The invention discloses a method for biologically synthesizing 1apos by utilizing yarrowia lipolytica. , 4apos; process for the preparation of-trans-abscisic acid glycols

Abstract

The invention provides a method for biologically synthesizing 1 ', 4'-trans-abscisic acid diol by utilizing yarrowia lipolytica. According to the invention, a strain of recombinant yarrowia lipolytica capable of producing the abscisic acid at high yield is used as a chassis cell, and the synthesis of the 1 ', 4'-trans-abscisic acid glycol to the abscisic acid is blocked by knocking out sesquiterpene dehydrogenase BcAba4 in the chassis cell, so that the yarrowia lipolytica capable of producing the 1 ', 4'-trans-abscisic acid glycol is obtained; a citric acid transporter 1 coding gene CEX1 is further knocked out, ATP citric acid lyase coding genes ACL1 and ACL2, a peroxisome carnitine acetyltransferase coding gene CAT2, lipase coding genes TGL3 and TGL4 and a peroxisome multifunctional enzyme coding gene MFE1 are overexpressed, and an ERG9 gene promoter is replaced by a growth dependent promoter PD19. The supply of the precursor acetyl CoA is improved, the path of the precursor acetyl CoA to grease synthesis is reduced, the metabolic flux from FPP to squalene is reduced, and the accumulation capacity of 1 ', 4'-trans-abscisic acid diol in the recombinant bacteria is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering and relates to a method for biosynthesizing 1',4'-trans-abscisic acid diol by utilizing Yarrowia lipolytica. Background Art

[0002] Abscisic acid (ABA) is an important sesquiterpenoid plant hormone, ranked alongside auxin, gibberellins, cytokinins, and ethylene as one of the five natural plant regulators. However, it is sensitive to ultraviolet light and is a highly photodegradable compound. Studies have shown that under sunlight, ABA rapidly undergoes cis-trans isomerization to form its trans form. Compared to the natural cis form, the trans form exhibits significantly reduced activity. This property severely limits the practical application of ABA, leading to the development of highly active and stable ABA analogs.

[0003] 1',4'-trans-abscisic acid diol (1',4'-trans-diolABA) is a structural analogue with plant growth regulating activity and similar structure to abscisic acid. The compound was first obtained by a research team from the State University of New York through chemical synthesis in 1972. Subsequently, scientists such as Hirai from Kyoto University in Japan identified the presence of this substance in the fermentation broth of Botrytis cinerea and confirmed that it is an endogenous precursor for abscisic acid biosynthesis. Physiological function studies have shown that 1',4'-trans-abscisic acid diol can significantly inhibit the growth of detached hypocotyls of soybeans and the germination of rice seeds. Experiments in tobacco seedlings have shown that this compound can produce physiological effects similar to exogenous abscisic acid by upregulating the expression of abiotic stress response genes and potential disease resistance-related genes.

[0004] Currently, the production of ABA analogs primarily relies on chemical total synthesis. However, due to long synthetic routes and low overall yields, production costs remain high, severely limiting their application in agricultural production. In contrast, the production of ABA analogs from microbial secondary metabolites offers significant advantages. The ABA biosynthetic pathway in Botrytis cinerea has been clearly characterized, and this process is regulated by a gene cluster consisting of four genes: BcABA1-BcABA4. In 2019, a Japanese research team identified for the first time the key enzyme in this pathway - the novel sesquiterpene synthase BcAba3, and through biotransformation and in vitro enzyme catalysis experiments, clarified the four-step catalytic process from farnesyl pyrophosphate (FPP) to abscisic acid: (1) BcAba3 catalyzes the cyclization of FPP to form the abscisic acid skeleton; (2) cytochrome P450 enzymes BcAba1 and BcAba2 catalyze the oxidation to produce α-agar-acid and 1',4'-abscisic acid diol in turn; (3) Finally, BcAba4 catalyzes the dehydrogenation of 1',4'-abscisic acid diol to produce abscisic acid.

[0005] The Chengdu Institute of Biology, Chinese Academy of Sciences, successfully constructed a strain that produces 1',4'-trans-abscisic acid diol by knocking out the BcAba4 gene in Botrytis cinerea through genetic engineering. However, the product yield from this strain was only 0.6 g / L, far below the requirements for industrial production. Therefore, screening and constructing efficient microbial chassis cells and establishing an efficient biosynthetic system for 1',4'-trans-abscisic acid diol are of great significance for achieving large-scale production of this compound. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to address the deficiencies in the prior art and provide a method for constructing and applying a recombinant Yarrowia lipolytica for synthesizing 1',4'-trans-abscisic acid diol, which is specifically achieved by constructing a recombinant Yarrowia lipolytica; in response to the problem of insufficient precursor supply in the 1',4'-trans-abscisic acid diol synthesis pathway, resulting in low biosynthesis efficiency, the efficient synthesis of 1',4'-trans-abscisic acid diol is achieved by enhancing the expression of acetyl CoA, a precursor of the mevalonate pathway, and weakening the metabolic flow from acetyl CoA to oils and from FPP to squalene synthesis pathways.

[0007] To address the above technical issues, the present invention discloses a method for constructing and using a recombinant Yarrowia lipolytica for synthesizing 1',4'-trans-abscisic acid diol. The recombinant Yarrowia lipolytica is capable of synthesizing 1',4'-trans-abscisic acid diol, thereby achieving efficient biosynthesis of 1',4'-trans-abscisic acid diol. The technical solutions of the present invention are as follows:

[0008] The present invention provides a method for constructing a recombinant Yarrowia lipolytica for synthesizing 1',4'-trans-abscisic acid diol. The starting strain of the recombinant Yarrowia lipolytica is a Yarrowia lipolytica that produces abscisic acid.

[0009] The recombinant Yarrowia lipolytica blocks the synthesis pathway from 1',4'-trans-abscisic acid diol to abscisic acid, and simultaneously undergoes at least one of the following modifications a1 to a6:

[0010] a1, knockout of the citrate transporter 1 encoding gene CEX1;

[0011] a2, introduction of ATP citrate lyase encoding genes ACL1 and ACL2;

[0012] a3, introduction of the peroxisomal carnitine acetyltransferase encoding gene CAT2;

[0013] a4, introduction of lipase encoding genes TGL3 and TGL4;

[0014] a5, introduction of the peroxisome multifunctional enzyme encoding gene MFE1;

[0015] a6, replaces the natural promoter of the ERG9 gene, reduces the flux of the sesquiterpene synthesis precursor FPP to squalene, and thereby increases the synthesis of 1',4'-trans-abscisic acid diol.

