A method for biosynthesis of tetraenemenadione by yarrowia lipolytica

Abstract

The application provides a method for biosynthesis of tetraenemenaquinone by Yarrowia lipolytica. The method is achieved by constructing recombinant Yarrowia lipolytica, and the construction method of the recombinant Yarrowia lipolytica for synthesizing tetraenemenaquinone comprises the following steps: intensively expressing a mevalonate pathway in Yarrowia lipolytica, and introducing a heterologous synthesis pathway of geranylgeranyl pyrophosphate to provide sufficient isoprene side chain donors for the biosynthesis of tetraenemenaquinone; on the basis, overexpressing a codon-optimized aromatic isopentenyltransferase gene with menaquinone substrate specificity in Yarrowia lipolytica. The construction method of Yarrowia lipolytica for biosynthesis of tetraenemenaquinone provided by the application is simple in operation, the constructed Yarrowia lipolytica can obtain a high-concentration tetraenemenaquinone product by using commercially available menaquinone as a substrate, the biosynthesis steps are short, and the method has high production application value.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a method for biosynthesizing tetraene-menaquinone using Yersinia lipolytica. Background Technology

[0002] In 1934, Danish biochemist Carl Peter Henrik Dam accidentally discovered a substance with blood-clotting properties during an experiment. Experiments proved it to be a fat-soluble vitamin found in alfalfa, and he named it "Kougalatian Vitamin," taking the first letter to name it Vitamin K. Three months later, another American scientist, Edward Doisv, isolated a compound with the same physiological function as Vitamin K from rotten fish and determined its structure. Based on the order of discovery, they were named Vitamin K1 and K2, respectively. The two scientists shared the 1943 Nobel Prize in Physiology or Medicine for their first discovery of the Vitamin K compound.

[0003] Subsequently, the structures of a series of K-group vitamins were discovered and elucidated. K-group vitamins are a collective term for a class of menadione (2-methyl-1,4-naphthoquinone) derivatives, mainly including K1 (Phylloquinone, PK), K2 (Menaquinone, MK), and K3 (Menadione). K1 and K2 are naturally occurring, found in plants and animals respectively, and are fat-soluble vitamins. Chemically, K1 and K2 are both derivatives of menadione, differing only in the R group of their side chains. K3, however, is menadione, synthesized artificially, and is a water-soluble vitamin. The specific chemical structural formulas of the K-group vitamins are shown in the figure below:

[0004]

[0005] The above analysis shows that vitamin K2 is not a single substance, but a collective term for a series of structurally similar compounds. Based on the different numbers of isoprene structural units in the molecular side chains, naturally occurring vitamin K2 includes more than ten compounds from MK-1 to MK-14. Among these compounds, MK-4 (tetraene-menadione) and MK-7 (heptaene-menadione) are currently the most widely used. Tetraene-menadione has important and extensive applications in the fields of medicine and food fortification. As early as 1972, tetraene-menadione was approved for marketing as a procoagulant; in 1995, Japan officially approved tetraene-menadione soft capsules developed by Eisai Co., Ltd. as a treatment for osteoporosis, with a clinically recommended dose of 45 mg / day. Tetraene-menadione is listed in the Japanese Pharmacopoeia JP16. In 2010, my country's National Medical Products Administration approved Eisai Co., Ltd.'s imported tetraenmenadione soft capsules as a treatment for osteoporosis, suitable for improving bone mass and pain associated with osteoporosis. It is also one of the recommended drugs in the "Guidelines for the Diagnosis and Treatment of Primary Osteoporosis" and the "Guidelines for the Diagnosis and Treatment of Osteoporosis Using Integrated Traditional Chinese and Western Medicine." In 2021, the United States Pharmacopeia (USP) approved tetraenmenadione as a dietary supplement, allowing its addition to health foods, functional foods, and fortified foods. Since then, the application of tetraenmenadione in the food industry has surged, with its wide applicability and target audience unmatched by other drugs. The reason why tetraene-menaquinone was approved by the USP as a dietary supplement and has emerged as a leader in the field of food fortification is mainly due to its good safety profile and excellent physiological activity. It plays a key role in maintaining human bone health and regulating calcium metabolism. It can activate osteocalcin, significantly promote bone formation, and inhibit bone resorption. It can promote bone development in children and adolescents and reduce the risk of osteoporosis and vascular calcification caused by vitamin K2 deficiency in middle-aged and elderly people.

[0006] Currently, the main source of tetraene menadione is chemical synthesis, which is quite difficult to achieve. Besides the menadione parent ring, the most crucial aspect is the precise synthesis of the isoprene side chain in its structure. Only by ensuring that the isoprene side chain is in the all-trans configuration can the synthesized tetraene menadione possess biological activity. For a long time, although new methods and processes for synthesizing the menadione parent ring have been continuously developed (Journal of Labelled Compounds and Radiopharmaceuticals, 1989, 27(11): 1293-1298), the chemical synthesis method cannot guarantee the high purity of the all-trans isomer of the side chain. To achieve the precise synthesis of the isoprene side chain, further separation and purification are often required, making the process very cumbersome. Compared to chemical synthesis, developing a biosynthetic method for the production of tetraene-menadione has significant advantages. Scientists in Japan and my country have successively discovered that the Gram-negative bacterium *Flavobacterium* sp. can naturally synthesize tetraene-menadione (Agricultural and Biological Chemistry, 1987, 51(1):2409–2415; CN103571897B). However, this genus relies on a carbon source to synthesize tetraene-menadione de novo, involving a complex naphthoquinone parent ring and an all-trans isoprene structure, resulting in an extremely complex metabolic pathway and very low yield. Moreover, this genus is an opportunistic pathogen that can cause pneumonia, meningitis, sepsis, and other infections, making it unsuitable for industrial production. Therefore, to achieve large-scale biosynthetic production of tetraene-menadione, it is necessary to find a safe microbial method to synthesize tetraene-menadione with fewer steps. Summary of the Invention

[0007] Objective of the Invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a method for biosynthesizing tetraene-menaquinone using the recognized safe microorganism Yersinia lipolytica with fewer steps, specifically by constructing recombinant Yersinia lipolytica; and to address the problem that the existing Flavobacterium biosynthesis of tetraene-menaquinone involves cumbersome reaction steps, resulting in low biosynthesis efficiency, by simplifying the biosynthesis steps and achieving efficient synthesis of tetraene-menaquinone.

