Construction method and application of recombinant yarrowia lipolytica for synthesizing eriocin

Abstract

The invention provides a construction method and an application of recombinant yarrowia lipolytica for synthesizing furithrolin. The recombinant yarrowia lipolytica is obtained by introducing a combination of a coding gene of tenosyl ether synthase, a coding gene of cytochrome P450 oxidase, a coding gene of cytochrome P450 reductase, a coding gene of acetyltransferase, a coding gene of cytochrome b5 and a coding gene of scaffold protein, and knocking out a coding gene of phosphatidic acid phosphatase at the same time. The efficient synthesis of the fuchsin is realized. The construction method of the yarrowia lipolytica for biosynthesis of the eriolarynx element, provided by the invention, is simple to operate, and the constructed recombinant yarrowia lipolytica can efficiently produce the eriolarynx element and has relatively high production and application values.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and relates to a method for constructing and applying a recombinant lipophilic yeast that synthesizes trichodin. Background Technology

[0002] Forskolin is a hemispheric diterpenoid compound accumulated in the roots of *Forskolinia granatum*. As an adenylate cyclase activator, it can significantly increase intracellular cyclic adenosine monophosphate (cAMP) levels, making it an important drug for treating cardiovascular and respiratory diseases and also widely used as a weight-loss supplement. Traditional production methods rely on extraction from the roots of *Forskolinia granatum*, which faces bottlenecks such as scarce plant resources and low content of active ingredients. To address the problem of limited plant resources, researchers have attempted to prepare forskolin through chemical synthesis. However, due to the complex structure of forskolin, containing multiple chiral centers and oxidation modification sites, the chemical synthesis route is cumbersome, the reaction conditions are harsh, and selectivity control is difficult. This not only results in low synthesis efficiency but also leads to high solvent and reagent consumption, making it difficult to achieve green and sustainable preparation.

[0003] In recent years, the rapid development of synthetic biology has provided new technological pathways for obtaining complex plant natural products. Compared with bacterial hosts, yeast has certain advantages in eukaryotic protein folding, membrane protein expression, and cofactor supply, and is considered a potential ideal platform for constructing plant natural product biosynthetic systems. *Yarrowia lipolytica* (Yarrowia lipolytica) Yarrowia lipolytica Trichoderma possesses unique physiological and biochemical characteristics, demonstrating significant value for both academic research and industrial applications, and has been used in the biosynthesis of various terpenes and their derivatives. However, the biosynthesis of trichoderma involves multiple cytochrome P450 monooxygenase catalytic steps. These enzymes generally suffer from low expression efficiency, insufficient catalytic activity, and limited electron transfer efficiency in heterologous hosts, resulting in low synthesis levels of the target product and limiting the application effectiveness of existing technologies. Therefore, current technologies still lack a method for efficiently reconstructing the trichoderma biosynthetic pathway in *Yersinia lipolytica* and effectively improving the catalytic efficiency of related key enzymes. Developing a novel method for constructing recombinant *Yersinia lipolytica* for the synthesis of trichoderma and its application is of great significance for overcoming the shortcomings of existing technologies and achieving economically feasible production of trichoderma. Summary of the Invention

[0004] Objective of the Invention: The technical problem to be solved by this invention is to address the shortcomings of existing technologies by providing a method for constructing and applying recombinant Yersinia lipolytica for synthesizing trichodin; and to address the problem of low biosynthetic efficiency of trichodin in existing technologies by utilizing the cytochrome b5 encoding gene from the roots of *Corydalis trichodinia*. CfCytB5A To enhance electron transfer between NADPH and P450 enzymes, the gene encoding phosphatidylphosphatase was knocked out.PAH1 Regulation of endoplasmic reticulum size and overexpression of scaffold proteins from Arabidopsis thaliana AtMSBP1 Enhanced endoplasmic reticulum localization enzyme anchoring, knockout of GTP-bound Ras2 protein inhibiting hyphal formation, and overexpression of *Mucor* (*Mucor*) Mucor circinelloides The malic acid oxidase (McE2) gene in the malic acid oxidase enhances the supply of NADPH, thereby achieving efficient synthesis of salivain.

[0005] To address the aforementioned technical problems, this invention discloses a method for constructing and applying a recombinant *Yersinia lipolytica* yeast for synthesizing trichodin. The constructed recombinant *Yersinia lipolytica* yeast can synthesize the diterpenoid compound trichodin, thereby achieving efficient biosynthesis of trichodin. The technical solution of this invention is as follows: In a first aspect, the present invention provides a method for constructing a recombinant *Yersinia lipolytica* strain for synthesizing trichodin, which introduces a combination of genes encoding tuldophene synthase, cytochrome P450 oxidase, cytochrome P450 reductase, acetyltransferase, cytochrome b5, and scaffold protein, while simultaneously knocking out the gene encoding phosphatidylphosphatase; wherein the genes encoding tuldophene synthase, cytochrome P450 oxidase, cytochrome P450 reductase, and acetyltransferase are all genes related to the trichodin biosynthesis pathway and are all derived from *Coleopterus xanthipes* (a type of *Trichodinia*). Coleus forskohlii ); The gene encoding the lacrimal ether synthase (including) CfTPS2 and CfTPS3 The nucleotide sequence of ) is shown in SEQ ID No. 1~2, and a combination of SEQ ID No. 1 and SEQ ID No. 2 is also introduced; the gene encoding cytochrome P450 oxidase ( CfCYP76AH15, CfCYP76AH11, CfCYP76AH16 The nucleotide sequences of the cytochrome P450 reductase are shown in SEQ ID No. 3-5, and a combination of SEQ ID No. 3-5 is also introduced; CfCPR The genes encoding ), acetyltransferases (CfACT1-8), and cytochrome b5 (CfACT1-8) are mentioned. CfCytB5A ) and the gene encoding the scaffold protein ( AtMSBP1 The nucleotide sequences of the enzymes are shown in SEQ ID Nos. 6-9, respectively; the gene number of the phosphatidyl phosphatase PAH1 is YALI1_D35593g. In some embodiments of the present invention, the introduction... CfTPS2 and CfTPS3 A strain capable of synthesizing 13R-thujone (13R-MO) was constructed and introduced into the culture medium. CfCYP76AH15, CfCYP76AH11, CfCYP76AH16 as well as CfCPR,7-Deacetylated pilocarpine was obtained, followed by CfACT1-8 transacetylation to yield pilocarpine. (Introduction) CfCytB5A To enhance electron transfer between NADPH and P450 enzymes; knockout of PAH1 regulates endoplasmic reticulum size and translocation. AtMSBP1 Enhance the anchoring of endoplasmic reticulum localizing enzymes.