[0016] Preferably, the Yarrowia lipolytica producing abscisic acid has the phosphatidic acid phosphatase and RAS2 sites knocked out, and expresses the abscisic acid synthesis gene cluster (BcABA1-BcABA4) encoding gene, 3-hydroxy-3-methylglutaryl CoA reductase encoding gene, farnesyl pyrophosphate synthase encoding gene, citrate transporter encoding gene, malic enzyme encoding gene and hemoglobin encoding gene. Preferably, the Yarrowia lipolytica producing abscisic acid is the recombinant strain 6 constructed in Chinese patent CN117778218A.

[0017] Wherein, the blocking is achieved by knocking out the gene encoding the sesquiterpene dehydrogenase. Preferably, the starting strain of the present invention overexpresses BcABA3 and BcABA4 at the A08 site via the recombinant plasmid pUC-leu-A08-BcABA3-BcABA4. Therefore, by replacing pUC-leu-A08-BcABA3-BcABA4 with the recombinant plasmid pUC-HUH-A08-BcABA3, the sesquiterpene dehydrogenase BcAba4 in the starting strain can be knocked out, thereby obtaining Yarrowia lipolytica capable of accumulating 1',4'-trans-abscisic acid diol.

[0018] The CEX1, ACL1, ACL2, CAT2, TGL3, TGL4, and MFE1 are all derived from Yarrowia lipolytica. The GenBank sequence number of CEX1 is CR382130.1, the GenBank sequence number of ACL1 is VBB77400.1, the GenBank sequence number of ACL2 is QNP98078.1, the GenBank sequence number of CAT2 is UTQH01000002.1, the GenBank sequence number of TGL3 is AOW04197.1, the GenBank sequence number of TGL4 is AOW06932.1, and the GenBank sequence number of MFE1 is VBB78987.1.

[0019] The natural promoter of the ERG9 gene is replaced by the growth-dependent promoter PD19, and the sequence of the PD19 is shown in SEQ ID No. 4;

[0020] The GenBank sequence number of the ERG9 gene is AOW00473.1.

[0021] Preferably, the present invention provides a method for constructing a recombinant Yarrowia lipolytica, wherein the starting strain of the recombinant Yarrowia lipolytica is a Yarrowia lipolytica that produces abscisic acid; the recombinant Yarrowia lipolytica blocks the synthesis pathway from 1',4'-trans-abscisic acid diol to abscisic acid, and the blocking is achieved by knocking out the gene encoding sesquiterpene dehydrogenase; the recombinant Yarrowia lipolytica also undergoes all of the following modifications a1 to a6;

[0022] a1, knockout of the citrate transporter 1 encoding gene CEX1;

[0023] a2, introduction of ATP citrate lyase encoding genes ACL1 and ACL2;

[0024] a3, introduction of the peroxisomal carnitine acetyltransferase encoding gene CAT2;

[0025] a4, introduction of lipase encoding genes TGL3 and TGL4;

[0026] a5, introduction of the peroxisome multifunctional enzyme encoding gene MFE1;

[0027] a6, Growth-dependent promoter PD19 replaces the native promoter of the ERG9 gene.

[0028] Among them, the GenBank sequence number of CEX1 is CR382130.1, the GenBank sequence number of ACL1 is VBB77400.1, the GenBank sequence number of ACL2 is QNP98078.1, the GenBank sequence number of CAT2 is UTQH01000002.1, the GenBank sequence number of TGL3 is AOW04197.1, the GenBank sequence number of TGL4 is AOW06932.1, the GenBank sequence number of MFE1 is VBB78987.1, and the sequence of PD19 is shown in SEQ ID No. 4.

[0029] Further preferably, the method for introducing or knocking out the relevant gene in the recombinant Yarrowia lipolytica is: constructing a gene expression cassette or a gene knockout cassette of the relevant gene, and inserting the gene expression cassette or the gene knockout cassette into the Yarrowia lipolytica genome by homologous recombination, and 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).

[0030] In a second aspect, the present invention provides a recombinant Yarrowia lipolytica constructed by the construction method described in the first aspect.

[0031] In a third aspect, the present invention provides the use of the recombinant Yarrowia lipolytica described in the second aspect in the fermentation production of 1',4'-trans-abscisic acid diol. Preferably, the recombinant Yarrowia lipolytica is inoculated into a fermentation medium, and after fermentation and culture at 20-30°C for 24-36 hours, 5-aminolevulinic acid (ALA) is added, and culture is continued for 120-144 hours. Preferably, after fermentation and culture at 28°C for 24-36 hours, ALA is added, and culture is continued for 120 hours.

[0032] Wherein, the added concentration of 5-aminolevulinic acid is 500-800 μg / L, preferably 500 μg / L.

[0033] Preferably, the fermentation method for preparing 1',4'-trans-abscisic acid diol comprises the following steps: inoculating the strain into YPD medium for cultivation as seed liquid, and waiting for OD 600When the pH reaches about 5.0, it is inoculated into the fermentation medium at an inoculum size of 5% v / v. After culturing for 24-36 hours, sterilized heme precursor 5-aminolevulinic acid (ALA) is added to make the final concentration 500 μg / L. After culturing at a suitable temperature for a period of time, fed-batch fermentation with feeding liquid is started.

[0034] Further preferably, the fermentation medium is composed of: 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 120 g / L glucose, 2.0 mL / L metal ion mother solution, and 1.0 mL / L vitamin mother solution (the composition and preparation method of the metal ion mother solution and the vitamin mother solution are described in Yeast, 1992, 8, 501–517).

[0035] Further preferably, during fermentation in a liquid culture medium, the carbon source and nitrogen source required for the synthesis of 1',4'-trans-abscisic acid diol can be provided to the bacteria by automatically adding the feed liquid, thereby maintaining a good carbon-nitrogen ratio required by the fermentation process, and increasing the cell concentration and product synthesis efficiency in the fermentation system, thereby increasing the yield of 1',4'-trans-abscisic acid diol.

[0036] Further preferably, the method for automatically adding feeding liquid is to link the stirring speed with the dissolved oxygen concentration (DO) and automatically adjust it between 300-1000 rpm to control the DO to maintain at 20%. When the DO is higher than 10%, indicating that the cell respiration is weakened and in a state of sugar deficiency, sugar is automatically added at a rate of 10 mL / h. Once the DO is lower than 10%, sugar addition stops. When the sugar is consumed and the DO rises to greater than 10%, sugar addition automatically restarts. This reciprocating process controls the glucose concentration within a stable range, providing a suitable environment for the growth and metabolism of the strain. On the one hand, it avoids the errors caused by manual calculation of the glucose rate, and on the other hand, it simplifies the fermentation process.