[0008] To address the aforementioned technical problems, this invention discloses a recombinant *Yersinia lipolytica* yeast, its construction method, and its applications. The constructed recombinant *Yersinia lipolytica* yeast can biosynthesize tetraene-menadione using commercially available and relatively inexpensive menadione as a substrate, avoiding the complex and lengthy biosynthesis of the naphthoquinone parent ring, thereby achieving efficient biosynthesis of tetraene-menadione. The technical solution of this invention is as follows:

[0009] A recombinant Yersinia lipolytica yeast expresses genes related to the mevalonate pathway, genes related to the biosynthesis of gerany-gerany pyrophosphate (GGPP), and genes encoding aromatic isopentenyltransferases.

[0010] The aromatic isopentenyltransferase encoding gene is derived from Homo sapiens, and its nucleotide sequence is shown in SEQ ID No. 7 (obtained by codon optimization of the amino acid sequence of NCBI RefSeq: NP_037451.1), which has substrate specificity for menaquinone.

[0011] The N-terminus of the aromatic isopentenyltransferase encoding gene is linked to a solubilization tag; the solubilization tag is any one of maltose-binding protein (MBP), thioredoxin A (Txa), and small ubiquitin-associated modified protein (SUMO), and their nucleotide sequences are shown in SEQ ID No. 8-10, respectively. Preferably, the solubilization tag is SUMO.

[0012] The mevalonic acid (MVA) pathway-related genes include any one or more combinations of the following: ERG10 encoding acetyl-CoA thiolase, IDI encoding isopentenyl diphosphate isomerase, HMGR encoding 3-hydroxy-3-methylglutaryl-CoA reductase, or a truncated HMGR gene; the MVA pathway provides sufficient precursors for the biosynthesis of the isoprene side-chain donor GGPP of tetraene-menadione. Preferably, the MVA pathway-related genes include ERG10, IDI, and a truncated HMGR gene.

[0013] The GGPP biosynthesis-related genes include any one or a combination of two of the GGPP synthase encoding genes (GGPPs) or the farnesyl pyrophosphate synthase encoding genes (FPS). Expression of the GGPP biosynthesis-related genes supplies the synthesis of isoprene side chains. A combination of GGPPs and FPS is preferred.

[0014] Preferably, both ERG10 and IDI are derived from Yarrowia lipolytica.

[0015] The HMGR mentioned above is derived from Yarrowia lipolytica or Silicibacter pomeroyi;

[0016] The GGPPs mentioned are derived from any one of the following: *Sulfolobus acidocaldarius*, *Phomopsis amygdali*, *Synechococcus sp.*, or *Yarrowia lipolytica*; the FPS mentioned are derived from the chicken Gallus gallus.

[0017] More preferably, the NCBI RefSeq of the ERG10 is NC_090774.1; the NCBI RefSeq of the IDI is NC_090775.1;

[0018] The NCBI RefSeq of HMGR derived from Yarrowia lipolytica is XP_503558.3. The truncated HMGR gene is a sequence obtained by truncating 500 amino acid residues from the N-terminus of the HMGR sequence with NCBI RefSeq of XP_503558.3, and is named tHMG1.

[0019] The nucleotide sequence of HMGR derived from *Silicibacter pomeroyi* is shown in SEQ ID No. 1 (obtained by codon optimization of the amino acid sequence from NCBI RefSeq: WP_011241944.1).

[0020] The nucleotide sequence of the GGPPs derived from *Sulfolobus acidocaldarius* is shown in SEQ ID No. 2 (obtained by codon optimization of the amino acid sequence from NCBI RefSeq: BAA43200.1); the nucleotide sequence of the GGPPs derived from *Phomopsis amygdali* is shown in SEQ ID No. 3 (PaGGPPs, obtained by codon optimization of amino acid sequences from positions 390 to 719 of the amino acid sequence from UniProtKB: A2PZA5.1).

[0021] The nucleotide sequence of the GGPPs derived from *Synechococcus* sp. is shown in SEQ ID No. 4 (SsGGPPs, obtained by codon optimization of the amino acid sequence of NCBI RefSeq: WP_011429285.1); the NCBI RefSeq of the GGPPs derived from *Yarrowia lipolytica* is XM_502923.1, which is fused with the ERG20 gene (RefSeq: NC_090774.1) from *Yarrowia lipolytica* for expression; preferably, the ERG20 gene of *Yarrowia lipolytica* is mutated to obtain the mutant protein Erg20. F87A Then Erg20 F87A The mutant is fused with GGPP synthase (YlGgpps) derived from Yersinia lipolytica, namely Erg20. F87A The nucleotide sequence of the mutant protein gene is shown in SEQ ID No. 5.

[0022] The nucleotide sequence of the FPS derived from domestic chicken Gallus gallus is shown in SEQ ID No. 6 (obtained by codon optimization after mutating amino acid residue 112 of the amino acid sequence of UniProtKB: P08836.2).

[0023] More preferably, the GGPPs are derived from *Phomopsis amygdali*.

