-HisG-Ura-HisG as the backbone and insert the upstream homologous arms PEX10-up, TGL4-up, DGA1-up, DGA2-up of the start codon and the downstream homologous arms PEX10-dm, TGL4-dm, DGA1-dm, DGA2-dm of the stop codon at the PEX10 (Genbank: YALI0C01023g), TGL4 (Genbank: YALI0F10010g), DGA1 (Genbank: YALI0E32769g), and DGA2 (Genbank: YALI0D07986g) loci in Yarrowia lipolytica Po1fΔku70, respectively. The above fragments amplify the upstream and downstream homologous arms of the sites through the primers described in Table 2.

[0071] Taking the recombinant plasmid pUC-HUH-PEX10 as an example, the recombinant plasmid pUC-HUH-PEX10 uses pUC57-HisG-Ura-HisG as the backbone and inserts a 2250-bp upstream homologous arm (PEX10-up) of the start codon and a 1500-bp downstream homologous arm (PEX10-dm) of the stop codon at the PEX10 locus in Yarrowia lipolytica Po1fΔku70. The orotidine-5'-phosphate decarboxylase-encoding gene URA expression cassette (including the promoter P TEFin and terminator T xpr2t ) of Yarrowia lipolytica is also between the upstream and downstream homologous arms.

[0072] Using PEX10::PEX10-up-F / R described in Table 2 as primers and the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template, the upstream homologous arm PEX10-up of the start codon at the PEX10 locus was amplified. After digesting the plasmid pUC57-HisG-Ura-HisG with the restriction endonuclease EcoRⅠ from NEB, the linearized pUC57-HisG-Ura-HisG plasmid was recovered by agarose gel electrophoresis and gel extraction. The linearized pUC57-HisG-Ura-HisG plasmid and the upstream homologous arm PEX10-up of the start codon at the PEX10 locus constructed in this example (with pUC57-HisG-Ura-HisG homologous arm sequences at both ends) were used to perform one-step cloning using the IIOne Step Cloning Kit from Nanjing Novoprotein Science and Technology Co., Ltd. to obtain a circular recombinant vector. The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, screened through an LB plate with ampicillin resistance, and verified by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-HUH-PEX10-up.

[0073] Using PEX10::PEX10-dm-F / R described in Table 2 as primers and the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template, the downstream homologous arm PEX10-dm of the stop codon at the PEX10 locus was amplified. After digesting the plasmid pUC-HUH-PEX10-up with the restriction endonuclease HindⅢ from NEB, the linearized pUC-HUH-PEX10-up plasmid was recovered by agarose gel electrophoresis and gel extraction. The linearized pUC-HUH-PEX10-up plasmid and the downstream homologous arm PEX10-dm of the stop codon at the PEX10 locus constructed in this example (with pUC-HUH-PEX10-up homologous arm sequences at both ends) were used to perform one-step cloning using the IIOne Step Cloning Kit from Nanjing Novoprotein Science and Technology Co., Ltd. to obtain the recombinant knockout plasmid pUC-HUH-PEX10. The plasmid map of this recombinant knockout plasmid is as shown in Figure 3 Figure. The construction methods of the recombinant knockout plasmids pUC-HUH-TGL4, pUC-HUH-DGA1, and pUC-HUH-DGA2 are the same as that of pUC-HUH-PEX10, except that different primers were used. Specifically, the corresponding homologous arms were amplified using the primers numbered 35-46 in Table 2, and they can be constructed according to the method of the above knockout plasmids.

[0074] Table 1 Information table of inserted sequences in each recombinant plasmid

[0075]

[0076] Table 2 Primer Sequences

[0077]

[0078]

[0079] Example 2 Construction of Recombinant Yarrowia lipolytica (I) Construction of Recombinant Strain 1 (Expressing CpFAH12)

[0080] The recombinant plasmid pUC-HUH-LIP1-CpFAH12 was introduced into Yarrowia lipolytica Po1fΔku70, and the CpFAH12 expression cassette was integrated into the genomic LIP1 locus by homologous recombination to express the Δ-12 hydroxylase gene. Then, after losing a hisG tag and the Ura selection marker under the 5-fluoroorotic acid screening pressure, recombinant strain 1 was obtained.