[0037] Beneficial effects:

[0038] The present invention is based on the knockout of the Yarrowia lipolytica yeast responsible for encoding the non-homologous recombination gene KU70, thereby enhancing its homologous recombination ability, realizing gene integration through the homologous recombination function of Yarrowia lipolytica itself, and greatly improving the genetic stability of the introduced gene. Specifically, the following four steps are taken to construct the recombinant Yarrowia lipolytica: 1) using the recombinant strain 6 of Yarrowia lipolytica with high abscisic acid production (see Chinese patent CN117778218A for details) as the starting strain, knocking out the short-chain dehydrogenase BcAba4 that catalyzes the conversion of 1',4'-trans-abscisic acid diol to abscisic acid, thereby resulting in a large accumulation of 1',4'-trans-abscisic acid diol in Yarrowia lipolytica; 2) knocking out the gene encoding the citrate transporter 1 CEX1, overexpressing the gene encoding the ATP citrate lyase, and The results showed that the overexpression of the genes encoding ACL1 and ACL2 and the peroxisomal carnitine acetyltransferase CAT2 enhanced the synthesis of acetyl-COA, a key precursor in the biosynthesis of 1',4'-trans-abscisic acid diol; 3) overexpression of the two lipase-encoding genes TGL3 and TGL4 and the peroxisomal multifunctional enzyme-encoding gene MFE1 reduced lipid accumulation and enhanced the β-oxidation pathway, thereby enhancing lipid degradation and producing more acetyl-COA, shifting the metabolic flux from lipid synthesis to the synthesis of 1',4'-trans-abscisic acid diol; 4) replacement of the natural promoter of the ERG9 gene with the growth-dependent promoter PD19 reduced the flow of the sesquiterpene synthesis precursor FPP to squalene, thereby increasing the synthesis of 1',4'-trans-abscisic acid diol.

[0039] The method for constructing Yarrowia lipolytica for biosynthesizing 1',4'-trans-abscisic acid diol provided by the present invention is simple to operate, and the constructed Yarrowia lipolytica can efficiently produce 1',4'-trans-abscisic acid diol and has high production and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] 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.

[0041] Figure 1 It is the chemical structural formula of 1',4'-trans-abscisic acid diol and abscisic acid, wherein A is the structural formula of 1',4'-trans-abscisic acid diol, and B is the structural formula of abscisic acid.

[0042] Figure 2 The figure is a synthetic pathway diagram of the biosynthesis of 1',4'-trans-abscisic acid diol based on recombinant Yarrowia lipolytica in the present invention.

[0043] Figure 3 This is the plasmid map of pUC-HUH-A08-BcABA3 in the present invention.

[0044] Figure 4This is the plasmid map of PUC-HUH-IntE2-ACL1-ACL2 in the present invention.

[0045] Figure 5 This is the plasmid map of pUC-HUH-PD19-ERG9 in the present invention. DETAILED DESCRIPTION

[0046] 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.

[0047] The high-abscisic acid-producing recombinant Yarrowia lipolytica strain 6 described in the following examples has phosphatidic acid phosphatase and RAS2 sites knocked out, and expressed the abscisic acid synthesis gene cluster (BcABA1-BcABA4), 3-hydroxy-3-methylglutaryl CoA reductase, farnesyl pyrophosphate synthase, citrate transporter, malic enzyme and hemoglobin encoding genes, achieving 5433.23 mg / L abscisic acid production. For details, see Chinese patent CN117778218A.

[0048] The present invention eliminates the sesquiterpene dehydrogenase BcAba4 in a recombinant strain of Yarrowia lipolytica that produces high abscisic acid, thereby blocking the synthesis of 1',4'-trans-abscisic acid diol to abscisic acid. Furthermore, the ability of the engineered strain to accumulate 1',4'-trans-abscisic acid diol is enhanced by eliminating the citrate transporter 1 encoding gene CEX1, overexpressing the ATP citrate lyases ACL1 and ACL2, overexpressing the peroxisomal carnitine acetyltransferase CAT2, the lipase encoding genes TGL3 and TGL4, and the peroxisomal multifunctional enzyme encoding gene MFE1, and replacing the natural promoter of the ERG9 gene with the growth-dependent promoter PD19.

[0049] The A08 site integration plasmid pUC-HUH-A08 is obtained by inserting a 2521 bp sequence (upstream homology arm) upstream of the start codon of the A08 site (YALI0_A08360g) on ​​the chromosome of the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site of the pUC-HUH vector, and a 2031 bp sequence (downstream homology arm) downstream of the stop codon of the A08 site into the HindIII restriction site of 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 A08 site.

[0050] The IntE2 site integration plasmid pUC-HUH-IntE2 is obtained by inserting a 2118 bp 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 2035 bp 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 IntE2 site.

[0051] 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 Yarrowia lipolytica 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.

[0052] 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 B in the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site of 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 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 IntB site.

[0053] The IntC3 site integration plasmid pUC-HUH-IntC3 is obtained by inserting a 1627 bp sequence (upstream homology arm) upstream of the start codon of the IntC3 site (YALI1_B07043g) on ​​chromosome C in the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site of the pUC-HUH vector, and a 1594 bp sequence (downstream homology arm) downstream of the stop codon of the IntC3 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 IntC3 site.

[0054] In the following examples, the YPD liquid medium has a formula of 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose. The YPD plate has a formula of 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, and 20 g / L agar.

[0055] The SD-Ura plate has a formula of 20 g / L glucose, 6.7 g / L YNB (amino-free yeast nitrogen source, purchased from BBI Life Sciences), 0.67 g / L CSM-Ura (complete supplement mixture minus uracil, purchased from MP Biomedicals), and 23 g / L agar powder.

[0056] The YPD plate of 5-fluoroorotic acid has the following formula: 1 g / L 5-fluoroorotic acid, 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose and 23 g / L agar powder.

[0057] In the following examples, the promoter P TEFin 、P GPDin and P TEF , the terminator T lip2t and T xpr2t The nucleotide sequence of the promoter is shown in Chinese patent CN118421490A. FBA 、P GPD The nucleotide sequences of PD19 are shown in SEQ ID No. 2 to 4, and the terminator T ACL1t 、T ACL2t and T OCTt The nucleotide sequences are shown in SEQ ID No.5 to 7.

[0058] The structural formula of the 1',4'-trans-abscisic acid diol synthesized by the present invention is as follows: Figure 1 The biosynthetic pathway is shown in Figure 2 shown.

