Construction method and application of recombinant yarrowia lipolytica for synthesizing parthenolide

Abstract

The invention provides a construction method and application of recombinant yarrowia lipolytica for synthesizing parthenolide. The construction method of the recombinant yarrowia lipolytica for synthesizing parthenolide comprises the following steps: overexpressing a parthenolide biosynthetic pathway optimized by codons in the yarrowia lipolytica; adaptive P450 enzyme reductase is screened, so that the electron transfer efficiency in a P450 system is improved; a heterologous non-oxidized phosphopentose pathway and an acetyl coenzyme A shuttle pathway are constructed to enhance the cytoplasmic acetyl-CoA level; the ATP-dependent transporter ABC-G1 is expressed to relieve the metabolic burden of the cells; the endoplasmic reticulum size regulation factor INO2 is expressed to restore the stable state of the endoplasmic reticulum, and the heme precursor ALA is added to improve the functional expression of P450 enzyme, so that the efficient synthesis of parthenolide is finally realized. The construction method of the yarrowia lipolytica for biosynthesis of parthenolide provided by the invention is simple to operate, and the constructed yarrowia lipolytica can efficiently produce parthenolide and has higher production and application values.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering and relates to a construction method and application of a recombinant Yarrowia lipolytica for synthesizing parthenolide. Background Art

[0002] Parthenolide is a sesquiterpene lactone compound with significant biological activity, primarily found in plants of the Asteraceae family (such as feverfew). It has attracted widespread attention for its pharmacological effects, including anti-inflammatory, anti-tumor, antimalarial, and neuroprotective properties. However, traditional plant extraction methods are limited by long plant growth cycles, low content, complex extraction processes, and environmental dependence, making it difficult to meet market demand. Although chemical synthesis has made some progress, its cumbersome steps, low yield, and the use of toxic reagents have restricted its industrial application.

[0003] Currently, the production of parthenolide primarily relies on plant extraction, but this method has significant limitations. First, the content of parthenolide in plant raw materials is generally low, with the content in dried feverfew (Tanacetum parthenium) leaves being only 0.14-0.74% (w / w), while the content in the root bark of Magnolia grandiflora is slightly higher (3.1-8.0%). Second, plant cultivation requires a large amount of land resources and has a long growth cycle, resulting in inefficient raw material supply. In terms of chemical synthesis, existing methods mainly use C10 compounds as starting materials. However, due to the susceptibility of parthenolide's carbon ring core structure to cyclization or rearrangement reactions under acidic, alkaline, and high temperature conditions, coupled with the difficulty of stereoselective control caused by the presence of multiple chiral centers in the molecule, the synthetic route faces technical bottlenecks such as difficult purification and low product yield. These factors collectively make existing production methods difficult to achieve large-scale application and unable to meet the continued growth of market demand. In recent years, synthetic biology technology has provided new ideas for the production of complex natural products. Although scientists have attempted to synthesize parthenolide using Saccharomyces cerevisiae as a base cell, the yield has only been in the milligram range, which is still relatively low overall. Therefore, finding more economical and feasible large-scale preparation and production technologies for parthenolide is of great significance for its promotion and application. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to address the deficiencies of the existing technology and provide a method for biosynthesizing parthenolide using Yarrowia lipolytica, which is specifically achieved by constructing a recombinant Yarrowia lipolytica; in order to address the problem of low efficiency of parthenolide biosynthesis in the existing technology, a matching cytochrome P450 enzyme reductase is screened, a heterologous non-oxidative pentose phosphate pathway and an acetyl-CoA shuttle pathway are constructed to enhance the cytoplasmic acetyl-CoA level, and the ATP-dependent transporter ABC-G1 is expressed to alleviate the metabolic burden of the cells, thereby achieving efficient synthesis of parthenolide.

[0005] In order to solve the above technical problems, the present invention discloses a recombinant Yarrowia lipolytica and its construction method and application, and the constructed recombinant Yarrowia lipolytica can synthesize parthenolide, thereby realizing efficient biosynthesis of parthenolide. The technical solution described in the present invention is as follows: The present invention provides a construction method of a recombinant Yarrowia lipolytica for synthesizing parthenolide, wherein the recombinant Yarrowia lipolytica is introduced with genes related to the biosynthetic pathway of parthenolide; wherein the genes related to the biosynthetic pathway of parthenolide include a combination of a gene encoding a germarene A synthase, a gene encoding a germarene A oxidase, a gene encoding a costunolide synthase, a lactone synthase, and a cytochrome P450 enzyme reductase; wherein the nucleotide sequences of the gene encoding a germarene A synthase (LTC2), a gene encoding a germarene A oxidase (CiGAO), a gene encoding a costunolide synthase (CiCOS), and a gene encoding a lactone synthase (TpPTS) are as shown in SEQ ID No. 1-4. The nucleotide sequence of the cytochrome P450 enzyme reductase encoding gene is any one of SEQ ID No. 5 to 8, preferably SEQ ID No. 5.

[0006] The recombinant Yarrowia lipolytica further introduces any one or more combinations of genes associated with the non-oxidative pentose phosphate pathway, genes associated with the acetyl-CoA shuttle pathway, genes encoding ATP-dependent transporters, and genes encoding endoplasmic reticulum size regulators. The non-oxidative pentose phosphate pathway genes include the phosphoketolase encoding gene PK from Leuconostoc mesenteroides and / or the phosphate acetyltransferase encoding gene PTA from Clostridium kluyveri; the acetyl-CoA shuttle pathway genes include the carnitine acetyltransferase encoding gene CAT2 from Saccharomyces cerevisiae; the ATP-dependent transporter encoding gene ABC-G1 is derived from Grosmania clavigera; and the endoplasmic reticulum size regulator encoding gene INO2 is derived from Yarrowia lipolytica.

[0007] Preferably, the nucleotide sequence of the phosphoketolase encoding gene is shown as SEQ ID No.9; the nucleotide sequence of the phosphate acetyltransferase encoding gene is shown as SEQ ID No.10; the nucleotide sequence of the carnitine acetyltransferase encoding gene is shown as SEQ ID No.11; the ATP-dependent transporter encoding gene ABC-G1, its nucleotide sequence is shown as SEQ ID No.12; the GenBank No. of the endoplasmic reticulum size regulatory factor encoding gene is AOW01275.1.