[0006] The recombinant Yersinia lipolyticis yeast described herein has had the gene encoding the GTP-binding protein knocked out and / or has had the gene encoding malic acid oxidase introduced.

[0007] The gene number of the GTP-binding protein Ras2 is YALI1_E35305g; the nucleotide sequence of the gene encoding malic acid oxidase (McE2) is shown in SEQ ID No. 10. Knocking out the Ras2 protein inhibits mycelial formation, and introducing the malic acid oxidase (McE2) gene enhances the supply of NADPH.

[0008] The recombinant Yersinia lipolyticis involved knocking out the gene encoding the GTP-binding protein and introducing the gene encoding malic acid oxidase.

[0009] The recombinant *Yersinia lipolytica* strain mentioned herein includes *Yersinia lipolytica* capable of synthesizing geraniylgeraniyl pyrophosphate. In some embodiments of the present invention, the starting strain includes *Yersinia lipolytica* SCL301. *Yersinia lipolytica* SCL301 can provide high levels of the universal diterpene precursor geraniylgeraniyl pyrophosphate (GGPP, C20), details of which can be found in reference [Sun, Mei Li, et al. Constructing a greenoleaginous yeast cell factory for sustainable production of the plant-derived diterpenoid sclareol. Green Chemistry 26.9(2024):10.].

[0010] In some embodiments of the present invention, the present invention provides a method for constructing a recombinant *Yersinia lipolytica* yeast for synthesizing lauryl ether synthase. The recombinant *Yersinia lipolytica* yeast is infused with a combination of genes encoding tretinoin synthase, cytochrome P450 oxidase, cytochrome P450 reductase, acetyltransferase, cytochrome b5, and a scaffold protein, while simultaneously knocking out the gene encoding phosphatidylphosphatase. The recombinant *Yersinia lipolytica* yeast also has the gene encoding GTP-binding protein knocked out and a gene encoding malic acid esterase introduced. The gene encoding tretinoin synthase (…) CfTPS2 and CfTPS3The nucleotide sequence of the cytochrome P450 oxidase is shown in SEQ ID No. 1~2. CfCYP76AH15, CfCYP76AH11, CfCYP76AH16 The nucleotide sequence of the cytochrome P450 reductase is shown in SEQ ID No. 3-5. CfCPR The genes encoding ), acetyltransferases (CfACT1-8), and cytochrome b5 (CfACT1-8) are mentioned. CfCytB5A ) and the gene encoding the scaffold protein ( AtMSBP1 The nucleotide sequences of the enzymes are shown in SEQ ID No. 6-9, respectively; the gene number of the phosphatidyl phosphatase PAH1 is YALI1_D35593g; the gene number of the GTP-binding protein (Ras2) is YALI1_E35305g; and the nucleotide sequence of the gene encoding the malic acid enzyme (McE2) is shown in SEQ ID No. 10.

[0011] In some other embodiments of the present invention, the method for introducing or knocking out related genes in recombinant Yersinia lipolytica is as follows: constructing a gene expression cassette or gene knockout cassette, inserting the gene expression cassette or gene knockout cassette into the Yersinia lipolytica genome through homologous recombination, wherein the introduction or knockout method is the integration method based on Ura3-blaster 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 Yersinia lipolytica reported (Biotechnology Journal, 2018, 13(9), 1700543).

[0012] Secondly, the present invention provides a recombinant Yersinia lipophila constructed by the construction method described in the first aspect.

[0013] Thirdly, the present invention provides the application of the recombinant Yersinia lipolytica described in the second invention in the fermentation production of saliva.

[0014] In this process, the recombinant *Yarrowia lipolyticis* is inoculated into a fermentation medium and fermented at 28-30°C for 24-36 hours. Then, 5-aminolevulinic acid is added, and the fermentation continues for 120-150 hours. In some embodiments of this invention, the fermentation medium comprises 20-80 g / L of carbon source and 5-10 g / L of nitrogen source.

[0015] The concentration of 5-aminolevulinic acid added is 500~800 µg / L, preferably 700 µg / L.

[0016] Beneficial effects: This invention specifically employs the following four steps to construct recombinant Yersinia lipolytica: 1) Overexpressing codon-optimized [product name] in Yersinia lipolytica. CfTPS2, CfTPS3, CfCYP76AH15, CfCYP76AH11, CfCYP76AH16, CfCPR and CfACT1-8 1) To achieve the production of trichosin from scratch; 2) The cytochrome b5 encoding gene from the roots of the cork of *Corydalis trichosinensis* ( CfCytB5A ), enhance electron transfer from NADPH to P450 enzymes; 3) knock out the gene encoding phosphatidylphosphatase. PAH1 and overexpression of scaffold proteins from Arabidopsis thaliana AtMSBP1 4) Regulate the size of the endoplasmic reticulum to restore its homeostasis; 5) Knock out RAS2 to obtain a hyphae-free phenotype, and introduce hyphae from Mucor ( Mucor circinelloides The malic acid oxidase-encoding gene (McE2) provides sufficient NADPH for the synthesis of tuftrin; thus, the recombinant Yersinia lipolytica constructed through the above four steps can efficiently biosynthesize tuftrin. Attached Figure Description

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0018] Figure 1 This is a schematic diagram of the biosynthesis pathway of trichodin based on recombinant Yersinia lipophila.