[0059] Example 1 Amplification of genetic elements and preparation of integration plasmids

[0060] (1) Preparation of target genes

[0061] According to the nucleotide sequence of the orotidine-5′-phosphate decarboxylase encoding gene URA3 in Yarrowia lipolytica provided by NCBI (GenBank No. AJ306421.1) and HisG tag (GenBank No. AF324729.1), Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the two HisG tag encoding gene sequences and inserted between the EcoRI and HindIII restriction sites of the plasmid pUC57 (purchased from Jinweizhi Company). An orotidine-5′-phosphate decarboxylase encoding gene expression cassette (driven by the endogenous promoter P of Yarrowia lipolytica) was inserted between the two HisG tag encoding gene sequences. TEFin , orotidine-5′-phosphate decarboxylase encoding genes Ura and T xpr2t composition) to achieve the recycling of URA3 marker and obtain the plasmid pUC57-HisG-URA3-HisG (pUC-HUH).

[0062] The sesquiterpene synthase BcABA3 from Botrytis cinerea (GenBank accession number: 5434511) was codon-optimized and commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the optimized BcABA3 (SEQ ID No. 1). The optimized sequence was inserted into the plasmid pUC57 (purchased from GenScript) to obtain the plasmid pUC57-BcABA3.

[0063] Using Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, corresponding primer pairs were designed and PCR amplified to amplify CEX1 (GenBank No. CR382130.1), ATP citrate lyases ACL1 (GenBank No. VBB77400.1) and ACL2 (GenBank No. QNP98078.1), peroxisomal carnitine acetyltransferase CAT2 (GenBank No. UTQH01000002.1), lipase-encoding genes TGL3 (GenBank No. AOW04197.1) and TGL4 (GenBank No. AOW06932.1), and peroxisomal multifunctional enzyme-encoding gene MFE1 (GenBank No. VBB78987.1). The native promoter of the ERG9 gene, as well as the promoter and terminator, were replaced by the growth-dependent promoter PD19 (sequence shown in SEQ ID No. 4). Details are provided in the construction process of the integration plasmid below.

[0064] (2) Construction of integration plasmid

[0065] 1) Construction of integration plasmid pUC-HUH-A08-BcABA3

[0066] The integration plasmid pUC-HUH-A08-BcABA3 is based on the pUC57-HisG-URA3-HisG backbone and is inserted into the upstream and downstream homology arms of the A08 site in the Yarrowia lipolytica Po1fΔku70 genome. Two HisG tag encoding genes are located between the upstream and downstream homology arms of the A08 site, and the BcABA3 expression cassette (P TEFin -BcABA3-T xpr2t ), the specific structure is shown in Figure 3 In the present invention, the expression cassette is represented as follows: promoter-target gene-terminator, for example, the BcABA3 expression cassette is represented as P TEFin -BcABA3-T xpr2t , the promoter is P TEFin , the target gene is BcABA3, and the terminator is T xpr2t .

[0067] The promoter region of the BcABA3 expression cassette was amplified using the primer pairs A08-TEFin-F / A08-TEFin-R and A08-Xpr2t-F / A08-Xpr2t-R described in Table 1, respectively, and the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template. TEFin and terminator Txpr2t .

[0068] The DNA of plasmid pUC57-BcABA3 was used as template and A08-BcABA3-F and A08-BcABA3-R described in Table 1 were used as primers to amplify the promoter P at both ends. TEFin and terminator T xpr2t Homology arms of the BcABA3 gene.

[0069] Each fragment was purified and recovered using TaKaRa MiniBEST DNA Fragment Purification Kit (Takara Biotechnology (Dalian) Co., Ltd.).

[0070] The A08 site integration plasmid pUC-HUH-A08 was digested with the restriction endonuclease PacI from NEB, and the linearized A08 site integration plasmid was recovered by agarose gel electrophoresis.

[0071] The linearized A08 site was integrated into the plasmid and the elements (promoter P TEFin , target gene BcABA3, terminator T xpr2t ) One-step cloning was achieved using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novozymes Biotechnology Co., Ltd. to obtain a circular recombinant vector.

[0072] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid pUC-HUH-A08-BcABA3 was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0073] The recombinant plasmid pUC-HUH-A08-BcABA3 was digested with restriction endonuclease NotI from NEB, and the linearized recombinant plasmid pUC-HUH-A08-BcABA3 was recovered by agarose gel electrophoresis.

[0074] Table 1 Primer sequences for the construction of integration plasmid pUC-HUH-A08-BcABA3

[0075]

[0076] 2) Construction of knockout plasmid pUC-HUH-CEX1 (i.e., CEX1 site integration plasmid)

[0077] The knockout plasmid pUC-HUH-CEX1 is based on the pUC57-HisG-URA3-HisG backbone, and is inserted with a 1517 bp homology arm (CEX1-up) upstream of the start codon and a 1557 bp homology arm (CEX1-dn) downstream of the stop codon of the CEX1 locus in the Yarrowialipolytica Po1fΔku70 genome (GENEBANK accession number: CR382130.1), a hisG tag encoding gene, and an orotidine-5'-phosphate decarboxylase encoding gene expression cassette (containing the Yarrowialipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) between the upstream and downstream homology arms.

[0078] Using the CEX1-up-F / CEX1-up-R primers described in Table 2 and the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template, the homology arm CEX1-up upstream of the start codon of the CEX1 locus was amplified.

[0079] The plasmid pUC57-HUH was digested with restriction endonuclease EcoRI from NEB, and the linearized pUC57-HisG-Ura-HisG plasmid was recovered by agarose gel electrophoresis.

[0080] The linearized pUC57-HisG-Ura-HisG plasmid and CEX1-up (with pUC57-hisG-Ura-hisG homology arm sequences at both ends) were cloned and cloned using the DNA sequence of Nanjing Novozyme Biotechnology Co., Ltd. IIOne Step Cloning Kit achieves one-step cloning to obtain a circular recombinant vector.

[0081] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid pUC-HUH-CEX1-up was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0082] Using the CEX1-dn-F / CEX1-dn-R primers described in Table 2 and the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template, the homology arm CEX1-dm downstream of the stop codon of the CEX1 locus was amplified.

[0083] The plasmid pUC-HUH-CEX1-up was digested with the restriction endonuclease PACI from NEB, and the linearized pUC-HUH-CEX1-up plasmid was recovered by agarose gel electrophoresis.

[0084] The linearized pUC-HUH-CEX1-up plasmid and CEX1-dm (with pUC-HUH-CEX1-up homology arm sequences at both ends) were used by Nanjing Novozyme Biotechnology Co., Ltd. IIOne Step Cloning Kit achieves one-step cloning to obtain a circular recombinant vector.

[0085] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid pUC-HUH-CEX1 was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0086] The plasmid pUC-HUH-CEX1 was digested with restriction endonuclease NotⅠ from NEB Company, and the linearized pUC-HUH-CEX1 plasmid was recovered by agarose gel electrophoresis.