[0008] The present invention provides a method for constructing a recombinant Yarrowia lipolytica for synthesizing parthenolide, wherein the recombinant Yarrowia lipolytica is introduced with genes related to the biosynthesis pathway of parthenolide; wherein the genes related to the biosynthesis pathway of parthenolide include a combination of a gene encoding a germarene A synthase, a gene encoding a germarene A oxidase, a gene encoding a costunolide synthase, a gene encoding a lactone synthase, and a gene encoding a cytochrome P450 enzyme reductase; wherein the nucleotide sequences of the gene encoding a germarene A synthase, the gene encoding a germarene A oxidase, the gene encoding a costunolide synthase, and the gene encoding a lactone synthase are shown in SEQ ID No. 1-4, respectively; the nucleotide sequence of the gene encoding a cytochrome P450 enzyme reductase is shown in SEQ ID No. As shown in No. 5, the recombinant Yarrowia lipolytica is further introduced with a combination of genes related to the non-oxidative pentose phosphate pathway, genes related to the acetyl-CoA shuttle pathway, genes encoding ATP-dependent transporters, and genes encoding endoplasmic reticulum size regulatory factors; the non-oxidative pentose phosphate pathway-related genes include a combination of genes encoding phosphoketase and genes encoding phosphate acetyltransferase; the nucleotide sequence of the phosphoketase encoding gene is shown in SEQ ID No. 9; the nucleotide sequence of the phosphate acetyltransferase encoding gene is shown in SEQ ID No. 10; the acetyl-CoA shuttle pathway-related genes include a carnitine acetyltransferase encoding gene, the nucleotide sequence of which is shown in SEQ ID No. 11; the nucleotide sequence of the ATP-dependent transporter encoding gene is shown in SEQ ID No. 12; and the GenBank No. of the endoplasmic reticulum size regulatory factor encoding gene is AOW01275.1.

[0009] Preferably, the method for introducing or knocking out related genes in the recombinant Yarrowia lipolytica is: constructing a gene expression cassette or a gene knockout cassette, and inserting the gene expression cassette or the gene knockout cassette into the Yarrowia lipolytica genome by homologous recombination, wherein the introduction or knockout method is the Ura3-blaster-based integration method reported in the literature (Green Chemistry, 2021, 23(2), 780-787; PLoS One, 2018, 13(3), e0194954; Applied and Environmental Microbiology, 2014, 80(5), 1660-1669), and the integration site of gene expression is the neutral site of Yarrowia lipolytica reported (Biotechnology Journal, 2018, 13(9), 1700543).

[0010] The starting strain of the recombinant Yarrowia lipolytica is Yarrowia lipolytica Po1f with the KU70 gene knocked out.

[0011] In a second aspect, the present invention provides a recombinant Yarrowia lipolytica constructed by the construction method described in the first aspect.

[0012] In a third aspect, the present invention provides use of the recombinant Yarrowia lipolytica described in the second aspect in the fermentation production of parthenolide. The recombinant Yarrowia lipolytica is inoculated into a fermentation medium, cultured at 28-30°C for 24-36 hours, and then 5-aminolevulinic acid is added. The culture is continued for 120-150 hours. Preferably, the 5-aminolevulinic acid is added at a concentration of 500-800 μg / L.

[0013] Beneficial effects:

[0014] The present invention specifically adopts the following four steps to construct recombinant Yarrowia lipolytica: 1) overexpressing codon-optimized LTC2, CiGAO, CiCOS, TpPTS and ATR2 in Yarrowia lipolytica to achieve the production of parthenolide from scratch; 2) constructing a heterologous non-oxidative pentose phosphate pathway and an acetyl-CoA shuttle pathway in Yarrowia lipolytica to enhance the level of acetyl-CoA, a precursor for the biosynthesis of parthenolide; 3) overexpressing the ATP-dependent transporter ABC-G1 to alleviate the metabolic burden of cells; 4) overexpressing INO2 to restore endoplasmic reticulum homeostasis and adding the heme precursor ALA to enhance the heme supply and improve the functional expression of P450 enzymes, thereby improving the synthesis of parthenolide. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0016] Figure 1 Schematic diagram of the method for realizing the biosynthesis of parthenolide based on recombinant Yarrowia lipolytica of the present invention.

[0017] Figure 2 This is the plasmid map of pUC-HUH-IntB-PK-PTA in the present invention.

[0018] Figure 3 This is the liquid chromatogram of the fermentation broth obtained by shake flask fermentation of the recombinant strain ML-PA-1. DETAILED DESCRIPTION

[0019] The present invention will be further described below by way of specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0020] The Yarrowia lipolytica Po1f, Yarrowia lipolytica Po1fΔku70, YPD liquid medium, YPD plates, SD-Ura plates, and YPD plates containing 5-fluoroorotic acid described in the following examples are detailed in Chinese Patent CN118421490A. The SD-Leu plates were formulated with 20 g / L glucose, 6.7 g / L YNB (amino-free yeast nitrogen source, purchased from BBI Life Sciences), 0.67 g / L CSM-Leu (complete supplement mixture minus leucine, purchased from MP Biomedicals), and 23 g / L agar powder.

[0021] According to the nucleotide sequence of the β-isopropylmalate dehydrogenase encoding gene LEU2 in Yarrowia lipolytica provided by NCBI (GenBank No. M37309.1), Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize LEU2 and the β-isopropylmalate dehydrogenase encoding gene expression cassette (driven by the endogenous promoter P TEFin , β-isopropylmalate dehydrogenase encoding gene LEU2 and terminator T xpr2t The plasmid pUC57-Leu was inserted between the KpnI and PacI restriction sites of the plasmid pUC57 to obtain the plasmid pUC57-Leu. The construction method of the plasmid pUC57-HisG-URA3-HisG (pUC-HUH) is detailed in Chinese patent CN118421490A.

[0022] In the following examples, the purification and recovery described above used the TaKaRa MiniBEST DNA Fragment Purification Kit (Takara Biotechnology (Dalian) Co., Ltd.) to purify and recover each fragment. The restriction endonucleases described were all purchased from NEB. The one-step cloning described above was achieved using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novozymes Biotechnology Co., Ltd. The Yarrowia lipolytica competent cells and yeast transformation were prepared and achieved using the Zymogen Frozen EZ Yeast Transformation Kit II from ZymoResearch Corporation. The promoter and terminator were obtained by amplification using the corresponding amplification primers using the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template.

[0023] The biosynthetic pathway of the parthenolide synthesized by the present invention is as follows Figure 1 shown.

[0024] Example 1 Construction of the biosynthetic pathway of parthenolide

[0025] The codon-optimized germarene A synthase (LTC2) from lettuce (Lactuca sativa), the germarene A oxidase (CiGAO) from chicory (Cichorium intybus), the costunolide synthase (CiCOS) from chicory (Cichorium intybus), and the lactone synthase (TpPTS) from feverfew (Tanacetum parthenium) were commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the optimized nucleotide sequences LTC2 (SEQ ID No. 1), CiGAO (SEQ ID No. 2), CiCOS (SEQ ID No. 3), and TpPTS (SEQ ID No. 4). The optimized sequences were inserted into the plasmid pUC57 (purchased from GenScript) to obtain plasmids pUC57-LTC2, pUC57-CiGAO, pUC57-CiCOS, and pUC57-TpPTS, respectively. LTC2, CiGAO, CiCOS and TpPTS were obtained after codon optimization from the amino acid sequences of NCBI RefSeq: AAM11627.1, AZI95585.1, AZI95586.1 and X2EVV9.1, respectively.