[0019] Figure 2 The plasmid map of pUC-HUH-IntE2-CfTPS2-CfTPS3. Detailed Implementation

[0020] The present invention will be further described below through specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0021] The following examples describe the lipophilic yeast. Yarrowia lipolyticaThe formulations of Po1f △ku70 and the aforementioned YPD liquid culture medium, YPD plates, SD-Ura plates, and YPD plates containing 5-fluoroorotic acid are detailed in Chinese Patent CN118421490A. The SD-Leu plate has the following formulation: 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 to remove leucine, purchased from MPBiomedicals), and 23 g / L agar powder.

[0022] In the following embodiments, the construction methods of plasmid pUC57-Leu and plasmid pUC57-HisG-URA3-HisG (pUC-HUH) are detailed in Chinese Patent CN120536475A.

[0023] In the following examples, the purification and recovery of each fragment was performed using the TaKaRa MiniBEST DNA Fragment Purification Kit (Takara Bio Engineering (Dalian) Co., Ltd.). The restriction endonucleases were all purchased from NEB. The one-step cloning was achieved using the ClonExpress MultiSOne Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. The *Yersinia 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 based on *Yersinia lipolytica* (… Yarrowia lipolytica Using Po1f △ku70 genomic DNA as a template, the sample can be amplified using the corresponding amplification primers.

[0024] In the following examples, the host bacterium, Yersinia lipophila ( Yarrowia lipolytica SCL301, whose detailed construction method can be found in the reference [Sun, Mei Li, et al. Constructing a green oleaginous yeast cell factory for sustainable production of the plant-derived diterpenoid sclareol. Green Chemistry 26.9(2024):10.].

[0025] The biosynthetic pathway of the trichodin synthesized in this invention is as follows: Figure 1 As shown.

[0026] Example 1: Construction of the trichodin biosynthetic pathway The company was commissioned to synthesize codon-optimized versions of *Coleopterus xanthipes* (a type of flower). Coleus forskohlii The gene encoding tretinoin synthase from ( ) CfTPS2 and CfTPS3 ), three genes encoding cytochrome P450 oxidase ( CfCYP76AH15 , CfCYP76AH11 and CfCYP76AH16 ), cytochrome P450 reductase encoding gene ( CfCPR ) and acetyltransferase ( CfACT1-8 The nucleotide sequences of ) are respectively CfTPS2 (SEQ ID No.1) CfTPS3 (SEQ ID No.2) CfCYP76AH15 (SEQ ID No.3) CfCYP76AH11 (SEQ ID No.4) CfCYP76AH16 (SEQ ID No.5) CfCPR (SEQ ID No. 6) and CfACT1-8 (SEQ ID No. 7), and the optimized sequence was inserted into plasmid pUC57 to obtain plasmid pUC57- CfTPS2 pUC57- CfTPS3 pUC57- CfCYP76AH15, pUC57- CfCYP76AH11, pUC57- CfCYP76A16, pUC57- CfCPR and pUC57- CfACT1-8 The aforementioned CfTPS2, CfTPS3, CfCYP76AH15 , CfCYP76AH11 , CfCYP76AH16, CfCPR and CfACT1-8 The nucleotide sequences were obtained by codon optimization of the amino acid sequences shown in NCBI RefSeq:KF444507.1, AHW04048.1, AMZ03402.1, AMZ03393.1, AMZ03403.1, AQZ55559.1 and AMZ03407.1, respectively.

[0027] (1) Construction of the integrative plasmid pUC-HUH-IntE2-CfTPS2-CfTPS3 The integrative plasmid pUC-HUH-IntE2-CfTPS2-CfTPS3 uses the IntE2 site integrative plasmid pUC-HUH-IntE2 as a backbone and inserts the CfTPS2-CfTPS3 expression cassette (P... TEF -CfTPS2-T xpr2t and P EXP -CfTPS3-T mig1tIn this invention, the expression cassette is represented as follows: promoter-target gene-terminator, for example, the CfTPS2 expression cassette is represented as P. TEF -CfTPS2-T xpr2t The promoter is P TEF The target gene is CfTPS2, and the terminator is T. xpr2t .by Yarrowia lipolytica Using Po1f △ku70 genomic DNA as a template, and with TEF-F / TEF-R and xpr2t-F / xpr2t-R primer pairs as described in Table 1, the CfTPS2 expression cassette promoter P was amplified. TEF and Termination T xpr2t Using EXP-F / EXP-R and mig1t-F / mig1t-R as two primer pairs, the CfTPS3 expression cassette promoter P was amplified. EXP and Termination T mig1t Using DNA from plasmids pUC57-CfTPS2 and pUC57-CfTPS3 as templates, and primer pairs CfTPS2-F / CfTPS2-R and CfTPS3-F / CfTPS3-R as described in Table 1, the DNA of each plasmid was amplified. TEF and Termination T xpr2t The CfTPS2 gene of the homologous arm and the promoters P at both ends are respectively EXP and Termination T mig1t The CfTPS3 gene with homologous arms was purified and fragments were recovered. The IntE2 site integration plasmid was digested with the restriction endonuclease PacI, and the linearized IntE2 site integration plasmid was recovered by agarose gel electrophoresis. The linearized IntE2 site integration plasmid and each element (promoter P) in the CfTPS2-CfTPS3 gene expression cassette constructed in this embodiment were then compared. TEF Target gene CfTPS2, terminator T xpr2t and promoter P EXP Target gene CfTPS3, terminator T mig1t A circular recombinant vector was obtained through one-step cloning. This circular recombinant vector was transformed into *E. coli* DH5α competent cells, and positive recombinant plasmids pUC-HUH-IntE2-CfTPS2-CfTPS3 were obtained through ampicillin-resistant plate selection and colony PCR and sequencing verification. Figure 2 The linearized recombinant plasmid pUC-HUH-IntE2-CfTPS2-CfTPS3 was obtained by digestion with the restriction endonuclease NotI and recovery by agarose gel electrophoresis.