[0087] Table 2 Primer sequences for the construction of knockout plasmid pUC-HUH-CEX1

[0088]

[0089] 3) Construction of integration plasmid pUC-HUH-IntE2-ACL1-ACL2

[0090] The integration plasmid pUC-HUH-IntE2-ACL1-ACL2 is based on the pUC57-HisG-URA3-HisG backbone, and is inserted into the upstream and downstream homology arms of the start codon of the IntE2 site in the Yarrowia lipolytica Po1fΔku70 genome. Two HisG tag encoding genes are located between the upstream and downstream homology arms of the IntE2 site, and the ACL2 gene expression cassette (P GPD -ACL2-T ACL2t ) and ACL1 gene expression cassette (P FBA -ACL1-T ACL1t ), URA expression cassette (containing Yarrowia lipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homology arms, the specific structure is shown in Figure 4 .

[0091] The promoter P of the ACL2 expression cassette was amplified using the two primer pairs IntE2-GPD-F / IntE2-GPD-R and IntE2-ACL2t-F / IntE2-ACL2t-R described in Table 3, respectively, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template.GPD and terminator T ACL2t Using IntE2-ACL2-F / IntE2-ACL2-R as primers and Yarrowia lipolyticaPo1fΔku70 genomic DNA as template, we amplified the fragments with P at both ends. GPD and T ACL2t Homology arms of the ACL2 gene.

[0092] The promoter P of the ACL1 expression cassette was amplified using the primer pairs IntE2-FBA-F / IntE2-FBA-R and IntE2-ACL1t-F / IntE2-ACL1t-R described in Table 3, respectively, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template. FBA and terminator T ACL1t Using IntE2-ACL1-F / IntE2-ACL1-R as primers and Yarrowia lipolyticaPo1fΔku70 genomic DNA as template, we amplified the fragments with P at both ends. FBA and T ACL1t Homology arms of the ACL1 gene.

[0093] The IntE2 site integration plasmid was digested with restriction endonuclease PacⅠ from NEB, and the linearized IntE2 site integration plasmid was recovered by agarose gel electrophoresis.

[0094] The linearized IntE2 site was integrated into the plasmid and the elements of the ACL2 and ACL1 gene expression cassettes constructed in this example (ACL2 expression cassette promoter P GPD , gene ACL2 and ACL2 expression cassette terminator T ACL2t , ACL1 expression cassette promoter P FBA , gene ACL1 and ACL1 expression cassette terminator T ACL1t ) One-step cloning was achieved using the ClonExpressMultiS One Step Cloning Kit from Nanjing Novozymes Biotechnology Co., Ltd. The ACL2 and ACL1 gene expression cassettes were inserted between the downstream homology arm and the hisG tag encoding gene of the IntE2 site integration plasmid to obtain a circular recombinant vector.

[0095] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid pUC-HUH-IntE2-ACL1-ACL2 was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0096] pUC-HUH-IntE2-ACL1-ACL2 was digested with restriction endonuclease FSPⅠ from NEB, and the linearized pUC-HUH-IntE2-ACL1-ACL2 plasmid was recovered by agarose gel electrophoresis.

[0097] Table 3 Primer sequences for the construction of integration plasmid pUC-HUH-IntE2-ACL1-ACL2

[0098]

[0099] 4) Construction of integration plasmid pUC-HUH-Lip1-CAT2

[0100] The integration plasmid pUC-HUH-Lip1-CAT2 is based on the pUC57-HisG-URA3-HisG backbone, and is inserted into the upstream and downstream homology arms of the Lip1 site in the Yarrowia lipolytica Po1fΔku70 genome. Two HisG tag encoding genes are located between the upstream and downstream homology arms of the Lip1 site, and a CAT2 expression cassette is inserted between the downstream homology arms and the HisG tag encoding gene (P TEF -CAT2-T OCTt ).

[0101] The CAT2 expression cassette promoter P was amplified using the two primer pairs Lip1-TEF-F / Lip1-TEF-R and Lip1-OCTt-F / Lip1-OCTt-R described in Table 4, respectively, and the genomic DNA of Yarrowia lipolytica Po1fΔku70 was used as a template. TEF and terminator T OCTt Using Yarrowia lipolytica Po1fΔku70 genomic DNA as a template and Lip1-CAT2-F and Lip1-CAT2-R as primers described in Table 4, we amplified the primers with promoter P at both ends. TEF and terminator T OCTt Homology arms of the CAT2 gene.

[0102] Each fragment was purified and recovered using TaKaRa MiniBEST DNA Fragment Purification Kit (Takara Biotechnology (Dalian) Co., Ltd.).

[0103] The Lip1 site integration plasmid was digested with the restriction endonuclease PacI from NEB, and the linearized Lip1 site integration plasmid was recovered by agarose gel electrophoresis.

[0104] The linearized Lip1 site integration plasmid and the various elements in the CAT2 gene expression cassette constructed in this example were cloned in one step using the ClonExpress MultiS One Step Cloning Kit produced by Nanjing Novozymes Biotechnology Co., Ltd. to obtain the recombinant plasmid pUC-HUH-Lip1-CAT2.

[0105] The recombinant plasmid pUC-HUH-Lip1-CAT2 was digested with restriction endonuclease NotI from NEB, and the linearized recombinant plasmid pUC-HUH-Lip1-CAT2 was recovered by agarose gel electrophoresis.

[0106] Table 4 Primer sequences for the construction of integration plasmid pUC-HUH-Lip1-CAT2

[0107]

[0108] 5) Construction of integration plasmid pUC-HUH-IntC3-TGL3-TGL4

[0109] The integration plasmid pUC-HUH-IntC3-TGL3-TGL4 is based on the pUC57-HisG-URA3-HisG backbone, and is inserted into the upstream and downstream homology arms of the start codon of the IntC3 site in the Yarrowia lipolytica Po1fΔku70 genome. Two HisG tag encoding genes are located between the upstream and downstream homology arms of the IntC3 site, and the TGL3 gene expression cassette (P GPD1in -TGL3-T lip2t ) and TGL4 gene expression cassette (P GPD1in -TGL4-T xpr2t ). URA expression cassette (containing the Yarrowia lipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) also between the upstream and downstream homology arms.

[0110] The promoter P of the TGL3 expression cassette was amplified using the primer pairs IntC3-GPD1in-F1 / IntC3-GPD1in-R1 and IntC3-Lip2t-F / IntC3-Lip2t-R described in Table 5, respectively, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template. GPD1in and terminator T lip2tUsing IntC3-TGL3-F / IntC3-TGL3-R as primers and Yarrowialipolytica Po1fΔku70 genomic DNA as template, the amplified fragments were cloned with P at both ends. GPD1in and T lip2t Homology arms of the TGL3 gene.