[0026] (1) Construction of integration plasmid pUC-HUH-IntA-LTC2

[0027] The integration plasmid pUC-HUH-IntA-LTC2 is based on the IntA site integration plasmid pUC-HUH-IntA as the backbone, and the LTC2 expression cassette (P H3 -LTC2-T lip2t In the present invention, the expression cassette is represented as follows: promoter-target gene-terminator, for example, the LTC2 expression cassette is represented as P H3 -LTC2-T lip2t , the promoter is P H3 , the target gene is LTC2, and the terminator is T lip2t The promoter region of the LTC2 expression cassette was amplified using the primer pairs IntA-H3-F / IntA-H3-R and IntA-Lip2t-F / IntA-Lip2t-R described in Table 1 and the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template. H3 and terminator T lip2t Using the DNA of plasmid pUC57-LTC2 as template and the primers IntA-LCT2-F and IntA-LCT2-R described in Table 1 as primers, the amplified fragments were cloned and the two ends of the fragments were cleaved. H3 and terminator T lip2t The LTC2 gene of the homology arm was purified and recovered. The IntA site integration plasmid was digested with restriction endonuclease PacI, and the linearized IntA site integration plasmid was recovered by agarose gel electrophoresis. The linearized IntA site integration plasmid and the various elements (promoter P) in the LTC2 gene expression cassette constructed in this example were combined. H3 , target gene LTC2, terminator T lip2t) A circular recombinant vector was obtained by one-step cloning. The circular recombinant vector was transformed into Escherichia coli DH5α competent cells. Plate screening for ampicillin resistance and verification by colony PCR and sequencing yielded the positive recombinant plasmid pUC-HUH-IntA-LTC2. The recombinant plasmid pUC-HUH-IntA-LTC2 was digested with the restriction endonuclease NotI, and the linearized recombinant plasmid pUC-HUH-IntA-LTC2 was recovered by agarose gel electrophoresis. The IntA site integration plasmid pUC-HUH-IntA is obtained by inserting a 1633 bp sequence (upstream homology arm) upstream of the start codon of the IntA site (YALI1_A15185g) on ​​chromosome A in the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site in the pUC-HUH vector, and a 1621 bp sequence (downstream homology arm) downstream of the stop codon of the IntA site into the HindIII restriction site in the pUC-HUH vector. A HisG tag encoding gene is connected to each end of the Ura3 gene, and the Ura3 gene connected to the HisG tag is located between the upstream and downstream homology arms of the IntA site.

[0028] (2) Construction of integration plasmid pUC-HUH-IntC-CiGAO

[0029] The integration plasmid pUC-HUH-IntC-CiGAO is based on the IntC site integration plasmid pUC-HUH-IntC as the backbone, and the CiGAO expression cassette (P H3 -CiGAO-T mig1t The CiGAO expression cassette promoter P was amplified using the primer pairs IntC-H3-F / IntC-H3-R and IntC-mig1t-F / IntC-mig1t-R described in Table 1, respectively, and the genomic DNA of Yarrowia lipolytica Po1fΔku70 was used as a template. H3 and terminator T mig1t Using the DNA of plasmid pUC57-CiGAO as template and the primers IntC-CiGAO-F and IntC-CiGAO-R described in Table 1 as primers, the amplified fragments were cloned and the two ends of the fragments were cleaved. H3 and terminator T mig1t The CiGAO gene of the homology arm was purified and recovered. The IntC site integration plasmid was digested with restriction endonuclease PacI, and the linearized IntC site integration plasmid was recovered by agarose gel electrophoresis. The linearized IntC site integration plasmid and the various elements (promoter P H3 , target gene CiGAO, terminator T mig1t) A circular recombinant vector was obtained by one-step cloning. The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, screened for ampicillin resistance on plates, and verified by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-HUH-IntC-CiGAO. The recombinant plasmid pUC-HUH-IntC-CiGAO was digested with the restriction endonuclease NotI from NEB, and the linearized recombinant plasmid pUC-HUH-IntC-CiGAO was recovered by agarose gel electrophoresis.

[0030] The IntC site integration plasmid pUC-HUH-IntC is obtained by inserting a 1402 bp sequence (upstream homology arm) upstream of the start codon of the IntC site (YALI1_C25990g) on ​​chromosome C in the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site in the pUC-HUH vector, and a 396 bp sequence (downstream homology arm) downstream of the stop codon of the IntC site into the HindIII restriction site in the pUC-HUH vector. A HisG tag encoding gene is connected to each end of the Ura3 gene, and the Ura3 gene connected to the Ura3 gene is located between the upstream and downstream homology arms of the IntC site.

[0031] (3) Construction of integration plasmid pUC57-Leu-A08-CiCOS

[0032] The recombinant plasmid pUC57-Leu-A08-CiCOS is based on the A08 site integration plasmid pUC-Leu-A08 as the backbone, and the CiCOS expression cassette (P H3 -CiCOS-T OCTt The promoter P of the CiCOS expression cassette was amplified using primers A08-H3-F / A08-H3-R and A08-OCTt-F / A08-OCTt-R as described in Table 1 and genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template. H3 and terminator T OCTt Using the DNA of plasmid pUC57-CiCOS as template and A08-CiCOS-F and A08-CiCOS-R described in Table 1 as primers, we amplified the two ends of the promoter P H3 and terminator T OCTt The CiCOS gene of the homology arm was purified and recovered. The A08 site integration plasmid was digested with restriction endonuclease PacI and the linearized A08 site integration plasmid was recovered by agarose gel electrophoresis. The linearized A08 site integration plasmid and the various elements (promoter P) in the CiCOS gene expression cassette constructed in this example were combined. H3, target gene CiCOS, terminator T OCTt ) A circular recombinant vector was obtained by one-step cloning. The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, screened for ampicillin resistance on plates, and verified by colony PCR and sequencing to obtain the positive recombinant plasmid pUC57-Leu-A08-CiCOS. The recombinant plasmid pUC57-Leu-A08-CiCOS was digested with the restriction endonuclease NotI, and the linearized recombinant plasmid pUC57-Leu-A08-CiCOS was recovered by agarose gel electrophoresis. The A08 site integration plasmid pUC-Leu-A08 is obtained by inserting the 2521bp sequence (upstream homology arm) upstream of the start codon of the A08 site (YALI1_A08073g) on ​​chromosome A in the Yarrowia lipolytica Po1fΔku70 genome into the PacI restriction site of the pUC57-Leu (pUC-Leu) vector, and inserting the 2031bp sequence (downstream homology arm) downstream of the stop codon of the A08 site into the HindIII restriction site of the pUC-Leu vector. The Leu expression cassette is located between the upper and downstream homology arms of the A08 site. The LEU expression cassette (containing the Yarrowia lipolytica endogenous promoter P TEFin , 3(β)-isopropylmalate dehydrogenase encoding gene Leu and terminator T xpr2t ) is also between the upstream and downstream homology arms. (4) Construction of integration plasmid pUC-HUH-Lip1-TpPTS