[0024] More preferably, the present invention provides a recombinant Yarrowia lipolytica that expresses genes related to the mevalonate pathway, genes related to the biosynthesis of gerany-gerany-pyrophosphate (GGPP), and genes encoding aromatic isopentenyltransferases; the mevalonate pathway-related genes include truncated genes ERG10, IDI, and HMGR, all derived from Yarrowia lipolytica; the GGPP biosynthesis-related genes include GGPPs derived from Phomopsisamygdali and FPS derived from Gallus gallus; the aromatic isopentenyltransferase encoding gene is derived from Homo sapiens, with a solubilization tag SUMO attached to its N-terminus.

[0025] Secondly, the present invention provides a method for constructing the recombinant *Yersinia lipolytica* described in the first aspect, which involves inserting gene expression cassettes encoding mevalonate pathway-related genes, GGPP biosynthesis-related genes, and aromatic isopentenyltransferase genes into the *Yersinia lipolytica* genome. Preferably, the 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 for gene expression is the reported neutral site of *Yersinia lipolytica* (Biotechnology Journal, 2018, 13(9), 1700543).

[0026] Thirdly, the present invention provides the application of the recombinant Yersinia lipolytica described in the first aspect in the biosynthesis of tetraene-menaquinone.

[0027] The recombinant Yersinia lipolyticis was inoculated into a fermentation medium and fermented at 28-30°C for 24-36 hours. Then, menaquinone was added and the culture was continued for another 24-90 hours.

[0028] Preferably, the concentration of the added menaquinone is 2-15 g / L, and more preferably 5.0 g / L.

[0029] More preferably, after obtaining the seed culture from the recombinant *Yersinia lipolyticis*, it is inoculated into the fermentation medium. The seed culture is prepared as follows: The recombinant *Yersinia lipolyticis* is streaked on YPD plates and cultured at 28°C for 48 hours. A single colony is picked and placed in 5 mL of YPD liquid medium, then incubated in a shaker at 28°C and 200 rpm for 16-24 hours until OD (digesterone) is obtained. 600 A value of around 5 is used as the seed liquid for fermentation.

[0030] More preferably, the fermentation seed liquid is transferred to a 250mL shake flask containing 50mL of fermentation culture medium to allow its initial OD to reach its target concentration. 600 The concentration was controlled at 0.5. Then it was placed in a shaker at 28°C and 200 rpm for fermentation.

[0031] More preferably, the fermentation medium consists 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 ion stock solution, and 1.0 mL / L vitamin stock solution (the composition and preparation method of the trace metal ion stock solution and vitamin stock solution are described in the literature Yeast, 1992, 8, 501–517). After culturing for 24–36 h, sterilized menadione stock solution is added to bring the final concentration to 2–15 g / L, and culturing continues for 24–90 h until the batch fermentation is complete. After fermentation, intracellular and extracellular tetraene menadione is isolated and extracted for HPLC detection.

[0032] Beneficial effects:

[0033] This invention is based on *Yersinia lipolytica*, which has its homologous recombination ability enhanced by knocking out the gene KU70 responsible for non-homologous recombination. Gene integration is achieved through the homologous recombination function of *Yersinia lipolytica* itself, significantly improving the genetic stability of the introduced gene. Specifically, the following three steps are used to construct recombinant *Yersinia lipolytica*: 1) Enhancing the expression of the mevalonate (MVA) pathway in *Yersinia lipolytica* to provide sufficient precursors for the biosynthesis of geranylgeranyl pyrophosphate (GGPP), the isoprene side chain donor for tetraene-menadiene; 2) Enhancing the biosynthesis of GGPP in *Yersinia lipolytica* to supply the synthesis of the isoprene side chain; 3) Overexpressing the codon-optimized Homo sapiens-derived aromatic isopentenyltransferase encoding gene (HsUBIAD1) with substrate specificity for tetraene-menadiene in *Yersinia lipolytica*. Thus, the recombinant *Yersinia lipolytica* constructed through these three steps can efficiently biosynthesize tetraene-menadiene using commercially available tetraene-menadiene as a substrate. The existing technology for the biosynthesis of tetraene-menadione involves complex naphthoquinone parent ring biosynthesis processes requiring up to 20 steps, resulting in extremely high difficulty and low efficiency. By using commercially available and relatively inexpensive menadione as a substrate (costing only 1 / 300 to 1 / 200 of tetraene-menadione), and utilizing a newly constructed menadione-based metabolic pathway from recombinant Yersinia lipolytica, the target product tetraene-menadione can be synthesized. This approach ensures the safety of the biosynthesis, simplifies the synthesis steps, avoids the influence of complex metabolic regulatory mechanisms, and significantly increases the yield of the target product, continuously meeting consumer demand. Attached Figure Description

[0034] 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.

[0035] Figure 1This is a schematic diagram illustrating the method for biosynthesizing tetraene-menaquinone based on recombinant Yersinia lipolytica. In this diagram, ERG10 is acetyl-CoA thiolytic enzyme, HMGR is 3-hydroxy-3-methyl-glutaryl-CoA reductase, tHMG1 is truncated 3-hydroxy-3-methyl-glutaryl-CoA reductase, GGPPs is gerany-gerany-1 pyrophosphate synthase, ERG20 is endogenous farnesyl pyrophosphate synthase, FPS is heterologous farnesyl pyrophosphate synthase, IDI is isopentenyl diphosphate isomerase, and BIAD1 is an aromatic isopentenyl transferase.

[0036] Figure 2 This is a plasmid map of pUC-HUH-IntA-PaGGPPs in this invention.

[0037] Figure 3 pUC-HUH-LIP1-FPS in this invention F112A The plasmid map.