[0111] The promoter P of the TGL4 expression cassette was amplified using the two primer pairs IntC3-GPD1in-F2 / IntC3-GPD1in-R2 and IntC3-Xpr2t-F / IntC3-Xpr2t-R described in Table 5, respectively, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template. GPD1in and terminator T xpr2t Using IntC3-TGL4-F / IntC3-TGL4-R as primers and Yarrowialipolytica Po1fΔku70 genomic DNA as template, the amplified fragments were cleaved with P at both ends. GPD1in and T xpr2t Homology arms of the TGL4 gene.

[0112] The IntC3 site integration plasmid was digested with restriction endonuclease PacⅠ from NEB, and the linearized IntC3 site integration plasmid was recovered by agarose gel electrophoresis.

[0113] The linearized IntC3 site was integrated into the plasmid and the elements of the TGL3 and TGL4 gene expression cassettes constructed in this example (TGL3 expression cassette promoter P GPD1in , gene TGL3 and TGL3 expression cassette terminator T lip2t , TGL4 expression cassette promoter P GPD1in , gene TGL4 and TGL4 expression cassette terminator T xpr2t ) One-step cloning was achieved using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novozymes Biotechnology Co., Ltd. The TGL3 and TGL4 gene expression cassettes were inserted between the downstream homology arm and the hisG tag encoding gene of the IntC3 site integration plasmid to obtain a circular recombinant vector.

[0114] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid pUC-HUH-IntC3-TGL3-TGL4 was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0115] After digesting pUC-HUH-IntC3-TGL3-TGL4 with restriction endonuclease FSPⅠ from NEB, the linearized pUC-HUH-IntC3-TGL3-TGL4 plasmid was recovered by agarose gel electrophoresis.

[0116] Table 5 Primer sequences for the construction of integration plasmid pUC-HUH-IntC3-TGL3-TGL4

[0117]

[0118] 6) Construction of integration plasmid pUC-HUH-IntB-MFE1

[0119] The integration plasmid pUC-HUH-IntB-MFE1 is based on the pUC57-HisG-URA3-HisG backbone, and is inserted into the upstream and downstream homology arms of the IntB site in the Yarrowia lipolytica Po1fΔku70 genome. Two HisG tag encoding genes are located between the upstream and downstream homology arms of the IntB site, and the MFE1 expression cassette (P TEF -MFE1-T xpr2t ).

[0120] The promoter region of the MFE1 expression cassette was amplified using the primer pairs IntB-TEF-F / IntB-TEF-R and IntB-Xpr2t-F / IntB-Xpr2t-R described in Table 6, respectively, and the genomic DNA of Yarrowia lipolytica Po1fΔku70 was used as a template. TEF and terminator T xpr2t Using Yarrowia lipolytica Po1fΔku70 genomic DNA as template and IntB-MFE1-F and IntB-MFE1-R as primers described in Table 6, we amplified the primers with promoter P at both ends. TEF and terminator T xpr2t Homology arms of the MFE1 gene.

[0121] Each fragment was purified and recovered using TaKaRa MiniBEST DNA Fragment Purification Kit (Takara Biotechnology (Dalian) Co., Ltd.).

[0122] The IntB site integration plasmid was digested with restriction endonuclease PacI from NEB, and the linearized IntB site integration plasmid was recovered by agarose gel electrophoresis.

[0123] The linearized IntB site integration plasmid and the various elements in the MFE1 gene expression cassette constructed in this example were cloned in one step using the ClonExpress MultiS One Step Cloning Kit produced by Nanjing Novozymes Biotechnology Co., Ltd. to obtain the recombinant plasmid pUC-HUH-IntB-MFE1.

[0124] The recombinant plasmid pUC-HUH-IntB-MFE1 was digested with restriction endonuclease SwaI from NEB, and the linearized recombinant plasmid pUC-HUH-IntB-MFE1 was recovered by agarose gel electrophoresis.

[0125] Table 6 Primer sequences for the construction of integration plasmid pUC-HUH-IntB-MFE1

[0126]

[0127] 7) Construction of promoter replacement plasmid pUC-HUH-PD19-ERG9

[0128] The promoter replacement plasmid pUC-HUH-PD19-ERG9 is based on the pUC57-HisG-URA3-HisG backbone and is inserted into the promoter PERG9 position of the ERG9 locus in the Yarrowia lipolytica Po1fΔku70 genome (GenBank accession number: AOW00473.1). The 1338 bp downstream of the ERG9 start codon is the upstream homology arm (ERG9-UP). The promoter PD19 is inserted upstream of the start codon, and the 1684 bp upstream of the ERG9 start codon is the downstream homology arm (ERG9-DN). The hisG tag encoding gene and the orotidine-5'-phosphate decarboxylase encoding gene expression cassette are located between the upper and downstream homology arms. The specific structure is shown in [ 1 ]. Figure 5 .

[0129] Using ERG9UP-F / ERG9UP-R and PD19-F / PD19-R described in Table 7 as primers and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, the homology arm ERG9-UP upstream of the start codon of the ERG9 locus and the promoter PD19 were amplified.

[0130] The plasmid pUC57-HUH was digested with restriction endonuclease EcoRI from NEB and linearized pUC57-HUH was recovered by agarose gel electrophoresis.

[0131] The linearized pUC57-HUH plasmid and fragments ERG9-UP and PD19 (with pUC57-HisG-Ura-HisG homology arm sequences at both ends) were cloned and cloned using the DNA sequencing technology of Nanjing Novozyme Biotechnology Co., Ltd. IIOne Step Cloning Kit achieves one-step cloning to obtain a circular recombinant vector.

[0132] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid pUC-HUH-ERG9-UP was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0133] Using ERG9DN-F / ERG9DN-R described in Table 7 as primers and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, 1684 bp upstream of the ERG9 start codon was amplified as the downstream homology arm ERG9-DN.

[0134] The plasmid pUC-HUH-ERG9-UP was digested with restriction endonuclease PacI from NEB, and the linearized pUC-HUH-ERG9-UP plasmid was recovered by agarose gel electrophoresis.

[0135] The linearized pUC-HUH-ERG9-UP plasmid and ERG9-DN (with pUC-HUH-ERG9-UP homology arm sequences at both ends) were used by Nanjing Novozyme Biotechnology Co., Ltd. IIOne Step Cloning Kit achieves one-step cloning to obtain a circular recombinant vector.

[0136] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid pUC-HUH-PD19-ERG9 was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0137] The plasmid pUC-HUH-PD19-ERG9 was digested with restriction endonuclease BstAPIⅠ from NEB, and the linearized pUC-HUH-PD19-ERG9 plasmid was recovered by agarose gel electrophoresis.