[0033] The integration plasmid pUC-HUH-Lip1-TpPTS is based on the integration plasmid pUC-HUH-Lip1 at the Lip1 site, and the TpPTS expression cassette (P TEFin -TpPTS-T xpr2t The TpPTS expression cassette promoter P was amplified using the two primer pairs Lip1-TEFin-F / Lip1-TEFin-R and Lip1-xpr2t-F / Lip1-xpr2t-R described in Table 1 and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template. TEFin and terminator T xpr2t Using the DNA of plasmid pUC57-TpPTS as template and the primers Lip1-TpPTS-F and Lip1-TpPTS-R described in Table 1 as primers, the amplified fragments were cloned and the two ends of the fragments were cleaved. TEFin and terminator T xpr2tThe TpPTS gene of the homology arm was purified and recovered. The Lip1 site integration plasmid was digested with restriction endonuclease PacI, and the linearized Lip1 site integration plasmid was recovered by agarose gel electrophoresis. The linearized Lip1 site integration plasmid and the various elements (promoter P TEFin , target gene TpPTS, terminator T xpr2t ) A circular recombinant vector was obtained by one-step cloning. The circular recombinant vector was transformed into Escherichia coli DH5α competent cells. Plate screening for ampicillin resistance and verification by colony PCR and sequencing yielded the positive recombinant plasmid pUC-HUH-Lip1-TpPTS. The recombinant plasmid pUC-HUH-Lip1-TpPTS was digested with the restriction endonuclease NotI, and the linearized recombinant plasmid pUC-HUH-Lip1-TpPTS was recovered by agarose gel electrophoresis. The Lip1 site integration plasmid pUC-HUH-Lip1 is obtained by inserting a 1458 bp sequence (upstream homology arm) upstream of the start codon of the Lip1 site (YALI1_E13541g) on ​​chromosome E in the Yarrowialipolytica Po1fΔku70 genome into the EcoRI restriction site of the pUC-HUH vector, and a 1438 bp sequence (downstream homology arm) downstream of the stop codon of the Lip1 site into the HindIII restriction site of the pUC-HUH vector. A HisG tag encoding gene is connected to both ends of the Ura3 gene, and the Ura3 gene connected to the HisG tag is located between the upstream and downstream homology arms of the Lip1 site.

[0034] Table 1 Primer sequences for constructing plasmids encoding genes related to the biosynthesis pathway of parthenolide

[0035]

[0036] (5) The linearized recombinant plasmids constructed in Examples (1) to (4) of this invention were sequentially transformed into Yarrowia lipolytica Po1fΔku70 for homologous recombination to obtain recombinant bacteria. The specific method is as follows: Yarrowia lipolytica Po1fΔku70 was cultured overnight in YPD liquid medium to prepare competent cells. The linearized pUC-HUH-IntA-LTC2 was transformed into Yarrowia lipolytica Po1fΔku70 competent cells by yeast and homologous recombination was performed. Positive clones were screened using SD-Ura plates and identified by PCR. The positive clones identified by PCR were spread on YPD plates containing 5-fluoroorotic acid and placed in a 30°C incubator for 3 days. Single colonies were streaked on YPD plates containing 5-fluoroorotic acid and SD-Ura plates at the same time to observe the growth of the bacteria. Single colonies that grew on YPD plates containing 5-fluoroorotic acid but failed to grow on SD-Ura plates (i.e., lost a hisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure) were selected and identified by PCR to obtain recombinant strain 1. Linearized pUC-HUH-IntC-CiGAO was transformed into competent cells of recombinant strain 1 via yeast culture. After losing a hisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure, recombinant strain 2 was obtained. The specific construction method was the same as that of recombinant strain 1. Linearized pUC57-Leu-A08-CiCOS was transformed into competent cells of recombinant strain 2 via yeast culture. Positive clones were screened on SD-Leu plates and identified by PCR to obtain recombinant strain 3. Linearized pUC-HUH-Lip1-TpPTS was transformed into competent cells of recombinant strain 3 via yeast culture. After losing a hisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure, ML-PA-1 was obtained. The specific construction method was the same as that of recombinant strain 1.

[0037] Example 2

[0038] Plant-derived cytochrome P450 reductases, including ATR2 from Arabidopsis thaliana, GuCPR from Glycyrrhiza uralensis, LjCPR from Lotus japonicus, and AaCPR1 from Artemisia annua, were codon-optimized and then commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the optimized nucleotide sequences ATR2 (SEQ ID No. 5), GuCPR (SEQ ID No. 6), LjCPR (SEQ ID No. 7), and AaCPR1 (SEQ ID No. 8). The optimized sequences were then inserted into plasmid pUC57 (purchased from GenScript) to obtain plasmids pUC57-ATR2, pUC57-GuCPR, pUC57-LjCPR, and pUC57-AaCPR1, respectively. ATR2, GuCPR, LjCPR, and CiCPR2 were obtained after codon optimization from the amino acid sequences of NCBI RefSeq: NC_003075.7, NC_003075.7, XP_057433390.1, and A0A2U1LIM9.2, respectively. The IntE site integration plasmid pUC-HUH-IntE is obtained by inserting a 1533bp sequence (upstream homology arm) upstream of the start codon of the IntE site (YALI1_E36641g) on ​​chromosome E in the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site of the pUC-HUH vector, and a 1521bp sequence (downstream homology arm) downstream of the stop codon of the IntE site into the HindIII restriction site of the pUC-HUH vector. A HisG tag encoding gene is connected to both ends of the Ura3 gene, and the Ura3 gene connected to the HisG tag is located between the upstream and downstream homology arms of the IntE site.