[0028] The IntE2 site integration plasmid pUC-HUH-IntE2 is used to integrate the plasmid with the IntE2 site. Yarrowia lipolyticaA 1533 bp sequence (upstream homologous arm IntE2-up) upstream of the start codon at the IntE2 site (YALI1_E36641g) on ​​chromosome E in the Po1f △ku70 genome was inserted into the pUC-HUH vector. EcoR At the I restriction site, a 1521 bp sequence downstream of the IntE2 stop codon (downstream homologous arm IntE2-dm) is inserted into the pUC-HUH vector. Hind The Ura3 gene, obtained from the III restriction site, has a HisG tag encoding gene attached to both ends. The Ura3 gene with the HisG tag is located between the upstream and downstream homologous arms of the IntE2 site.

[0029] (2) Construction of the integrative plasmid pUC57-Leu-A08-AH15-AH11-AH16 The recombinant plasmid pUC57-Leu-A08-AH15-AH11-AH16 uses the A08 site integration plasmid pUC-Leu-A08 as its backbone and inserts the CfCYP76AH15, CfCYP76AH11, and CfCYP76AH16 expression cassettes (P... H3 -CfCYP76AH15-T lip2t P H3 -CfCYP76AH11-T Cyc1t and P TEFin -CfCYP76AH16-T xpr2t ).by Yarrowia lipolytica Using Po1f △ku70 genomic DNA as a template, and with H3(AH15)-F / H3(AH15)-R and lip2t-F / lip2t-R primer pairs as described in Table 1, the promoter P of the CfCYP76AH15 expression cassette was amplified. H3 and Termination T lip2t Using H3(AH11)-F / H3(AH11)-R and cyc1t-F / cyc1t-R as primer pairs, the promoter P of the CfCYP76AH11 expression cassette was amplified. H3 and Termination T Cyc1t ; using plasmid pUC57- CfCYP76AH15 Using DNA as a template and AH15-F and AH15-R as primers as described in Table 1, amplification was performed using P promoters at both ends. H3 and Termination T lip2t homologous arms CfCYP76AH15 Gene; using plasmid pUC57- CfCYP76AH11 Using DNA as a template and AH11-F and AH11-R as primers as described in Table 1, amplification was performed using P promoters at both ends. H3 and Termination T Cyc1t homologous armsCfCYP76AH11 Gene; using plasmid pUC57- CfCYP76AH16 Using DNA as a template and AH16-F / AH16-R as primers as described in Table 1, amplification was performed using P-terminals at both ends. TEFin and T xpr2t homologous arms CfCYP76AH16 Genes. Purification and recovery of fragments.

[0030] Using restriction endonucleases Pac The A08 site integration plasmid was digested with enzymes, and the linearized A08 site integration plasmid was recovered by agarose gel electrophoresis. The linearized A08 site integration plasmid and the elements (P) in the CfCYP76AH15, CfCYP76AH11, and CfCYP76AH16 gene expression cassettes constructed in this embodiment were then compared. H3 - CfCYP76AH15 -T lip2t P H3 - CfCYP76AH11 -T Cyc1t , CfCYP76AH16 A circular recombinant vector was obtained through one-step cloning. This vector was transformed into *E. coli* DH5α competent cells, and positive recombinant plasmids pUC57-Leu-A08-AH15-AH11-AH16 were obtained through ampicillin-resistant plate selection and colony PCR and sequencing verification. Not The plasmid pUC57-Leu-A08-AH15-AH11-AH16 was recovered by agarose gel electrophoresis after enzyme digestion. The construction method of the A08 site integration plasmid pUC-Leu-A08 is detailed in Chinese Patent CN120536475A.