[0038] Figure 4 This is the plasmid map of pUC-Leu-A08-SUMO-YlHsUBID1 in this invention. Detailed Implementation

[0039] 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.

[0040] The Yarrowia lipolytica Po1f described in the following examples was purchased from the U.S. Culture Collection Center, catalog number ATCC MYA-2613.

[0041] The Yarrowia lipolytica Po1fΔku70 described in the following examples was constructed by knocking out the KU70 gene responsible for non-homologous recombination in the Yarrowia lipolytica Po1f strain (published in Ji Q, et al., Metabolic Engineering Communications, 2021, 11, e00152).

[0042] The integrative plasmids of Yersinia lipophila described in the following examples were all constructed based on neutral sites reported in the literature (Biotechnology Journal, 2018, 13(9), 1700543). The neutral sites include A08, SCP2, LIP1, IntA, IntC and IntF, as shown in Table 1.

[0043] Table 1. Neutral sites and their integrative plasmids used in this invention.

[0044]

[0045] The A08 site integration plasmid pUC-Leu-A08 is obtained by inserting a 2521 bp sequence (upstream homologous arm) upstream of the start codon at the A08 site (YALI1_A08073g) on ​​chromosome A in the Yarrowia lipolytica Po1fΔku70 genome into the PacI restriction site of the pUC57-Leu (pUC-Leu, construction method see Example 1) vector, and inserting a 2031 bp sequence (downstream homologous arm) downstream of the stop codon at the A08 site into the HindIII restriction site of the pUC-Leu vector. The Leu expression cassette is located between the upstream and downstream homologous arms at the A08 site.

[0046] The SCP2 site integration plasmid pUC-HUH-SCP2 is obtained by inserting a 1523bp sequence (upstream homologous arm) upstream of the start codon of the SCP2 site (YALI1_E01865g) on ​​chromosome E of the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site of the pUC57-HisG-Ura3-HisG (pUC-HUH, construction method see Example 1) vector, and a 1524bp sequence (downstream homologous arm) downstream of the stop codon of the SCP2 site into the HindIII restriction site of the pUC-HUH vector. The Ura3 gene is linked to a HisG tag encoding gene at both ends, and the Ura3 gene with the HisG tag is located between the upstream and downstream homologous arms of the SCP2 site.

[0047] The LIP1 site integration plasmid pUC-HUH-LIP1 is obtained by inserting a 1458 bp sequence (upstream homologous arm) upstream of the start codon of the LIP1 site (YALI1_E13541g) on ​​chromosome E of the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site of the pUC-HUH vector, and a 1438 bp sequence (downstream homologous arm) downstream of the stop codon of the LIP1 site into the HindIII restriction site of the pUC-HUH vector. The Ura3 gene is linked to a HisG tag encoding gene at both ends, and the Ura3 gene with the HisG tag is located between the upstream and downstream homologous arms of the LIP1 site.

[0048] The IntA site integration plasmid pUC-HUH-IntA is obtained by inserting a 1633bp sequence (upstream homologous arm) upstream of the start codon of the IntA site (YALI1_A15185g) on ​​chromosome A in the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site of the pUC-HUH vector, and a 1621bp sequence (downstream homologous arm) downstream of the stop codon of the IntA site into the HindIII restriction site of the pUC-HUH vector. The Ura3 gene is linked to a HisG tag encoding gene at both ends, and the Ura3 gene with the HisG tag is located between the upstream and downstream homologous arms of the IntA site.

[0049] The IntC site integration plasmid pUC-HUH-IntC is obtained by inserting a 1402 bp sequence (upstream homologous arm) upstream of the IntC site (YALI1_C25990g) on ​​chromosome C of the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site of the pUC-HUH vector, and a 396 bp sequence (downstream homologous arm) downstream of the IntC site stop codon into the HindIII restriction site of the pUC-HUH vector. The Ura3 gene is linked to a HisG tag encoding gene at both ends, and the Ura3 gene linked to the IntC site is located between the upstream and downstream homologous arms.

[0050] The IntF site integration plasmid pUC-HUH-IntF is obtained by inserting a 1610 bp sequence (upstream homologous arm) upstream of the start codon of the IntF site (YALI0_F24167g) on ​​chromosome F of the Yarrowia lipolytica Po1fΔku70 genome into the EcoRI restriction site of the pUC-HUH vector, and a 1852 bp sequence (downstream homologous arm) downstream of the stop codon of the IntF site into the HindIII restriction site of the pUC-HUH vector. The Ura3 gene is linked to a HisG tag encoding gene at both ends, and the Ura3 gene linked to the HisG tag is located between the upstream and downstream homologous arms of the IntF site.

[0051] In the following embodiments, the promoter P TEFin The nucleotide sequence is shown in SEQ ID No. 11, and the terminator T... xpr2t The nucleotide sequence is shown in SEQ ID No. 12.

[0052] In the following examples, the YPD liquid culture medium is formulated with 20 g / L peptone, 10 g / L yeast extract and 20 g / L glucose.

[0053] The YPD plate has the following formulation: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose and 20 g / L agar.

[0054] The SD-Ura 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-Ura (complete supplement mixture to remove uracil, purchased from MPBiomedicals), and 23 g / L agar powder.

[0055] 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.

[0056] The YPD plate containing 5-fluoroorotic acid has the following formulation: 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] Example 1: Amplification of Gene Elements and Preparation of Integrative Plasmids

[0058] (I) Preparation of target genes

[0059] Based on the nucleotide sequence of the β-isopropylmalate dehydrogenase encoding gene LEU2 from Yarrowia lipolytica (GenBank No. M37309.1) provided on NCBI, Suzhou Genewise Biotech Co., Ltd. was commissioned to synthesize LEU2. This cassette expresses the β-isopropylmalate dehydrogenase encoding gene (Leu) using the endogenous promoter P of Yarrowia lipolytica. TEFin The gene encoding β-isopropylmalate dehydrogenase, LEU2, and the terminator T xpr2t The plasmid pUC57-Leu was obtained by inserting it between the KpnI and PacI restriction sites of plasmid pUC57.