[0039] (1) Construction of integration plasmid pUC-HUH-IntE-ATR2

[0040] The integration plasmid pUC-HUH-IntE-ATR2 is based on the IntE site integration plasmid pUC-HUH-IntE as the backbone, and the ATR2 gene expression cassette (P EXP -ATR2-T lip2t The ATR2 expression cassette promoter P was amplified using the primer pairs IntE-EXP-F / IntE-EXP-R1 and IntE-Lip2t-F1 / IntE-Lip2t-R described in Table 2, respectively, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template.EXP and terminator T lip2t Using the DNA of plasmid pUC57-ATR2 as template and ATR2-F and ATR2-R as primers described in Table 2, the amplified fragments were cloned and the promoters P EXP and terminator T lip2t The ATR2 gene of the homology arm was purified and recovered. The IntE site integration plasmid was digested with the restriction endonuclease PacI, and the linearized IntE site integration plasmid was recovered by agarose gel electrophoresis. The linearized IntE site integration plasmid and the various elements in the ATR2 gene expression cassette constructed in this example were cloned in one step to obtain the recombinant plasmid pUC-HUH-IntE-ATR2. The recombinant plasmid pUC-HUH-IntE-ATR2 was digested with the restriction endonuclease NotI, and the linearized recombinant plasmid pUC-HUH-IntE-ATR2 was recovered by agarose gel electrophoresis.

[0041] (2) Construction of integration plasmid pUC-HUH-IntE-GuCPR

[0042] The integration plasmid pUC-HUH-IntE-GuCPR is based on the IntE site integration plasmid pUC-HUH-IntE as the backbone, and the GuCPR gene expression cassette (P EXP -GuCPR-T lip2t The ATR2 expression cassette promoter P was amplified using the primer pairs IntE-EXP-F / IntE-EXP-R2 and IntE-Lip2t-F2 / IntE-Lip2t-R described in Table 2, respectively, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template. EXP and terminator T lip2t Using the DNA of plasmid pUC57-GuCPR as template and GuCPR-F and GuCPR-R as primers described in Table 2, the amplified fragments were cloned and cloned. EXP and terminator T lip2t The GuCPR gene of the homology arm was purified and each fragment was recovered. The linearized IntE site integration plasmid prepared in step (1) of this example and each element in the GuCPR gene expression cassette constructed in this example were cloned in one step to obtain the recombinant plasmid pUC-HUH-IntE-GuCPR. The recombinant plasmid pUC-HUH-IntE-GuCPR was digested with the restriction endonuclease NotI, and the linearized recombinant plasmid pUC-HUH-IntE-GuCPR was recovered by agarose gel electrophoresis. (3) Construction of integration plasmid pUC-HUH-IntE-LjCPR

[0043] The integration plasmid pUC-HUH-IntE-LjCPR is based on the IntE site integration plasmid pUC-HUH-IntE as the backbone, and the LjCPR gene expression cassette (P EXP -LjCPR-T lip2t The promoter of the LjCPR expression cassette P was amplified using the primer pairs IntE-EXP-F / IntE-EXP-R3 and IntE-Lip2t-F3 / IntE-Lip2t-R described in Table 2, respectively, and the genomic DNA of Yarrowia lipolytica Po1fΔku70 was used as a template. EXP and terminator T lip2t Using the DNA of plasmid pUC57-LjCPR as template and LjCPR-F and LjCPR-R as primers described in Table 2, we amplified the two ends of the promoter P EXP and terminator T lip2t The LjCPR gene of the homology arm was purified and recovered. The linearized IntE site integration plasmid prepared in step (1) of this example and the various elements in the LjCPR gene expression cassette constructed in this example were cloned in one step to obtain the recombinant plasmid pUC-HUH-IntE-LjCPR. The recombinant plasmid pUC-HUH-IntE-LjCPR was digested with the restriction endonuclease NotI, and the linearized recombinant plasmid pUC-HUH-IntE-LjCPR was recovered by agarose gel electrophoresis.

[0044] (4) Construction of integration plasmid pUC-HUH-IntE-AaCPR1

[0045] The integration plasmid pUC-HUH-IntE-AaCPR1 is based on the IntE site integration plasmid pUC-HUH-IntE as the backbone, and the AaCPR1 gene expression cassette (P EXP -AaCPR1-T lip2t The AaCPR1 expression cassette promoter P was amplified using the primer pairs IntE-EXP-F / IntE-EXP-R4 and IntE-Lip2t-F4 / IntE-Lip2t-R described in Table 2, respectively, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template. EXP and terminator T lip2t. Using the DNA of pUC57-AaCPR1 as a template and IntE-AaCPR1-F and IntE-AaCPR1-R described in Table 2 as primers, the AaCPR1 gene with homology arms at both ends was amplified, and each fragment was purified and recovered. The linearized IntE site integration plasmid prepared in step (1) of this example and each element in the AaCPR1 gene expression cassette were cloned in one step to obtain the recombinant plasmid pUC-HUH-IntE-AaCPR1. The recombinant plasmid pUC-HUH-IntE-AaCPR1 was digested with the restriction endonuclease NotI, and the linearized recombinant plasmid pUC-HUH-IntE-AaCPR1 was recovered by agarose gel electrophoresis.

[0046] Table 2 Primer sequences for constructing recombinant plasmids encoding cytochrome P450 enzyme reductase genes

[0047]

[0048] The linearized recombinant plasmids pUC-HUH-IntE-ATR2, pUC-HUH-IntE-GuCPR, pUC-HUH-IntE-LjCPR, and pUC-HUH-IntE-AaCPR1 constructed in Examples (1) to (4) were respectively transformed into the recombinant strain ML-PA-1 of Example 1 for homologous recombination to obtain recombinant strains ML-PA-2, ML-PA-3, ML-PA-4, and ML-PA-5. The specific construction method was the same as that for recombinant strain 1 in Example 1. Specifically, ML-PA-2 was obtained by transforming pUC-HUH-IntE-ATR2 into the recombinant bacteria ML-PA-1, ML-PA-3 was obtained by transforming pUC-HUH-IntE-GuCPR into the recombinant bacteria ML-PA-1, ML-PA-4 was obtained by transforming pUC-HUH-IntE-LjCPR into the recombinant bacteria ML-PA-1, and ML-PA-5 was obtained by transforming pUC-HUH-IntE-AaCPR1 into the recombinant bacteria ML-PA-1.

[0049] Example 3

[0050] The phosphoketolase PK from Leuconostoc mesenteroides, the phosphate acetyltransferase PTA from Clostridium kluyveri, the carnitine acetyltransferase Cat2 from Saccharomyces cerevisiae, and the ATP-dependent transporter ABC-G1 from Grosmania clavigera were codon-optimized and then commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the optimized nucleotide sequences PK (SEQ ID No. 9), PTA (SEQ ID No. 10), Cat2 (SEQ ID No. 11), and ABC-G1 (SEQ ID No. 12). The optimized sequences were inserted into the plasmid pUC57 (purchased from GenScript) to obtain plasmids pUC57-PK, pUC57-PTA, pUC57-CAT2, and pUC57-ABC-G1, respectively. Using the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template, corresponding primer pairs were designed and the endoplasmic reticulum size regulatory factor encoding gene INO2 (GenBank No. AOW01275.1) was amplified by PCR.