[0031] (3) Construction of the integration plasmid pUC-HUH-IntC3-CfCPR-CfACT1-8 The integrative plasmid pUC-HUH-IntC3-CfCPR-CfACT1-8 uses the IntC3 site integrative plasmid pUC-HUH-IntC3 as its backbone and inserts the CfCPR and CfACT1-8 expression cassettes (P... GPD1 -CfCPR-T xpr2t and P FBA1 -CfACT1-8-T lip2t ).by Yarrowia lipolytica Using Po1f △ku70 genomic DNA as a template, and employing the primer pairs GPD1-F / GPD1-R and xpr2t-F1 / xpr2t-R1 as described in Table 1, amplification was performed. CfCPR Expression box promoter P GPD1 and Termination T xpr2tUsing FBA1-F / FBA1-R and lip2t-F1 / lip2t-R1 as two primer pairs, amplification was performed. CfACT1-8 Expression box promoter P FBA1 and Termination T lip2t Using plasmid pUC57-CfCPR DNA as a template and the primers CfCPR-F / CfCPR-R as described in Table 1, amplification was performed using promoters P at both ends. GPD1 and Termination T xpr2t homologous arms CfCPR Gene; using plasmid pUC57- CfACT1-8 Using DNA as a template and CfACT-F / CfACT-R as primers as described in Table 1, amplification was performed using P promoters at both ends. FBA1 and Termination T lip2t homologous arms CfACT1-8 Genes were purified and fragments recovered. Restriction endonucleases were used. Pac The IntC3 site integration plasmid was digested with enzymes, and the linearized IntC3 site integration plasmid was recovered by agarose gel electrophoresis. The linearized IntC3 site integration plasmid and each element (promoter P) in the CfCPR and CfACT1-8 gene expression cassettes constructed in this embodiment were then compared. GPD1 Target gene CfCPR and terminator T xpr2t ; promoter P FBA1 The target gene CfACT1-8 and the terminator T lip2t A circular recombinant vector was obtained through one-step cloning. This vector was transformed into *E. coli* DH5α competent cells, and positive recombinant plasmids pUC-HUH-IntC3-CfCPR-CfACT1-8 were obtained through ampicillin-resistant plate selection and colony PCR and sequencing verification. Restriction endonucleases were then used to... Not I was digested with enzymes, and the linearized recombinant plasmid pUC-HUH-IntC3-CfCPR-CfACT1-8 was obtained by agarose gel electrophoresis. The IntC3 site integration plasmid pUC-HUH-IntC3 is... Yarrowia lipolytica A 1402 bp sequence (upstream homologous arm) upstream of the IntC3 site (YALI1_C25990g) on ​​chromosome C in the Po1f △ku70 genome was inserted into the pUC-HUH vector. EcoR At the I restriction site, a 1395 bp sequence (downstream homologous arm) downstream of the IntC3 stop codon is inserted into the pUC-HUH vector. Hind The Ura3 gene, obtained from the III restriction site, has a HisG tag encoding gene attached to both ends. The Ura3 gene with the HisG tag is located between the upstream and downstream homologous arms of the IntC3 site.

[0032] Table 1 Primer sequences for constructing plasmids related to the trichodin biosynthesis pathway

[0033] The linearized recombinant plasmids constructed in (1) to (3) of this embodiment were sequentially transformed into the host bacterium *Yarrowia lipophila* SCL301 for homologous recombination to obtain recombinant bacteria. The specific method is as follows: SCL301 was cultured overnight in YPD liquid medium to prepare competent cells. The linearized pUC-HUH-IntE2-CfTPS2-CfTPS3 was transformed into SCL301 competent cells via yeast for homologous recombination. Positive clones were screened using SD-Ura plates and identified by PCR. Positive clones identified by PCR were plated on YPD plates containing 5-fluoroorotic acid and incubated at 30 ℃ for 3 days. Single colonies were streaked simultaneously on both YPD plates and SD-Ura plates containing 5-fluoroorotic acid to observe bacterial growth. Single colonies that grew on YPD plates containing 5-fluoroorotic acid but failed to grow on SD-Ura plates (i.e., losing one hisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure) were selected for PCR identification, yielding recombinant strain 1. Linearized pUC-Leu-A08-AH15-AH11-AH16 was transformed into competent cells of recombinant strain 1 via yeast. Positive clones were selected using SD-Leu plates and identified by PCR, yielding recombinant strain 2. Linearized pUC-HUH-IntC3-CfCPR-CfACT1-8 was transformed into competent cells of recombinant strain 2 via yeast. After losing one hisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure, ML-For-1 was obtained.

[0034] Example 2: Enhancing electron transport of NADPH to P450 enzymes The company was commissioned to synthesize codon-optimized versions of *Coleopterus xanthipes* (a type of flower). Coleus forskohlii The cytochrome b5 encoding gene derived from cork roots ( CfCytB5A Its nucleotide sequence is shown in SEQ ID No. 8, and the sequence was inserted into plasmid pUC57 to obtain plasmid pUC57- CfCytB5A The IntB site integration plasmid pUC-HUH-IntB is used to integrate the plasmid with the IntB site. Yarrowia lipolytica The sequence 2024 bp upstream of the start codon of the IntB site (YALI1_B07043g) on ​​chromosome B in the Po1f △ku70 genome (upstream homologous arm) was inserted into the pUC-HUH vector. EcoR At the I restriction site, a 2091 bp sequence (downstream homologous arm) is inserted into the pUC-HUH vector downstream of the IntB stop codon. Hind The Ura3 gene, obtained from the III restriction site, has a HisG tag encoding gene attached to each end. The Ura3 gene with the HisG tag is located between the upstream and downstream homologous arms of the IntB site.

[0035] The integrative plasmid pUC-HUH-IntB-CfCytB5A is based on the IntB site integrative plasmid pUC-HUH-IntB, with the CfCytB5A gene expression cassette inserted into it. EXP -CfCytB5A-T lip2t Using IntB-EXP-F / IntB-EXP-R and IntB-Lip2t-F / IntB-Lip2t-R as two primer pairs as described in Table 2, respectively, Yarrowia lipolytica Using Po1f △ku70 genomic DNA as a template, the CfCytB5A expression cassette promoter P was amplified. EXP and Termination T lip2t Using plasmid pUC57- CfCytB5A Using DNA as a template, as described in Table 2 CfCytB5A -F and CfCytB5A -R is a primer, with promoters P at both ends of the amplification. EXP and Termination T lip2t homologous arms CfCytB5A Genes were purified and fragments recovered. Restriction endonucleases were used. Pac The IntB site integration plasmid was digested with enzymes, and the linearized IntB site integration plasmid was recovered by agarose gel electrophoresis. The linearized IntB site integration plasmid and the plasmid constructed in this embodiment were then compared. CfCytB5A The elements in the gene expression cassette were cloned in one step to obtain the recombinant plasmid pUC-HUH-IntB-CfCytB5A, and then used with restriction endonucleases. Not I was digested with enzymes, and the linearized recombinant plasmid pUC-HUH-IntB-CfCytB5A was recovered by agarose gel electrophoresis.

[0036] The constructed linearized recombinant plasmid pUC-HUH-IntB-CfCytB5A was transformed into the recombinant bacterium ML-For-1 from Example 1 for homologous recombination, resulting in recombinant bacterium ML-For-2. The specific construction method was the same as that used for recombinant bacterium 1 in Example 1.