[0138] Table 7 Primer sequences for the construction of integration plasmid pUC-HUH-PD19-ERG9

[0139]

[0140] Example 2 Construction of recombinant Yarrowia lipolytica for synthesis of 1',4'-trans-abscisic acid diol

[0141] The linearized recombinant plasmids pUC-HUH-A08-BcABA3, pUC-HUH-CEX1, pUC-HUH-IntE2-ACL1-ACL2, pUC-HUH-Lip1-CAT2, pUC-HUH-IntC3-TGL3-TGL4, pUC-HUH-IntB-MFE1 and pUC-HUH-PD19-ERG9 constructed in Example 1 were sequentially transformed into a recombinant Yarrowia lipolytica strain with high abscisic acid production (see Chinese Patent CN117778218A for details). ), homologous recombination was performed, a BcABA3 expression cassette was inserted at the A08 site (the BcABA3 expression cassette was used to replace the BcABA3 gene expression cassette - BcABA4 gene expression cassette in recombination 6), the CEX1 site was knocked out, the ACL1 and ACL2 expression cassettes were inserted at the IntE2 site, the CAT2 expression cassette was inserted at the Lip1 site, the TGL3 and TGL4 expression cassettes were inserted at the IntC3 site, the MFE1 expression cassette was inserted at the IntB site, and the ERG9 promoter was replaced with the growth-dependent promoter PD19 to obtain the recombinant Yarrowia lipolytica of the present invention.

[0142] The specific method is as follows:

[0143] (1) The recombinant strain 6, which produces high-ABA, was cultured overnight in YPD liquid medium to prepare competent cells. The linearized recombinant plasmid pUC-HUH-A08-BcABA3 was transformed into competent cells of the recombinant strain 6 using the Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination.

[0144] (2) Use SD-Ura plates to screen positive clones and identify them by PCR. The positive clones identified correctly 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 could grow on YPD plates containing 5-fluoroorotic acid but could not grow on SD-Ura plates were selected for PCR identification, and the obtained strain was named recombinant Yarrowia lipolytica ML-IAA-1.

[0145] (3) The positive clones identified by PCR in step (2) were cultured overnight in YPD liquid medium to prepare competent cells. The linearized recombinant plasmid pUC-HUH-CEX1 was transformed into the competent cells of the positive clones identified by PCR in step (2) using Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination.

[0146] (4) Use SD-Ura plates to screen positive clones and identify them by PCR using the same method as step (2).

[0147] (5) The positive clones identified by PCR in step (4) were cultured overnight in YPD liquid medium to prepare competent cells. The linearized recombinant plasmid pUC-HUH-IntE2-ACL1-ACL2 was transformed into the competent cells of the positive clones identified by PCR in step (4) using the Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination.

[0148] (6) Positive clones were screened using SD-Ura plates and identified by PCR using the same method as in step (2). The resulting strain was named recombinant Yarrowia lipolytica ML-IAA-2.

[0149] (7) The positive clones identified by PCR in step (6) were cultured overnight in YPD liquid medium to prepare competent cells. The linearized recombinant plasmid pUC-HUH-Lip1-CAT2 was transformed into the competent cells of the positive clones identified by PCR in step (6) using Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination.

[0150] (8) Use SD-Ura plates to screen positive clones and identify them by PCR using the same method as step (2).

[0151] (9) The positive clones identified by PCR in step (8) were cultured overnight in YPD liquid medium to prepare competent cells. The linearized recombinant plasmid pUC-HUH-IntC3-TGL3-TGL4 was transformed into the competent cells of the positive clones identified by PCR in step (8) using Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination.

[0152] (10) Use SD-Ura plates to screen positive clones and identify them by PCR using the same method as step (2).

[0153] (11) The positive clone identified by PCR in step (10) was cultured overnight in YPD liquid medium to prepare competent cells. The linearized recombinant plasmid pUC-HUH-IntB-MFE1 was transformed into the competent cells of the positive clone identified by PCR in step (10) using the Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination.

[0154] (12) Positive clones were screened using SD-Ura plates and identified by PCR using the same method as step (2). The resulting strain was named recombinant Yarrowia lipolytica ML-IAA-3.

[0155] (13) The positive clones identified by PCR in step (12) were cultured overnight in YPD liquid medium to prepare competent cells. The linearized recombinant plasmid pUC-HUH-PD19-ERG9 was transformed into the competent cells of the positive clones identified by PCR in step (12) using the Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination.

[0156] (14) Positive clones were screened using SD-Ura plates and identified by PCR using the same method as step (2). The resulting strain was named recombinant Yarrowia lipolytica ML-IAA-4.

[0157] Example 3 Fermentation of 1',4'-trans-abscisic acid diol by recombinant Yarrowia lipolytica

[0158] (1) Seed culture: The recombinant bacteria 6 with high abscisic acid production and ML-IAA-1 to 4 constructed in Example 2 were streaked on YPD plates and cultured at 28°C for 48 hours. A single colony was picked and placed in 5 mL of YPD liquid medium, and then cultured in a shaker at 28°C and 200 rpm for 16-24 hours. 600 A value of around 5 is used as fermentation seed liquid.

[0159] (2) Shake flask fermentation: The above seed solution was transferred to a 250 mL shake flask containing 50 mL shake flask fermentation medium to make its initial OD 600The fermentation temperature was controlled at 0.5. The culture was then placed in a shaker at 28°C and 200 rpm for 24-36 hours, and sterilized heme precursor 5-aminolevulinic acid (ALA) was added to a final concentration of 500 μg / L. The culture was continued for 120 hours, and the fermentation was terminated. The composition of the shake flask fermentation medium was: 80 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract.

[0160] This example also involved fed-batch fermentation of the recombinant strain ML-IAA-4. The specific process was as follows: the recombinant strain ML-IAA-4 was inoculated into 50 mL of YPD liquid medium and cultured at 30°C and 220 rpm for 24 hours to obtain a primary seed solution. This primary seed solution was then transferred to a 5L fermentor at a 5% v / v inoculum. 3 L of fermentor medium was added to the 5L fermentor. After 24-36 hours of incubation, sterilized heme precursor 5-aminolevulinic acid (ALA) was added to a final concentration of 500 μg / L. After incubation at 30°C for 48 hours, fed-batch fermentation with automatic feed addition was initiated. The automatic feed addition method involved positively correlating the stirring speed with the dissolved oxygen concentration (DO) between 300 and 1000 rpm to maintain the DO at 20%. The pH was maintained at 5.5 until the end of fermentation. The culture lasted for 5 days. The fermentation medium in the fermenter was composed of: 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 120 g / L glucose, 2.0 mL / L metal ion stock solution, and 1.0 mL / L vitamin stock solution. The feed solution was formulated as: 800 g / L glucose, 11.11 g / L ammonium sulfate, 10.0 mL / L metal ion stock solution, and 10.0 mL / L vitamin stock solution.