[0060] Based on the nucleotide sequence of the orotate nucleoside-5′-phosphate decarboxylase encoding gene URA3 (GenBank No. AJ306421.1) and the HisG tag (GenBank No. AF324729.1) from Yarrowia lipolytica provided on NCBI, Suzhou Genewise Biotechnology Co., Ltd. synthesized the gene. The two HisG tag encoding gene sequences were inserted between the EcoRI and HindIII restriction sites of plasmid pUC57 (purchased from Genewise). An orotate nucleoside-5′-phosphate decarboxylase encoding gene expression cassette (derived from the endogenous promoter P of Yarrowia lipolytica) was inserted between the two HisG tag encoding gene sequences. TEFin The genes encoding orotic nucleoside-5′-phosphate decarboxylase, Ura and T, are... xpr2t The composition was optimized to enable the recycling of URA3 markers, resulting in plasmid pUC57-HisG-URA3-HisG(pUC-HUH).

[0061] Based on the amino acid sequence of fusicoccadiene synthase (UniProtKB:A2PZA5.1) from *Phomopsis amygdali* provided on NCBI, after codon optimization, the optimized nucleotide sequence encoding the gene PaGGPPs (nucleotide sequence shown in SEQ ID No. 3) was synthesized by Suzhou Genewise Biotechnology Co., Ltd., and inserted into plasmid pUC57 to obtain plasmid pUC57-PaGGPPs.

[0062] Based on the amino acid sequence of the farnesyl pyrophosphate synthase mutant encoding gene (UniProtKB: P08836.2) from domestic chicken (Gallus gallus) provided on NCBI, after mutating amino acid residue 112 and optimizing the codons, Suzhou Genewise Biotechnology Co., Ltd. was commissioned to synthesize the optimized farnesyl pyrophosphate synthase mutant encoding gene FPS. F112A (The nucleotide sequence is shown in SEQ ID No. 6), and it is inserted into plasmid pUC57 to obtain plasmid pUC57-FPS. F112A .

[0063] Based on the aromatic isopentenyltransferase (HsUBIAD1, NCBI RefSeq: NP_037451.1) encoding gene from Homo sapiens (as provided on NCBI), and after codon optimization, Suzhou Genewiz Biotechnology Co., Ltd. synthesized the optimized human aromatic isopentenyltransferase encoding gene YlHsUBIAD1 (nucleotide sequence shown in SEQ ID No. 7). A lysosome-associated small molecule ubiquitin-related modification protein (SUMO) encoding gene was ligated to its N-terminus. The codon-optimized nucleotide sequence of this lysosome-associated protein encoding gene is shown in SEQ ID No. 10. The entire nucleotide sequence of the SUMO tag was then ligated to the N-terminus of YlHsUBIAD1 and inserted into plasmid pUC57 (purchased from Genewiz), resulting in plasmid pUC57-SUMO-YlHsUBIAD1.

[0064] Using Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, corresponding primer pairs were designed and amplified by PCR using the following gene sequences: tHMG1 (full-length sequence of 3-hydroxy-3-methylglutaryl-CoA reductase truncated to the N-terminus 500 amino acid residues), ERG10 (NCBI RefSeq: NC_090774.1), IDI (NCBI RefSeq: NC_090775.1), and IDI (NCBI RefSeq: NC_090775.1), along with their promoters and terminators. See the following section for details on the construction of the integrative plasmid.

[0065] (II) Construction of integrative plasmids

[0066] 1) Construction of the integrative plasmid pUC-HUH-IntC-tHMG1

[0067] The integrative plasmid pUC-HUH-IntC-tHMG1 uses pUC57-HisG-URA3-HisG as its backbone and inserts homologous arms upstream and downstream of the IntC site in the Yarrowia lipolytica Po1fΔku70 genome. Two HisG tag-encoding genes are located between the upstream and downstream homologous arms of the IntC site, and the tHMG1 expression cassette (P) is inserted between the downstream homologous arm and the HisG tag-encoding gene. TEFin -tHMG1-T xpr2t In this invention, the expression cassette is represented as follows: promoter-target gene-terminator, for example, the tHMG1 expression cassette is represented as P.TEFin -tHMG1-T xpr2t The promoter is P TEFin The target gene is tHMG1, and the terminator is T. xpr2t .

[0068] Using IntC-TEFin-F / IntC-TEFin-R and IntC-xpr2t-F / IntC-xpr2t-R as primer pairs as described in Table 2, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, the tHMG1 expression cassette promoter P was amplified. TEFin and Termination T xpr2t .

[0069] Using Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, and IntC-tHMG1-F and IntC-tHMG1-R as primers as described in Table 2, amplification was performed with promoters P at both ends. TEFin and Termination T xpr2t The tHMG1 gene of the homologous arm.

[0070] Each fragment was purified and recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit (Takara Bio Engineering (Dalian) Co., Ltd.).

[0071] The IntC site integration plasmid was digested with the restriction endonuclease PacI from NEB, and the linearized IntC site integration plasmid was recovered by agarose gel electrophoresis.

[0072] The linearized IntC site integration plasmid and the elements in the tHMG1 gene expression cassette constructed in this embodiment were cloned in one step using the ClonExpress MultiS One Step Cloning Kit of Nanjing Novizan Biotechnology Co., Ltd., to obtain the recombinant plasmid pUC-HUH-IntC-tHMG1.