[0037] Table 2 Primer sequences for constructing recombinant plasmids of the cytochrome b5 encoding gene.

[0038] Example 3: Adjusting the size of the endoplasmic reticulum to restore endoplasmic reticulum homeostasis This embodiment constructs the recombinant knockout plasmid pUC-HUH-PAH1 to knock out the phosphatidylphosphatase encoding gene. PAH1 .

[0039] In this embodiment, a company was commissioned to synthesize codon-optimized Arabidopsis thaliana. Arabidopsis thaliana Scaffold protein of origin AtMSBP1 Its nucleotide sequence is shown in SEQ ID No. 9, and the optimized sequence was inserted into plasmid pUC57 to obtain plasmid pUC57- AtMSBP1。 For details on the construction method of the SCP2 site integration plasmid pUC-HUH-SCP2, please refer to Chinese Patent CN120536475A.

[0040] (1) Construction of the recombinant knockout plasmid pUC-HUH-PAH1 (i.e., PAH1 site integration plasmid) The recombinant plasmid pUC-HUH-PAH1 uses pUC57-HUH as its backbone. EcoR I. Inserted at the restriction site Yarrowia lipolytica Po1f In the △ku70 genome, a 1501 bp homologous arm (PAH1-up) upstream of the start codon at the PAH1 site (gene number YALI1_D35593g) is located in... Pac A 1502 bp homologous arm (PAH1-dn) was inserted downstream of the PAH1 stop codon at the I restriction site, containing the hisG tag encoding gene and the orotate nucleoside-5'-phosphate decarboxylase encoding gene expression cassette (containing...). Yarrowia lipolytica Endogenous promoter P TEFin orotate nucleoside-5'-phosphate decarboxylase and terminator T xpr2t Between the upstream and downstream homologous arms. Using PAH1-Up-F / PAH1-Up-R as primers as described in Table 3, with Yarrowia lipolytica Po1f Using △ku70 genomic DNA as a template, the homologous arm PAH1-up upstream of the start codon at the PAH1 site was amplified. Restriction endonuclease was used. EcoR I. After digesting the plasmid pUC57-HUH with enzymes, the linearized pUC57-HUH plasmid was recovered by agarose gel electrophoresis. The linearized pUC57-HUH plasmid and its upstream homologous arm PAH1-up were then cloned in one step to obtain the circular recombinant vector pUC-HUH-PAH1-up. Using PAH1-Dw-F / PAH1-Dw-R as primers as described in Table 3, ... Yarrowia lipolytica Po1f Using △ku70 genomic DNA as a template, the downstream homologous arm PAH1-dn of the PAH1 stop codon was amplified. Restriction endonuclease was used. Pac After digesting the plasmid pUC-HUH-PAH1-up with restriction enzymes, the linearized pUC-HUH-PAH1-up plasmid was recovered by agarose gel electrophoresis. The linearized pUC-HUH-PAH1-up plasmid and PAH1-dn were cloned in one step to obtain a circular recombinant vector. The circular recombinant vector was transformed into *E. coli* DH5α competent cells, and the recombinant plasmid pUC-HUH-PAH1 was obtained through ampicillin-resistant plate selection and colony PCR and sequencing verification. The recombinant plasmid pUC-HUH-PAH1 was then digested with restriction endonucleases. Not I. Enzyme digestion was performed, and the linearized pUC-HUH-PAH1 plasmid was obtained by agarose gel electrophoresis.

[0041] (2) Integral plasmid pUC-HUH-SCP2- AtMSBP1 Construction Integral plasmid pUC-HUH-SCP2- AtMSBP1 It uses the SCP2 site integration plasmid pUC-HUH-SCP2 as a backbone and inserts... AtMSBP1 Gene expression cassette (P TEFin - AtMSBP1 -T pex10t Using SCP2-TEFin-F / SCP2-TEFin-R and SCP2-pex10t-F / SCP2-pex10t-R as primer pairs as described in Table 3, respectively, Yarrowia lipolytica Using Po1f△ku70 genomic DNA as a template, amplification was performed. AtMSBP1 Expression box promoter P TEFin and Termination T pex10t Using plasmid pUC57- AtMSBP1 Using DNA as a template, as described in Table 3 AtMSBP1 -F and AtMSBP1 -R is a primer, with promoters P at both ends of the amplification. TEFin and Termination T pex10t homologous arms AtMSBP1 Genes were purified and fragments recovered. Restriction endonucleases were used. Hind III. The SCP2 site integration plasmid was digested with enzymes, and the linearized SCP2 site integration plasmid was recovered by agarose gel electrophoresis. The linearized SCP2 site integration plasmid and the plasmid constructed in this embodiment were then compared. AtMSBP1 The recombinant plasmid pUC-HUH-SCP2- was obtained by cloning the elements in the gene expression cassette in one step. AtMSBP1 Using restriction endonucleases Not I was subjected to enzyme digestion, and the linearized recombinant plasmid pUC-HUH-SCP2- was recovered by agarose gel electrophoresis. AtMSBP1 .

[0042] The linearized recombinant plasmid pUC-HUH-PAH1 was transformed into recombinant bacteria ML-For-2 to obtain intermediate bacteria 1. Then, the linearized recombinant plasmid pUC-HUH-SCP2- was... AtMSBP1 ML-For-3 was obtained by transformation into intermediate strain 1. The construction method of the strain is the same as that of recombinant strain 1 in Example 1.

[0043] Table 3 Primer sequences for constructing recombinant plasmids as described in Example 3

[0044] Example 4: Enhancing NADPH Supply and Morphological Modification This embodiment constructs the recombinant knockout plasmid pUC-HUH-RAS2 to obtain a hyphae-free phenotype and introduces a mycorrhizal fungus (Mucor). Mucor circinelloides The malic acidase-encoding gene (McE2) provides sufficient NADPH for the synthesis of trichomoniasis.