[0161] The vitamin mother solution 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 niacin, 0.2 g / L p-aminobenzoic acid and 25 g / L inositol.

[0162] The metal ion mother solution 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.

[0163] After the shake flask or fermentation tank fermentation, the target product was isolated and analyzed by HPLC according to the following method. The extraction and HPLC analysis of 1',4'-trans-abscisic acid diol were performed as follows: 4 mL of fermentation broth was transferred to a 10 mL centrifuge tube and 4 mL of ethyl acetate supplemented with formic acid (the volume of formic acid was 0.5% of the volume of ethyl acetate). Vortex for 30 minutes, centrifuge (3000 × g, 4°C, 15 minutes), transfer 3 mL of the supernatant to a new tube, and rotary evaporate for 2 hours (45°C, 2 hours). The residue was redissolved in methanol and centrifuged at 8000 rpm for 2 minutes. The supernatant was filtered through an organic nylon filter (0.22 μm) and transferred to a glass bottle for HPLC analysis. HPLC detection conditions were as follows: mobile phase: a mixture of methanol and water (adjusted to pH 3 with phosphoric acid) in a 70:30 volume ratio; column: Agilent EC-C18 (4.6×150 mm, 4 μm, Agilent); microinjection volume: 50 μL; column temperature: 30°C; mobile phase flow rate: 0.6 mL / min; detection wavelength: 260 nm; injection volume: 20 μL. Standard solutions of varying concentrations (1-400 mg / L) prepared with 1',4'-trans-abscisic acid diol were used to construct a calibration curve based on the peak area and concentration of the standard solutions, and the product yield was calculated.

[0164] After the shake flask fermentation, the yield of 1',4'-trans-abscisic acid diol of the starting bacteria (recombinant bacteria 6) was 12.23 mg / L as determined by HPLC. The yields of 1',4'-trans-abscisic acid diol of ML-IAA-1, ML-IAA-2, ML-IAA-3 and ML-IAA-4 were 212.21 mg / L, 298 mg / L, 355.24 mg / L and 487.45 mg / L, respectively, as determined by HPLC. Among them, the yield of 1',4'-trans-abscisic acid diol produced by the recombinant bacteria ML-IAA-4 was the highest. The yield of 1',4'-trans-abscisic acid diol obtained by the recombinant bacteria ML-IAA-4 in a 5L fermentor by fed-batch fermentation could reach 2.15 g / L, that is, 2.15 g of 1',4'-trans-abscisic acid diol could be obtained per liter of fermentation broth. Currently reported microbial methods for synthesizing 1',4'-trans-abscisic acid diol have yields in the milligram range. For example, the Chengdu Institute of Biology, Chinese Academy of Sciences, achieved a yield of only 54.4 mg / L of 1',4'-trans-abscisic acid diol in engineered Botrytis cinerea (Microbial Cell Factories, 2024, 23:185). The method described herein for biosynthesizing 1',4'-trans-abscisic acid diol using Yarrowia lipolytica is more efficient and has promising industrial applications.

[0165] The present invention provides a method for biosynthesizing 1',4'-trans-abscisic acid diol using Yarrowia lipolytica. There are many methods and approaches for implementing this technical solution. The above is merely 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. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A method for constructing a recombinant Yarrowia lipolytica for synthesizing 1',4'-trans-abscisic acid diol, characterized in that: The starting strain of the recombinant Yarrowia lipolytica is Yarrowia lipolytica that produces abscisic acid; The recombinant Yarrowia lipolytica blocks the synthesis pathway from 1',4'-trans-abscisic acid diol to abscisic acid, and simultaneously undergoes at least one of the following modifications a1 to a6: a1, knockout of the citrate transporter 1 encoding gene CEX1; a2, introduction of ATP citrate lyase encoding genes ACL1 and ACL2; a3, introduction of the peroxisomal carnitine acetyltransferase encoding gene CAT2; a4, introduction of lipase encoding genes TGL3 and TGL4; a5, introduction of the peroxisome multifunctional enzyme encoding gene MFE1; a6, replace the native promoter of the ERG9 gene.

2. The construction method according to claim 1, characterized in that The blocking is achieved by knocking out the gene encoding sesquiterpene dehydrogenase.

3. The construction method according to claim 1, characterized in that The CEX1, ACL1, ACL2, CAT2, TGL3, TGL4 and MFE1 are all derived from Yarrowia lipolytica.

4. The construction method according to claim 3, characterized in that The GenBank sequence number of CEX1 is CR382130.1, the GenBank sequence number of ACL1 is VBB77400.1, the GenBank sequence number of ACL2 is QNP98078.1, the GenBank sequence number of CAT2 is UTQH01000002.1, the GenBank sequence number of TGL3 is AOW04197.1, the GenBank sequence number of TGL4 is AOW06932.1, and the GenBank sequence number of MFE1 is VBB78987.

1.

5. The construction method according to claim 1, characterized in that The natural promoter of the ERG9 gene is replaced by the growth-dependent promoter PD19, and the nucleotide sequence of the PD19 is shown in SEQ ID No. 4; The GenBank sequence number of the ERG9 gene is AOW00473.

1.

6. The construction method according to claim 1, characterized in that The blocking is achieved by knocking out the gene encoding sesquiterpene dehydrogenase; the recombinant Yarrowia lipolytica undergoes all the following modifications a1 to a6; a1, knockout of the citrate transporter 1 encoding gene CEX1; a2, introduction of ATP citrate lyase encoding genes ACL1 and ACL2; a3, introduction of the peroxisomal carnitine acetyltransferase encoding gene CAT2; a4, introduction of lipase encoding genes TGL3 and TGL4; a5, introduction of the peroxisome multifunctional enzyme encoding gene MFE1; a6, Growth-dependent promoter PD19 replaces the native promoter of the ERG9 gene. Among them, the GenBank sequence number of CEX1 is CR382130.1, the GenBank sequence number of ACL1 is VBB77400.1, the GenBank sequence number of ACL2 is QNP98078.1, the GenBank sequence number of CAT2 is UTQH01000002.1, the GenBank sequence number of TGL3 is AOW04197.1, the GenBank sequence number of TGL4 is AOW06932.1, the GenBank sequence number of MFE1 is VBB78987.1, the GenBank sequence number of ERG9 gene is AOW00473.1, and the nucleotide sequence of PD19 is shown in SEQ ID No.

4.

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 fermentative production of 1',4'-trans-abscisic acid diol.

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 20-30° C. for 24-36 hours, 5-aminolevulinic acid is added, and the culture is continued for 120-144 hours.

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

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