[0073] The recombinant plasmid pUC-HUH-IntC-tHMG1 was digested with the restriction endonuclease EcoRI from NEB, and the linearized recombinant plasmid pUC-HUH-IntC-tHMG1 was recovered by agarose gel electrophoresis.

[0074] Table 2 Primer sequences for constructing the integrative plasmid pUC-HUH-IntC-tHMG1

[0075]

[0076] 2) Construction of the integrative plasmid pUC-HUH-SCP2-ERG10

[0077] The integrative plasmid pUC-HUH-SCP2-ERG10 uses pUC57-HisG-URA3-HisG as its backbone and inserts it into the upstream and downstream homologous arms of the SCP2 site in the Yarrowia lipolytica Po1fΔku70 genome. Two HisG tag-encoding genes are located between the upstream and downstream homologous arms of the SCP2 site, and an ERG10 expression cassette (P) is inserted between the downstream homologous arm and the HisG tag-encoding gene. TEFin -ERG10-T xpr2t ERG10 expression cassette is represented as P. TEFin -ERG10-T xpr2t The promoter is P TEFin The target gene is ERG10, and the terminator is T. xpr2t .

[0078] Using SCP2-TEFin-F / SCP2-TEFin-R and SCP2-xpr2t-F / SCP2-xpr2t-R as primer pairs as described in Table 3, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, the ERG10 expression cassette promoter P was amplified. TEFin and Termination T xpr2t .

[0079] Using Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, and SCP2-ERG10-F and SCP2-ERG10-R as primers as described in Table 3, the amplified molecules with promoters P at both ends were amplified. TEFin and Termination T xpr2t The ERG10 gene of the homologous arm.

[0080] Each fragment was purified and recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit (Takara Bio Engineering (Dalian) Co., Ltd.).

[0081] The SCP2 site integration plasmid was digested with HindIII restriction endonuclease from NEB, and the linearized SCP2 site integration plasmid was recovered by agarose gel electrophoresis.

[0082] The linearized SCP2 site integration plasmid and the elements in the ERG10 gene expression cassette constructed in this embodiment were cloned in one step using the ClonExpress MultiS One Step Cloning Kit of Nanjing Novizan Biotechnology Co., Ltd., to obtain the recombinant plasmid pUC-HUH-SCP2-ERG10.

[0083] The recombinant plasmid pUC-HUH-SCP2-ERG10 was digested with the restriction endonuclease EcoRI from NEB, and the linearized recombinant plasmid pUC-HUH-SCP2-ERG10 was recovered by agarose gel electrophoresis.

[0084] Table 3 Primer sequences for constructing the integrative plasmid pUC-HUH-SCP2-ERG10

[0085]

[0086]

[0087] 3) Construction of the integration plasmid pUC-HUH-IntF-IDI

[0088] The integrative plasmid pUC-HUH-IntF-IDI uses pUC57-HisG-URA3-HisG as its backbone and inserts homologous arms upstream and downstream of the IntF site in the Yarrowialipolytica Po1fΔku70 genome. Two HisG tag-encoding genes are located between the upstream and downstream homologous arms of the IntF site, and an IDI expression cassette (P) is inserted between the downstream homologous arm and the HisG tag-encoding gene. TEFin -IDI-T xpr2t IDI expression box is represented as P. TEFin -IDI-T xpr2t The promoter is P TEFin The target gene is IDI, and the terminator is T. xpr2t .

[0089] Using the primer pairs IntF-TEFin-F / IntF-TEFin-R and IntF-xpr2t-F / IntF-xpr2t-R as described in Table 4, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, the IDI expression cassette promoter P was amplified. TEFin and Termination T xpr2t .

[0090] Using Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, and IntF-IDI-F and IntF-IDI-R as primers as described in Table 4, amplification was performed with promoters P at both ends. TEFin and Termination T xpr2t IDI gene of homologous arm.

[0091] Each fragment was purified and recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit (Takara Bio Engineering (Dalian) Co., Ltd.).

[0092] The IntF site integration plasmid was digested with the restriction endonuclease PacI from NEB, and the linearized IntF site integration plasmid was recovered by agarose gel electrophoresis.

[0093] The linearized IntF site integration plasmid and the elements in the IDI gene expression cassette constructed in this embodiment were cloned in one step using the ClonExpress MultiS One Step Cloning Kit of Nanjing Novizan Biotechnology Co., Ltd., to obtain the recombinant plasmid pUC-HUH-IntF-IDI.

[0094] The recombinant plasmid pUC-HUH-IntF-IDI was digested with the restriction endonuclease NotI from NEB, and the linearized recombinant plasmid pUC-HUH-IntF-IDI was recovered by agarose gel electrophoresis.

[0095] Table 4 Primer sequences for constructing the integrative plasmid pUC-HUH-IntF-IDI

[0096]

[0097] 4) Construction of the integration plasmid pUC-HUH-IntA-PaGGPPs

[0098] The integrative plasmid pUC-HUH-IntA-PaGGPPs uses pUC57-HisG-URA3-HisG as its backbone and inserts homologous arms upstream and downstream of the IntA site in the Yarrowia lipolytica Po1fΔku70 genome. Two HisG tag-encoding genes are located between the upstream and downstream homologous arms of the IntA site, and the PaGGPPs expression cassette (P) is inserted between the downstream homologous arm and the HisG tag-encoding genes. TEFin -PaGGPPs-T xpr2t For details, see [link / details]. Figure 2 PaGGPPs expression boxes are represented as P TEFin -PaGGPPs-T xpr2t The promoter is PTEFin The target gene is PaGGPPs, and the terminator is T. xpr2t .