[0081] The specific method is as follows:

[0082] Yarrowia lipolytica Po1fΔku70 was inoculated into YPD liquid medium and cultured overnight at 30 °C to prepare competent cells and then transferred into the recombinant plasmid: The recombinant plasmid pUC-HUH-LIP1-CpFAH12 was transformed into Yarrowia lipolytica Po1fΔku70 using the Zymogen Frozen EZYeast Transformation Kit II from Zymo Research Corporation for homologous recombination. Positive clones were screened using the screening medium SD-Ura, and then identified by PCR.

[0083] The positive clone single colonies identified correctly by PCR were streaked simultaneously on YPD plates containing 5-fluoroorotic acid and SD-Ura plates. Single colonies that could grow on the YPD plates containing 5-fluoroorotic acid but could not grow on the SD-Ura plates were named recombinant strain 1.

[0084] (II) Construction of Recombinant Strain 2 (Expressing CpFAH12 and Knocking out DGA1)

[0085] The recombinant plasmid pUC-HUH-DGA1 was introduced into recombinant strain 1, and the hisG-ura-hisG fragment was integrated into the genomic DGA1 locus by homologous recombination to knock out the acyl-CoA diacylglycerol acyltransferase 1 gene. Then, after losing a hisG tag and the Ura selection marker under the 5-fluoroorotic acid screening pressure, recombinant strain 2 was obtained. The specific construction method was the same as that of recombinant strain 1.

[0086] (3) Construction of Recombinant Strain 3 (expressing CpFAH12, knocking out DGA1 and DGA2)

[0087] The recombinant plasmid pUC-HUH-DGA2 was introduced into recombinant strain 2, and the hisG-ura-hisG fragment was integrated into the genomic DGA2 locus by homologous recombination, thereby knocking out the acyl-CoA diacylglycerol acyltransferase 2 gene. Then, after losing one hisG tag and the Ura selection marker under the selection pressure of 5-fluoroorotic acid, recombinant strain 3 was obtained. The specific construction method was the same as that of recombinant strain 1.

[0088] (4) Construction of Recombinant Strain 4 (expressing CpFAH12, knocking out DGA1, DGA2, and TGL4)

[0089] The recombinant plasmid pUC-HUH-TGL4 was introduced into recombinant strain 3, and the hisG-ura-hisG fragment was integrated into the genomic TGL4 locus by homologous recombination, thereby knocking out the triacylglycerol lipase 4 gene. Then, after losing one hisG tag and the Ura selection marker under the selection pressure of 5-fluoroorotic acid, recombinant strain 4 was obtained. The specific construction method was the same as that of recombinant strain 1. (5) Construction of Recombinant Strain 5 (expressing CpFAH12, knocking out DGA1, DGA2, TGL4, and PEX10)

[0090] The recombinant plasmid pUC-HUH-PEX10 was introduced into recombinant strain 4, and the hisG-ura-hisG fragment was integrated into the genomic PEX10 locus by homologous recombination, thereby knocking out the peroxisome biogenesis factor 10 gene. Then, after losing one hisG tag and the Ura selection marker under the selection pressure of 5-fluoroorotic acid, recombinant strain 5 was obtained. The specific construction method was the same as that of recombinant strain 1.

[0091] (6) Construction of Recombinant Strain 6 (expressing CpFAH12, knocking out DGA1, DGA2, TGL4, and PEX10, expressing RcLPCAT and CPT)

[0092] The recombinant plasmid pUC-HUH-IntC-RcLPCAT-CPT was introduced into recombinant strain 5, and the RcLPCAT expression cassette and CPT expression cassette were integrated into the genomic IntC locus by homologous recombination, thereby expressing the lysophosphatidylcholine acyltransferase gene and choline phosphotransferase gene. Then, after losing one hisG tag and the Ura selection marker under the selection pressure of 5-fluoroorotic acid, recombinant strain 6 was obtained. The specific construction method was the same as that of recombinant strain 1.

[0093] (7) Construction of Recombinant Strain 7 (expressing CpFAH12, knocking out DGA1, DGA2, TGL4, and PEX10, expressing RcLPCAT, CPT, and LRO1)

[0094] The recombinant plasmid pUC-HUH-PEX10-LRO1 was introduced into the recombinant bacterium 6, and the LRO1 expression cassette was integrated into the genomic PEX10 locus by homologous recombination, thereby expressing the phosphatidyl-diacylglycerol acyltransferase 1 gene. Then, after losing one hisG tag and the Ura screening marker under the selection pressure of 5-fluoroorotic acid, the recombinant bacterium 7 was obtained. The specific construction method was the same as that of the recombinant bacterium 1.