[0051] (1) Construction of integration plasmid pUC-HUH-IntB-PK-PTA

[0052] The integration plasmid pUC-HUH-IntB-PK-PTA is based on pUC-HUH-IntB, and the PK expression cassette (P EXP -PK-T cyc1t ) and PTA expression cassette (P TEFin -PTA-T pex10t ), the specific structure is shown in Figure 2 The promoter P of the PK expression cassette was amplified using the two primer pairs IntB-EXP-F / IntB-EXP-R and IntB-CYC1t-F / IntB-CYC1t-R described in Table 3, respectively, and the genomic DNA of Yarrowia lipolytica Po1fΔku70 was used as a template. EXP and terminator T cyc1t Using IntB-PK-F / IntB-PK-R as primers and pUC57-PK as template, the amplified fragments were cloned with P at both ends. EXP and T cyc1tThe promoter P of the PTA expression cassette was amplified using the two primer pairs IntB-TEFin-F / IntB-TEFin-R and IntB-Pex10t-F / IntB-Pex10t-R described in Table 3, using Yarrowia lipolytica Po1fΔku70 genomic DNA as a template. TEFin and terminator T Pex10t Using IntB-PTA-F / IntB-PTA-R as primers and pUC57-PTA as template, the amplified fragments were cloned with P at both ends. TEFin and T Pex10t The PTA gene of the homology arm was purified and recovered. The IntB site integration plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized IntB site integration plasmid was recovered by agarose gel electrophoresis. The linearized IntB site integration plasmid and the various elements in the PK and PTA gene expression cassette constructed in this example (PK expression cassette promoter P EXP , gene PK, PK expression cassette terminator T cyc1t , PTA expression cassette promoter P TEFin , gene PTA and PTA expression cassette terminator T Pex10t ) was cloned in one step, inserting the PK and PTA gene expression cassettes between the downstream homology arms and the hisG tag-encoding gene of the IntB site integration plasmid to generate a circular recombinant vector. The circular recombinant vector was transformed into Escherichia coli DH5α competent cells. The positive recombinant plasmid pUC-HUH-IntB-PK-PTA was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing. pUC-HUH-IntB-PK-PTA was digested with the restriction endonuclease Swa I from New England Biolabs, and the linearized pUC-HUH-IntB-PK-PTA plasmid was recovered by gel electrophoresis.

[0053] The IntB site integration plasmid pUC-HUH-IntB is obtained by inserting a 2024 bp sequence (upstream homology arm) upstream of the start codon of the IntB site (YALI1_B07043g) on ​​chromosome A in the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site in the pUC-HUH vector, and a 2091 bp sequence (downstream homology arm) downstream of the stop codon of the IntB site into the HindIII restriction site in the pUC-HUH vector. A HisG tag encoding gene is connected to each end of the Ura3 gene, and the Ura3 gene connected to the HisG tag is located between the upstream and downstream homology arms of the IntB site.

[0054] (2) Construction of integration plasmid pUC-HUH-IntD-Cat2

[0055] Integration plasmid pUC-HUH-IntD-Cat2 is based on pUC-HUH- I ntD is the skeleton. I A CAT2 expression cassette (P TEF -CAT2-T OCTt ). The CAT2 expression cassette promoter P was amplified using the IntD-TEF-F / IntD-TEF-R and IntD-OCT-F / IntD-OCT-R primer pairs described in Table 3 and the Yarrowia lipolytica Po1fΔku70 genomic DNA as a template. TEF and terminator T OCTt Using pUC57-CAT2 as template and IntD-CAT2-F and IntD-CAT2-R as primers, the amplified fragments were cloned with promoters P at both ends. TEF and terminator T OCTt The CAT2 gene of the homology arm. Purify and recover each fragment. The IntD site integration plasmid was digested with the restriction endonuclease PacI from NEB, and the linearized IntD site integration plasmid was recovered by agarose gel electrophoresis. The linearized IntD site integration plasmid and each element in the IntD gene expression cassette constructed in this example were cloned in one step to obtain the recombinant plasmid pUC-HUH-IntD-Cat2. The recombinant plasmid pUC-HUH-IntD-Cat2 was digested with the restriction endonuclease NotI from NEB, and the linearized recombinant plasmid pUC-HUH-IntD-Cat2 was recovered by gel electrophoresis.

[0056] The IntD site integration plasmid pUC-HUH-IntD is obtained by inserting a 1443 bp sequence (upstream homology arm) upstream of the start codon of the IntD site (YALI1_D21938g) on ​​chromosome D in the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site in the pUC-HUH vector, and a 1189 bp sequence (downstream homology arm) downstream of the stop codon of the IntD site into the HindIII restriction site in the pUC-HUH vector. A HisG tag encoding gene is connected to each end of the Ura3 gene, and the Ura3 gene connected to the HisG tag is located between the upstream and downstream homology arms of the IntD site.

[0057] (3) Construction of integration plasmid pUC-HUH-IntE5-ABC-G1

[0058] The integration plasmid pUC-HUH-IntE5-ABC-G1 is based on pUC-HUH-IntE5, and the ABC-G1 expression cassette is inserted between the downstream homology arm of IntE5 and the HisG tag encoding gene (P TEFin -ABC-G1-T xpr2t ). The ABC-G1 expression cassette promoter P was amplified using the IntE5-TEFin-F / IntE5-TEFin-R and IntE5-Xpr2t-F / IntE5-Xpr2t-R primers described in Table 3, respectively, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template. TEFin and terminator T xpr2t Using pUC57-ABC-G1 as template and IntE5-ABCG1-F and IntE5-ABCG1-R as primers, the amplified fragments were cloned with promoters P at both ends. TEFin and terminator T xpr2t The ABC-G1 gene of the homology arm was purified and recovered. The IntE5 site integration plasmid was digested with the restriction endonuclease PacI, and the linearized IntE5 site integration plasmid was recovered by agarose gel electrophoresis. The linearized IntE5 site integration plasmid and the various elements of the ABC-G1 gene expression cassette constructed in this example were cloned in one step to obtain the recombinant plasmid pUC-HUH-IntE5-ABC-G1. The recombinant plasmid pUC-HUH-IntE5-ABC-G1 was digested with the restriction endonuclease SwaI, and the linearized recombinant plasmid pUC-HUH-IntE5-ABC-G1 was recovered by agarose gel electrophoresis. The IntE5 site integration plasmid pUC-HUH-IntE5 is obtained by inserting a 2118 bp sequence (upstream homology arm) upstream of the start codon of the IntE site (YALI1_E36641g) in the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site in the pUC-HUH vector, and a 2035 bp sequence (downstream homology arm) downstream of the stop codon of the IntE5 site into the HindIII restriction site in the pUC-HUH vector. A HisG tag encoding gene is connected to each end of the Ura3 gene, and the Ura3 gene connected to the HisG tag is located between the upstream and downstream homology arms of the IntE5 site.