[0045] The company was commissioned to synthesize codons optimized from Mucor (Rhizopus spp.) Mucor circinelloides The malic acid oxidase encoding gene McE2 was obtained, and its nucleotide sequence is shown in SEQ ID No. 10. The optimized sequence was inserted into plasmid pUC57 to obtain plasmid pUC57-McE2.

[0046] (1) Construction of the knockout plasmid pUC-HUH-RAS2 (i.e., the RAS2 site integration plasmid) The knockout plasmid pUC-HUH-RAS2 uses pUC57-HUH as its backbone and inserts... Yarrowia lipolytica In the Po1f△ku70 genome, the RAS2 site (gene ID YALI1_E35305g) contains a 1532 bp homologous arm upstream of the start codon (RAS2-up) and a 1612 bp homologous arm downstream of the stop codon (RAS2-dm). The expression cassette contains the hisG tag encoding gene and the orotate nucleoside-5'-phosphate decarboxylase encoding gene. Yarrowia lipolytica Endogenous promoter P TEFin orotate nucleoside-5'-phosphate decarboxylase and terminator T xpr2t Between the upstream and downstream homologous arms. Using RAS2-Up-F / RAS2-Up-R primers as described in Table 4, with... Yarrowia lipolytica Po1f Using △ku70 genomic DNA as a template, the homologous arm RAS2-up upstream of the start codon at the RAS2 site was amplified. Restriction endonuclease was used. EcoR I. After digesting plasmid pUC57-HUH with enzymes, the linearized pUC57-HUH plasmid was recovered by agarose gel electrophoresis. The linearized pUC57-HUH plasmid and RAS2-up were cloned in one step to obtain the positive recombinant plasmid pUC-HUH-RAS2-up. Using RAS2-Dw-F / RAS2-Dw-R as primers as described in Table 4, ... Yarrowia lipolytica Using Po1f △ku70 genomic DNA as a template, the downstream homologous arm RAS2-dm of the RAS2 stop codon was amplified. Restriction endonuclease was used. Pac After digesting the plasmid pUC-HUH-RAS2-up with restriction enzymes, the linearized pUC-HUH-RAS2-up plasmid was recovered by agarose gel electrophoresis. The linearized pUC-HUH-RAS2-up plasmid and RAS2-dm were then cloned in one step to obtain the positive recombinant plasmid pUC-HUH-RAS2, which was then processed with restriction endonucleases. Not I. Enzyme digestion was performed, and the linearized pUC-HUH-RAS2 plasmid was recovered by agarose gel electrophoresis.

[0047] (2) Construction of the integrative plasmid pUC-HUH-RAS2-McE2 The integrative plasmid pUC-HUH-RAS2-McE2 is based on the RAS2 site integrative plasmid pUC-HUH-RAS2, with the McE2 gene expression cassette inserted into it. TEFin -McE2-T xpr2t Using the RAS2-TEFin-F / RAS2-TEFin-R and RAS2-XPR2-F / RAS2-XPR2-R primer pairs as described in Table 4, respectively, Yarrowia lipolytica Using Po1f △ku70 genomic DNA as a template, the McE2 expression cassette promoter P was amplified. TEFin and Termination T xpr2t Using plasmid pUC57-McE2 DNA as a template, and MCE2-F and MCE2-R as primers as described in Table 4, amplification was performed using promoters P at both ends. TEFin and Termination T xpr2t The McE2 gene of the homologous arm was purified and the fragments were recovered.

[0048] Using restriction endonucleases Pac The RAS2 site integration plasmid was digested with enzymes, and the linearized RAS2 site integration plasmid was recovered by agarose gel electrophoresis. The linearized RAS2 site integration plasmid and the plasmid constructed in this embodiment were then compared. McE2 The elements in the gene expression cassette were cloned in one step to obtain the recombinant plasmid pUC-HUH-RAS2-McE2, and then used with restriction endonucleases. NotI was digested with enzymes, and the linearized recombinant plasmid pUC-HUH-RAS2-McE2 was obtained by agarose gel electrophoresis.

[0049] Table 4 Primer sequences for constructing recombinant plasmids as described in Example 4

[0050] The linearized recombinant plasmid pUC-HUH-RAS2 was transformed into recombinant bacteria ML-For-3 to obtain intermediate bacteria 2.

[0051] The linearized recombinant plasmid pUC-HUH-RAS2-McE2 was transformed into recombinant bacteria ML-For-3 to obtain recombinant bacteria ML-For-4. The specific construction method of the strain is the same as that of recombinant bacteria 1 in Example 1.

[0052] Example 5: Production of Trichosin by Fermentation of Recombinant Yersinia Lipolysaccharide (1) Seed culture: Strains Po1f △ku70, SLC301, and ML-For-1, ML-For-2, ML-For-3, and ML-For-4 constructed in Examples 1-4 were cultured on YPD plates at 28 °C for 48 h. Single colonies were picked and placed in 5 mL of YPD liquid medium and cultured at 28 °C and 200 rpm for 16-24 h until OD was obtained. 600 A value of around 5 is used as the seed liquid for fermentation.