[0095] (VIII) Construction of recombinant bacterium 8 (expressing CpFAH12, knocking out DGA1, DGA2, TGL4, PEX10, expressing RcLPCAT, CPT, LRO1, RcPDAT1)

[0096] The recombinant plasmid pUC-HUH-7H-RcPDAT1 was introduced into the recombinant bacterium 7, and the RcPDAT1 expression cassette was integrated into the genomic 7H locus by homologous recombination, thereby expressing the phosphatidyl-diacylglycerol acyltransferase 1 gene. Then, after losing one hisG tag and the Ura screening marker under the selection pressure of 5-fluoroorotic acid, the recombinant bacterium 8 was obtained. The specific construction method was the same as that of the recombinant bacterium 1.

[0097] Example 3 Application of recombinant Yarrowia lipolytica in the production of ricinoleic acid (I) Cultivation of engineering bacteria and product extraction

[0098] Yarrowia lipolytica Po1f-Δku70, the initial bacterium, and the recombinant bacteria 1-8 in Example 2 were respectively used to produce ricinoleic acid. The specific method was as follows: The initial bacterium and the recombinant bacteria were activated and cultured in YPD liquid medium at 30 °C and 220 rpm for 16 h to obtain seed solutions. The seed solutions were inoculated into 50 mL of fermentation medium YPD60 at an inoculation amount of 1% v / v and cultured with shaking at 30 °C and 220 rpm for 5 days. After fermentation, the fermentation broth was transferred to a 50 mL centrifuge tube and centrifuged at 5000 rpm for 15 min. The supernatant was removed and dried to a constant weight in an oven (75 °C).

[0099] The formula of the fermentation medium YPD60 was: 20 g / L peptone, 10 g / L yeast extract, and 60 g / L glucose, and the solvent was water.

[0100] (II) Qualitative and quantitative analysis of ricinoleic acid

[0101] 1. Fatty acid methylation

[0102] Weigh about 1.5 g of dry bacterial cells, add 10 mL of 4 M hydrochloric acid, and react for 20 min; transfer to a boiling water bath and heat for 10 min; place at -80 °C for freezing for 15 min; add 10 mL of chloroform and 5 mL of methanol, and shake at 200 rpm for 30 min; take the lower lipid-soluble layer into a 10 mL centrifuge tube, and dry it with nitrogen in a fume hood; vacuum dry for 2 h to obtain the oil. Weigh 0.1 g of the oil, add 1 mL of n-hexane and 0.1 mL of 1 M potassium hydroxide / methanol solution, and shake rapidly for 1 min to mix completely. Let the reaction solution stand at room temperature for 15 min for the methylation reaction. After standing, set the centrifugation conditions to centrifuge at 5000 rpm for 5 min, take 200 μL of the supernatant and place it in a 2 mL centrifuge tube for gas phase analysis.

[0103] 2. Detection of ricinoleic acid

[0104] Detection conditions: FID detector, inlet temperature 250 °C, injection volume 1 μL, split ratio: 50:1, chromatographic column: DB23 (60 m * 0.25 μm * 0.15 μm). Chromatographic conditions: The initial temperature is 100 °C, rise to 196 °C at a rate of 25 °C / min, then rise to 220 °C at a rate of 2 °C / min, and hold for about 2 min. Qualitative and quantitative analysis is carried out using a fatty acid mixed standard from Sigma-Aldrich.

[0105] After 5 days of fermentation, the ricinoleic acid production of recombinant bacterium 8 was the highest, reaching 0.91 g / L, that is, 0.91 g of ricinoleic acid was produced per liter of fermentation broth. The initial bacterium could not synthesize ricinoleic acid, and the ricinoleic acid production of recombinant bacteria 1 - 8 after 5 days of fermentation is shown in Table 3 and Figure 4 as follows.

[0106] Table 3 Ricinoleic acid production of the initial bacterium and recombinant bacteria

[0107] Fermentation method Strain Biomass (g / L) Ricinoleic acid production (g / L) Erlenmeyer flask Initial bacteria 5.04 0 Erlenmeyer flask Recombinant bacterium 1 5.29 0.03 Erlenmeyer flask Recombinant bacterium 2 5.08 0.05 Erlenmeyer flask Recombinant bacterium 3 4.77 0.07 Erlenmeyer flask Recombinant bacterium 4 4.87 0.15 Erlenmeyer flask Recombinant bacterium 5 5.09 0.23 Erlenmeyer flask Recombinant bacterium 6 5.09 0.74 Erlenmeyer flask Recombinant bacterium 7 5.22 0.83 Erlenmeyer flask Recombinant bacterium 8 5.28 0.91 Fermenter Recombinant bacterium 8 78.53 4.23