[0059] (4) Construction of integration plasmid pUC-HUH-IntF-INO2

[0060] The integration plasmid pUC-HUH-IntF-INO2 is based on pUC-HUH-IntF, and the INO2 expression cassette is inserted between the downstream homology arm of IntF and the HisG tag encoding gene (PGPAT -INO2-T lip2t The promoter region of the INO2 expression cassette was amplified using the primer pairs IntF-GPAT-F / IntF-GPAT-R and IntF-Lip2t-F / IntF-Lip2t-R described in Table 3, respectively, and the genomic DNA of Yarrowialipolytica Po1fΔku70 as a template. GPAT and terminator T lip2t Using Yarrowia lipolytica Po1fΔku70 genomic DNA as template and IntF-INO2-F and IntF-INO2-R as primers described in Table 3, we amplified the primers with promoter P at both ends. GPAT and terminator T lip2t The INO2 gene of the homology arms was purified and recovered. The IntF site integration plasmid was digested with the restriction endonuclease PacI, and the linearized IntF site integration plasmid was recovered by gel electrophoresis. The linearized IntF site integration plasmid and the elements of the INO2 gene expression cassette constructed in this example were cloned in one step to obtain the recombinant plasmid pUC-HUH-IntF-INO2. The recombinant plasmid pUC-HUH-IntF-INO2 was digested with the restriction endonuclease NotI, and the linearized recombinant plasmid pUC-HUH-IntF-INO2 was recovered by gel electrophoresis.

[0061] The IntF site integration plasmid pUC-HUH-IntF is obtained by inserting a 1610 bp sequence (upstream homology arm) upstream of the start codon of the IntF site (YALI0_F24167g) on ​​chromosome F in the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site in the pUC-HUH vector, and inserting a 1852 bp sequence (downstream homology arm) downstream of the stop codon of the IntF site into the HindIII restriction site in the pUC-HUH vector. A HisG tag encoding gene is connected to each end of the Ura3 gene, and the Ura3 gene connected to the HisG tag is located between the upstream and downstream homology arms of the IntF site.

[0062] Table 3 Primer sequences for constructing the recombinant plasmid described in Example 3

[0063]

[0064]

[0065] The linearized recombinant plasmids pUC-HUH-IntB-PK-PTA, pUC-HUH-IntD-Cat2, pUC-HUH-IntE5-ABC-G1, and pUC-HUH-IntF-INO2 constructed in Examples (1) to (4) were sequentially transformed into the recombinant bacteria ML-PA-2 constructed in Example 2 to obtain recombinant Yarrowia lipolytica ML-PA-10. The specific construction method was the same as the construction of recombinant bacteria 1 in Example 1. Specifically, the plasmid pUC-HUH-IntB-PK-PTA was transformed into the recombinant bacteria ML-PA-2 to obtain intermediate bacteria 1, pUC-HUH-IntD-Cat2 was transformed into intermediate bacteria 1 to obtain intermediate bacteria 2, pUC-HUH-IntE5-ABC-G1 was transformed into intermediate bacteria 2 to obtain intermediate bacteria 3, and pUC-HUH-IntF-INO2 was transformed into intermediate bacteria 3 to obtain ML-PA-10.

[0066] Example 4 Fermentation of recombinant Yarrowia lipolytica to produce parthenolide

[0067] (1) Seed culture: Po1fΔku70 and ML-PA-1, ML-PA-2, ML-PA-3, ML-PA-4, ML-PA-5, and ML-PA-10 constructed in Examples 1-3 were cultured on YPD plates at 28°C for 48 h. A single colony was picked and placed in 5 mL of YPD liquid medium and cultured at 28°C, 200 rpm for 16-24 h. 600 A value of around 5 is used as fermentation seed liquid.

[0068] (2) Shake flask fermentation: The above seed solution was transferred to a 250 mL shake flask containing 50 mL fermentation medium to make its initial OD 600The fermentation medium is controlled at 0.5. The fermentation medium is then placed in a shaker at 28°C and 200 rpm for fermentation. After culturing for 30 hours, sterilized 5-aminolevulinic acid (ALA) is added to a final concentration of 500 μg / L. The culture is continued for 120 hours, and the fermentation is completed to obtain a fermentation broth. The fermentation medium is composed of: 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 22 g / L glucose, 2.0 mL / L trace metal mother liquor, and 1.0 mL / L vitamin mother liquor. The vitamin mother liquor is an aqueous solution containing the following ingredients: 0.05 g / L biotin, 1 g / L calcium pantothenate, 1 g / L thiamine, 1 g / L pyridoxine, 1 g / L nicotinic acid, 0.2 g / L para-aminobenzoic acid, and 25 g / L inositol. The trace metal mother liquor is an aqueous solution containing the following ingredients: 15 g / L EDTA, 4.5 g / L zinc sulfate, 0.3 g / L cobalt chloride, 1 g / L manganese chloride, 0.1 g / L copper sulfate, 4.5 g / L calcium chloride, 3 g / L ferrous sulfate, 0.4 g / L sodium molybdate, 1 g / L boric acid, and 0.1 g / L potassium iodide.

[0069] This example also involved fed-batch fermentation of the recombinant strain: recombinant strain ML-PA-10 was inoculated into 50 mL of YPD liquid medium and cultured at 30°C and 220 rpm for 24 hours to obtain a seed solution. This solution was then inoculated into a fermentor at a 5% v / v inoculum. 3 L of fermentation medium was added to a 5 L fermentor. After 24 hours of incubation, sterilized heme precursor 5-aminolevulinic acid (ALA) was added to a final concentration of 500 μg / L. The dissolved oxygen concentration was maintained at 20% for 0-48 hours of fermentation; if the fermentation time exceeded 48 hours, the dissolved oxygen concentration was maintained at 30%. The pH was maintained at 5.5 throughout the fermentation process until the end of the fermentation. Feeding was performed throughout the fermentation process to maintain a stable glucose concentration of 5 g / L in the fermentor. The fermentation temperature was 30°C, and the agitation speed was adjusted between 300 and 1000 rpm based on the dissolved oxygen concentration (DO). The incubation time was up to 120 hours. The composition of the fermentation medium is the same as that of the fermentation medium described in step (2) of this embodiment.