[0053] (2) Shake flask fermentation culture: The above seed culture was transferred to a 250 mL shake flask containing 50 mL of fermentation medium to allow its initial OD to reach 50 mL. 600The concentration was controlled at 0.5. The mixture was then placed in a shaker at 28 °C and 200 rpm for fermentation. After 30 h of culture, sterilized 5-aminolevulinic acid (ALA) was added to a final concentration of 700 µg / L, and the mixture was cultured for another 120 h. The fermentation broth was then obtained. The fermentation medium consisted 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 stock solution, and 1.0 mL / L vitamin stock solution. The vitamin mother liquor is an aqueous solution containing the following components: biotin 0.05 g / L, calcium pantothenate 1 g / L, thiamine 1 g / L, pyridoxine 1 g / L, nicotinic acid 1 g / L, para-aminobenzoic acid 0.2 g / L, and inositol 25 g / L; the trace metal mother liquor is an aqueous solution containing the following components: EDTA 15 g / L, zinc sulfate 4.5 g / L, cobalt chloride 0.3 g / L, manganese chloride 1 g / L, copper sulfate 0.1 g / L, calcium chloride 4.5 g / L, ferrous sulfate 3 g / L, sodium molybdate 0.4 g / L, boric acid 1 g / L, and potassium iodide 0.1 g / L.

[0054] This embodiment also included fed-batch fermentation of the recombinant bacteria: ML-For-4 recombinant bacteria were inoculated into 50 mL of YPD liquid medium and cultured at 30 °C and 220 rpm for 24 h to obtain a seed culture. This seed culture was then inoculated into a 5 L fermenter containing 3 L of fermentation medium at a 5% v / v inoculation rate. After 24 h of culture, sterilized heme precursor 5-aminolevulinic acid (ALA) was added to achieve a final concentration of 700 µg / L, and the dissolved oxygen concentration was controlled at 30%. During fermentation, the pH was consistently maintained at 5.5 until the fermentation was complete. During fermentation, glucose was fed in to maintain a glucose concentration below 10 g / L in the fermenter. The fermentation temperature was 30 °C, and the stirring speed was adjusted according to the dissolved oxygen concentration (DO), varying between 300 and 1000 rpm, for a total culture time of 120 h. The composition of the fermentation medium was the same as that described in step (2) of this embodiment.

[0055] After fermentation, the target product was separated and detected by HPLC according to the following method. The extraction and analysis method for trichodin was as follows: After fermentation, the bacterial culture was collected, centrifuged at 5000 r / min for 5 min, and 1 mL of the supernatant was aspirated. 1 mL of dodecane was added, and the mixture was vortexed for 20 min. The upper organic phase was aspirated into a 1.5 mL EP tube, and 100 μL of anhydrous sodium sulfate was added. The mixture was vortexed thoroughly to remove trace amounts of water from the organic phase. After centrifugation at 12000 r / min for 2 min, the upper extract was filtered through a 0.22 μM organic filter and placed in an HPLC vial for HPLC detection. Trichodin 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 1260 VWD UV detector (Agilent), the mobile phase was acetonitrile-water (5:5 v / v), the total flow rate was 0.8 mL / min, and the detector wavelength was set to 210 nm. Standard solutions of different concentrations (1-600 mg / L) were prepared using the standard of saliva-containing saliva, and a standard curve was plotted based on the peak area and concentration of the standard solution to calculate the yield of the product.

[0056] After fermentation, the production of tuftol from strains Po1f △ku70 and SLC301 was 0.00 mg / L, as determined by HPLC. After shake-flask fermentation, the production of tuftol from strains ML-For-1, ML-For-2, ML-For-3, and ML-For-4 reached 72.12 mg / L, 123.54 mg / L, 165.37 mg / L, and 286.32 mg / L, respectively, as determined by HPLC. Among them, the recombinant strain ML-For-4 produced the highest production of tuftol, with a tuftol content of 3.64 g / L obtained through fed-batch fermentation, meaning that 3.64 g of tuftol could be obtained per liter of fermentation broth.

[0057] This invention provides a method for constructing and applying a recombinant *Yersinia lipolytica* strain for synthesizing trichodin. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for constructing a recombinant lipophilic yeast strain for synthesizing trichodin, characterized in that, The recombinant Yersinia lipolytica contains a combination of genes encoding tretinoin synthase, cytochrome P450 oxidase, cytochrome P450 reductase, acetyltransferase, cytochrome b5, and scaffold protein, while simultaneously knocking out the gene encoding phosphatidyl phosphatase. The nucleotide sequence of the gene encoding the lacrimal ether synthase is shown in SEQ ID No. 1~2; The nucleotide sequences of the gene encoding the cytochrome P450 oxidase are shown in SEQ ID No. 3-5; The nucleotide sequences of the genes encoding cytochrome P450 reductase, acetyltransferase, cytochrome b5, and scaffold protein are shown in SEQ ID No. 6-9, respectively. The gene number of the phosphatidyl phosphatase is YALI1_D35593g.

2. The construction method according to claim 1, characterized in that, The recombinant Yersinia lipolyticis yeast described herein has had the gene encoding the GTP-binding protein knocked out and / or has had the gene encoding malic acid oxidase introduced.

3. The construction method according to claim 2, characterized in that, The gene number of the GTP-binding protein is YALI1_E35305g; the nucleotide sequence of the gene encoding the malic acid enzyme is shown in SEQ ID No.

10.

4. The construction method according to claim 2, characterized in that, The recombinant Yersinia lipolyticis described above had the gene encoding the GTP-binding protein knocked out, while the gene encoding malic acid oxidase was introduced.

5. The construction method according to claim 1, characterized in that, The recombinant Yersinia lipolytica, whose originating strain includes Yersinia lipolytica capable of synthesizing geraniylgeraniyl pyrophosphate.

6. The construction method according to claim 5, characterized in that, The starting strain mentioned includes Yersinia lipophila SCL301.

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

8. The application of the recombinant Yersinia lipophila according to claim 7 in the fermentation production of saliva.

9. The application according to claim 8, characterized in that, The recombinant Yersinia lipolytica was inoculated into the fermentation medium and fermented at 28-30°C for 24-36 h. Then, 5-aminolevulinic acid was added and the culture was continued for 120-150 h.

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

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