[0099] Using the primer pairs IntA-TEFin-F / IntA-TEFin-R and IntA-xpr2t-F / IntA-xpr2t-R as described in Table 5, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, the PaGGPPs expression cassette promoter P was amplified. TEFin and Termination T xpr2t .

[0100] Using plasmid pUC57-PaGGPPs DNA as a template, and IntA-PaGGPPs-F and IntA-PaGGPPs-R as primers as described in Table 5, amplification was performed with promoters P at both ends. TEFin and Termination T xpr2t PaGGPPs genes of homologous arms.

[0101] Each fragment was purified and recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit (Takara Bio Engineering (Dalian) Co., Ltd.).

[0102] The IntA site integration plasmid was digested with the restriction endonuclease PacI from NEB, and the linearized IntA site integration plasmid was recovered by agarose gel electrophoresis.

[0103] The linearized IntA site integration plasmid and each element in the PaGGPPs gene expression cassette constructed in this embodiment were cloned in one step using the ClonExpress MultiS One Step Cloning Kit of Nanjing Novizan Biotechnology Co., Ltd., to obtain the recombinant plasmid pUC-HUH-IntA-PaGGPPs.

[0104] The recombinant plasmid pUC-HUH-IntA-PaGGPPs was digested with the restriction endonuclease NotI from NEB, and the linearized recombinant plasmid pUC-HUH-IntA-PaGGPPs was recovered by agarose gel electrophoresis.

[0105] Table 5 Primer sequences for constructing the integrative plasmid pUC-HUH-IntA-PaGGPPs

[0106]

[0107] 5) Integrating plasmid pUC-HUH-LIP1-FPS F112A Construction

[0108] Integral plasmid pUC-HUH-LIP1-FPS F112A Using pUC57-HisG-URA3-HisG as the backbone, homologous arms upstream and downstream of the LIP1 site in the Yarrowia lipolytica Po1fΔku70 genome were inserted. Two HisG tag-encoding genes were located between the upstream and downstream homologous arms of the LIP1 site, and an FPS was inserted between the downstream homologous arm and the HisG tag-encoding gene. F112A Expression Box (P) TEFin -FPS F112A -T xpr2t For details, see [link / details]. Figure 3 FPS F112A The expression box is represented by P TEFin -FPS F112A -T xpr2t The promoter is P TEFin The target gene is FPS. F112A The terminator is T. xpr2t .

[0109] Using LIP1-TEFin-F / LIP1-TEFin-R and LIP1-xpr2t-F / LIP1-xpr2t-R as primer pairs as described in Table 6, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, FPS was amplified. F112A Expression box promoter P TEFin and Termination T xpr2t .

[0110] With plasmid pUC57-FPS F112A Using DNA as a template, and with the LIP1-FPS described in Table 6 F112A -F and LIP1-FPS F112A -R is a primer, with promoters P at both ends of the amplification. TEFin and Termination T xpr2t FPS of the homogeneous arm F112A Gene.

[0111] Each fragment was purified and recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit (Takara Bio Engineering (Dalian) Co., Ltd.).

[0112] 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.

[0113] The linearized LIP1 site integration plasmid and the FPS constructed in this embodiment were used. F112AThe components of the gene expression cassette were cloned in one step using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd., to obtain the recombinant plasmid pUC-HUH-LIP1-FPS. F112A .

[0114] The recombinant plasmid pUC-HUH-LIP1-FPS was mediated using the restriction endonuclease BamHI from NEB. F112A Enzyme digestion was performed, and the linearized recombinant plasmid pUC-HUH-LIP1-FPS was recovered by agarose gel electrophoresis. F112A .

[0115] Table 6. Integrative plasmid pUC-HUH-LIP1-FPS F112A Constructing primer sequences

[0116]

[0117] 6) Construction of the integration plasmid pUC-Leu-A08-SUMO-YlHsUBID1

[0118] The integrative plasmid pUC-Leu-A08-SUMO-YlHsUBID1 uses pUC-Leu-A08 as a backbone and inserts a SUMO-YlHsUBID1 expression cassette (P... TEFin -SUMO-YlHsUBID1-T xpr2t The structural diagram of the integrative plasmid pUC-Leu-A08-SUMO-YlHsUBID1 is shown below. Figure 4 .

[0119] Using the primer pairs A08-TEFin-F / A08-TEFin-R and A08-xpr2t-F / A08-xpr2t-R as described in Table 7, and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, the SUMO-YlHsUBID1 expression cassette promoter P was amplified. TEFin and Termination T xpr2t .

[0120] Using plasmid pUC57-SUMO-YlHsUBIAD1 DNA as a template, and primers A08-SUMO-YlHsUBID1-F and A08-SUMO-YlHsUBID1-R as described in Table 7, the DNA of plasmids pUC57-SUMO-YlHsUBID1-F and A08-SUMO-YlHsUBID1-R were used to amplify the DNA of plasmids pUC57-SUMO-YlHsUBID1-F, which have promoters P at both ends. TEFin and Termination T xpr2t The SUMO-YlHsUBID1 gene of the homologous arm.

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

[0122] The A08 site integration plasmid was digested with the restriction endonuclease AfeI from NEB, and the linearized A08 site integration plasmid was recovered by agarose gel electrophoresis.

[0123] The linearized A08 site integration plasmid and each element in the SUMO-YlHsUBID1 gene expression cassette constructed in this embodiment were cloned in one step using the ClonExpress MultiS One Step Cloning Kit of Nanjing Novizan Biotechnology Co., Ltd., to obtain the recombinant plasmid pUC-Leu-A08-SUMO-YlHsUBID1.