[0070] After fermentation, the target product was isolated and analyzed by HPLC according to the following method. The extraction and analysis of parthenolide were as follows: After fermentation, the bacterial broth was collected and centrifuged at 5000 rpm for 5 minutes. 1 mL of the fermentation supernatant was aspirated, 1 mL of dodecane was added, and the mixture was vortexed for 20 minutes. The upper organic phase was aspirated and transferred to a 1.5 mL EP tube. Then, 100 μL of anhydrous sodium sulfate was added to the EP tube. The organic phase was thoroughly vortexed to remove trace moisture from the organic phase. The mixture was centrifuged at 12000 rpm for 2 minutes. The upper extract was aspirated and filtered through a 0.22 μM organic filter membrane before being placed in an HPLC injection vial for HPLC analysis. Parthenolide detection conditions: The chromatographic column was a C18 column (4.6×150 mm, 4 μm, Agilent), the column temperature was 25°C, the detector was a 1260VWD UV detector (Agilent), the mobile phase A was acetonitrile, the mobile phase B was 0.1% (v / v%) formic acid in water, and the total flow rate was 1 mL / min; the detector wavelength was set at 210 nm. The chromatographic program was as follows: initial concentration was 65% phase A and 35% phase B, hold for 5 minutes; from 5 to 12 minutes, the gradient was adjusted to 100% phase A and 0% phase B, hold for 3 minutes; from 15 to 17 minutes, the gradient was adjusted back to 65% phase A and 35% phase B, hold for 3 minutes. Standard solutions of varying concentrations (1-500 mg / L) of parthenolide standards were prepared, and a standard curve was constructed based on the peak area and concentration of the standard solutions. The product yield was calculated from this curve.

[0071] After the fermentation, the yield of parthenolide of the starting strain Po1fΔku70 was 0.00 mg / L after HPLC detection. The yield of parthenolide of ML-PA-1, ML-PA-2, ML-PA-3, ML-PA-4, ML-PA-5 and ML-PA-10 after shake flask fermentation culture was 51.21 mg / L, 112.65 mg / L, 89.32 mg / L, 73.76 mg / L, 84.32 mg / L and 625.32 mg / L respectively after HPLC detection. Among them, the yield of parthenolide produced by the recombinant strain ML-PA-10 fermentation was the highest, and the parthenolide obtained by fed-batch fermentation could reach 2.32 g / L, that is, 2.32 g of parthenolide could be obtained per liter of fermentation liquid. Figure 3 As shown in the figure, the peak time of parthenolide is about 10.4 min.

[0072] The present invention provides a method for constructing and using a recombinant Yarrowia lipolytica for synthesizing parthenolide. While numerous methods and approaches exist for implementing this technical solution, the foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A method for constructing a recombinant Yarrowia lipolytica for synthesizing parthenolide, characterized in that: The recombinant Yarrowia lipolytica is introduced with genes related to the biosynthesis pathway of parthenolide; Wherein, the genes related to the parthenolide biosynthesis pathway include a combination of a gene encoding a germarene A synthase, a gene encoding a germarene A oxidase, a gene encoding a costunolide synthase, a gene encoding a lactone synthase, and a gene encoding a cytochrome P450 enzyme reductase; The nucleotide sequences of the gene encoding the germarene A synthase, the gene encoding the germarene A oxidase, the gene encoding the costunolide synthase, and the gene encoding the lactone synthase are shown in SEQ ID No. 1-4, respectively; The nucleotide sequence of the cytochrome P450 enzyme reductase encoding gene is any one of SEQ ID No. 5-8.

2. The construction method according to claim 1, characterized in that The recombinant Yarrowia lipolytica is introduced with any one or more combinations of genes related to the non-oxidative pentose phosphate pathway, genes related to the acetyl-CoA shuttle pathway, genes encoding ATP-dependent transporters, and genes encoding endoplasmic reticulum size regulatory factors.

3. The construction method according to claim 2, characterized in that The non-oxidative pentose phosphate pathway-related genes include a phosphoketase encoding gene derived from Leuconostoc mesenteroides and / or a phosphate acetyltransferase encoding gene derived from Clostridium kluyveri; The acetyl-CoA shuttle pathway-related genes include carnitine acetyltransferase encoding genes from Saccharomyces cerevisiae; The ATP-dependent transporter encoding gene is derived from Grosmania clavigera; The endoplasmic reticulum size regulatory factor encoding gene is derived from Yarrowia lipolytica.

4. The construction method according to claim 3, characterized in that The nucleotide sequence of the phosphoketolase encoding gene is shown in SEQ ID No. 9; the nucleotide sequence of the phosphate acetyltransferase encoding gene is shown in SEQ ID No. 10; the nucleotide sequence of the carnitine acetyltransferase encoding gene is shown in SEQ ID No. 11; the nucleotide sequence of the ATP-dependent transporter encoding gene is shown in SEQ ID No. 12; and the GenBank sequence number of the endoplasmic reticulum size regulatory factor encoding gene is AOW01275.

1.

5. The construction method according to claim 1, characterized in that The nucleotide sequence of the cytochrome P450 enzyme reductase encoding gene is shown in SEQ ID No.

5. The recombinant Yarrowia lipolytica further introduces a combination of genes related to the non-oxidative pentose phosphate pathway, genes related to the acetyl-CoA shuttle pathway, genes encoding ATP-dependent transporters, and genes encoding endoplasmic reticulum size regulatory factors; The non-oxidative pentose phosphate pathway-related genes include a combination of a phosphoketase encoding gene from Leuconostoc mesenteroides and a phosphate acetyltransferase encoding gene from Clostridium kluyveri; the nucleotide sequence of the phosphoketase encoding gene is shown in SEQ ID No. 9; the nucleotide sequence of the phosphate acetyltransferase encoding gene is shown in SEQ ID No. 10; The acetyl-CoA shuttle pathway-related gene includes a carnitine acetyltransferase encoding gene from Saccharomyces cerevisiae, and the nucleotide sequence of the carnitine acetyltransferase encoding gene is shown in SEQ ID No. 11; The ATP-dependent transporter encoding gene is derived from Grosmania clavigera, and its nucleotide sequence is shown in SEQ ID No. 12; The endoplasmic reticulum size regulatory factor encoding gene is derived from Yarrowia lipolytica, and its GenBank sequence number is AOW01275.

1.

6. The construction method according to claim 1, characterized in that The starting strain of the recombinant Yarrowia lipolytica is Yarrowia lipolytica Po1f with the KU70 gene knocked out.

7. The recombinant Yarrowia lipolytica constructed by the construction method according to any one of claims 1 to 6.

8. Use of the recombinant Yarrowia lipolytica according to claim 7 in the fermentation production of parthenolide.

9. The use according to claim 8, characterized in that The recombinant Yarrowia lipolytica is inoculated into a fermentation medium, and after fermentation culture at 28-30° C. for 24-36 hours, 5-aminolevulinic acid is added, and the culture is continued for 120-150 hours.

10. The use according to claim 9, characterized in that The added concentration of the 5-aminolevulinic acid is 500-800 μg / L.

Images