[0108] The strain 8 was inoculated into 50 mL of seed culture medium (YPD medium) and cultured for 24 h, and then inoculated into a fermenter at an inoculation amount of 5% v / v. 2.5 L of fermentation medium was added to a 5 L fermenter. During fermentation, the dissolved oxygen was controlled to be greater than 20% (0 - 48 h) and 0 - 5% (more than 48 h). The pH value was constantly controlled at 5.5 during the fermentation process until the fermentation ended. 800 g / L of glucose was fed twice at 40 h and 96 h. The temperature was controlled at 28 °C and cultured for 6 days. Among them, the composition of the fermentation medium was 150 g / L of glucose, 11 g / L of ammonium sulfate, 3 g / L of yeast extract, 0.1 g / L of corn peptone, 4 g / L of potassium dihydrogen phosphate, 2 g / L of magnesium sulfate, 0.8 g / L of calcium sulfate, 0.4 g / L of sodium chloride, 12 mg / L of thiamine hydrochloride, 1 mg / L of biotin, 160 mg / L of sodium molybdate, 0.2 mg / L of copper sulfate, 40 mg / L of boric acid, 180 mg / L of manganese sulfate, and 75 mg / L of ferrous chloride. After 5 days of fermentation, the highest yield of ricinoleic acid of the recombinant strain 8 reached 4.23 g / L, that is, 4.23 g of nervonic acid was produced per liter of fermentation broth, which was significantly higher than that of the initial strain and the recombinant strain 1.

[0109] The present invention provides a method for constructing a recombinant Yarrowia lipolytica with high yield of ricinoleic acid and the idea and method of its application. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by the prior art.

Claims

1. A method for constructing a recombinant Yarrowia lipolytica with high ricinoleic acid production, characterized in that: The recombinant Yarrowia lipolytica expresses the delta-12 hydroxylase gene FAH12, and also expresses any one or more combinations of the lysophosphatidylcholine acyltransferase gene LPCAT, the phospholipid diacylglycerol acyltransferase gene PDAT, and the choline phosphotransferase CPT; The recombinant Yarrowia lipolytica simultaneously knocks out any one or more combinations of acyl-CoA diacylglycerol acyltransferase 1 gene DGA1, acyl-CoA diacylglycerol acyltransferase 2 gene DGA2, peroxisome biogenesis factor 10 gene PEX10 and triacylglycerol lipase 4 gene TGL4.

2. The construction method according to claim 1, characterized in that: The recombinant Yarrowia lipolytica expresses FAH12, LPCAT, PDAT and CPT, and knocks out DGA1, DGA2, PEX10 and TGL4.

3. The construction method according to claim 1, characterized in that: The FAH12 is derived from Claviceps purpurea, and its nucleotide sequence is shown in SEQ ID No.

1.

4. The construction method according to claim 1, characterized in that: The LPCAT is derived from Ricinus communis, and its nucleotide sequence is shown in SEQ ID No.2; The PDAT is derived from Ricinus communis and / or Yarrowia lipolytica; wherein the nucleotide sequence of the PDAT derived from Ricinus communis is shown as SEQ ID No. 3, and the GenBank accession number of the PDAT derived from Yarrowia lipolytica is YALI0E16797g.

5. The construction method according to claim 4, characterized in that: The recombinant Yarrowia lipolytica simultaneously expresses PDAT derived from Ricinus communis and PDAT derived from Yarrowia lipolytica.

6. The construction method according to claim 1, characterized in that: The CPT is derived from Yarrowia lipolytica, and its GenBank accession number is YALI0C10989g.

7. The construction method according to claim 1, characterized in that: The GenBank accession number of the DGA1 is YALI0E32769g; the GenBank accession number of the DGA2 is YALI0D07986g; the GenBank accession number of the PEX10 is YALI0C01023g; and the GenBank accession number of the TGL4 is YALI0F10010g.

8. The construction method according to claim 1, characterized in that: The recombinant Yarrowia lipolytica is derived from a strain of Yarrowia lipolytica Po1f in which the KU70 gene is knocked out.

9. The recombinant Yarrowia lipolytica constructed by the construction method according to any one of claims 1 to 8.

10. Use of the recombinant Yarrowia lipolytica according to claim 9 in the fermentation production of ricinoleic acid.

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