[0124] The recombinant plasmid pUC-Leu-A08-SUMO-YlHsUBID1 was digested with the restriction endonuclease NotI from NEB, and the linearized recombinant plasmid pUC-Leu-A08-SUMO-YlHsUBID1 was recovered by agarose gel electrophoresis.

[0125] Table 7 Primer sequences for constructing the integrative plasmid pUC-Leu-A08-SUMO-YlHsUBID1

[0126]

[0127] Example 2: Construction of recombinant Yersinia lipophila for the synthesis of tetraene-menaquinone

[0128] The linearized recombinant plasmids pUC-HUH-IntC-tHMG1, pUC-HUH-SCP2-ERG10, pUC-HUH-IntF-IDI, pUC-HUH-IntA-PaGGPPs, and pUC-HUH-LIP1-FPS constructed in Example 1 were used. F112A pUC-Leu-A08-SUMO-YlHsUBID1 was sequentially transformed into Yarrowia lipolytica Po1fΔku70 for homologous recombination. The tHMG1 expression cassette was inserted at the IntC site, the ERG10 expression cassette at the SCP2 site, the IDI expression cassette at the IntF site, the PaGGPPs expression cassette at the IntA site, and the FPS expression cassette at the LIP1 site. F112A The expression cassette was inserted at site A08 into the SUMO-YlHsUBID1 expression cassette to obtain recombinant Yersinia lipophila.

[0129] The specific method is as follows:

[0130] (1) Competent cells of Yarrowia lipolytica Po1fΔku70 were prepared after overnight culture in YPD liquid medium. pUC-HUH-IntC-tHMG1 was transformed into Yarrowia lipolytica Po1fΔku70 competent cells using Zymo Research Corporation's Zymogen Frozen EZ Yeast Transformation Kit II for homologous recombination.

[0131] (2) 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°C for 3 days. Single colonies were streaked simultaneously on both YPD and SD-Ura plates containing 5-fluoroorotic acid, and bacterial growth was observed. Single colonies that grew on YPD plates containing 5-fluoroorotic acid but not on SD-Ura plates were selected for PCR identification.

[0132] (3) After culturing the positive clones identified by PCR in step (2) overnight in YPD liquid medium, competent cells were prepared. pUC-HUH-SCP2-ERG10 was transformed into the competent cells of the positive clones identified by PCR in step (2) using Zymogen Frozen EZ Yeast Transformation Kit II of Zymo Research Corporation for homologous recombination.

[0133] (4) Positive clones were screened using SD-Ura plates and identified by PCR, using the same method as step (2).

[0134] (5) Prepare competent cells by culturing the positive clones identified by PCR in step (4) in YPD liquid medium overnight. Transform pUC-HUH-IntF-IDI into the competent cells of the positive clones identified by PCR in step (4) using Zymogen Frozen EZ Yeast Transformation Kit II of Zymo Research Corporation for homologous recombination.

[0135] (6) Positive clones were screened using SD-Ura plates and identified by PCR, using the same method as step (2).

[0136] (7) Prepare competent cells by culturing the positive clones identified by PCR in step (6) in YPD liquid medium overnight. Transform pUC-HUH-IntA-PaGGPPs into the competent cells of the positive clones identified by PCR in step (6) using Zymogen Frozen EZ Yeast Transformation Kit II of Zymo Research Corporation for homologous recombination.

[0137] (8) Positive clones were screened using SD-Ura plates and identified by PCR, using the same method as step (2).

[0138] (9) After culturing the correctly identified positive clones from step (8) in YPD liquid medium overnight, competent cells were prepared. The pUC-HUH-LIP1-FPS cells were then cultured using the Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation. F112A Transformed into competent cells of the positive clones identified by PCR in step (8) for homologous recombination. Positive clones were screened using SD-Ura plates and identified by PCR.

[0139] (10) After overnight culture of the positive clones identified by PCR in step (9) in YPD liquid medium, competent cells were prepared. pUC-Leu-A08-SUMO-YlHsUBID1 was transformed into the competent cells of the positive clones identified by PCR in step (9) using the Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination. Positive clones were screened using SD-Leu plates and identified by PCR. The obtained strain was named recombinant Yersinia lipophila XJ-MK-4.

[0140] Example 3: Production of tetraene-menaquinone by fermentation of recombinant Yersinia lipophila

[0141] (1) Seed culture: Recombinant Yersinia lipolyticis was streaked onto YPD plates and cultured at 28℃ for 48 h. Single colonies were picked and placed in 5 mL of YPD liquid medium, then incubated in a shaker at 28℃ 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.

[0142] (2) Fermentation culture: The above-mentioned fermentation seed culture was transferred to a 250 mL shake flask containing 50 mL of fermentation medium to allow its initial OD to rise. 600The concentration was controlled at 0.5. Then it was placed in a shaker at 28°C and 200 rpm for fermentation. 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 ion stock solution, and 1.0 mL / L vitamin stock solution.

[0143] 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.

[0144] 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.

[0145] After 30 hours of fermentation, sterilized menadione mother liquor (water as solvent) was added to a final concentration of 5.0 g / L. Fermentation continued for 48-90 hours until the batch fermentation was complete, yielding the fermentation broth. After fermentation, the target product was separated and analyzed by HPLC according to the following method. The extraction and HPLC analysis method for tetraene-menadione is as follows: The fermentation medium was centrifuged to collect the supernatant. The cell particles were mixed with the same volume of sterile water as the supernatant collected from the fermentation medium. Tetraene-menadione was extracted from the fermentation broth (including the fermentation supernatant and cell precipitate) using 2-propanol and n-hexane (1:2, v / v) at a ratio of 4:1 (organic solvent: liquid). The mixture was vortexed for 15 minutes, then centrifuged at 10,000 rpm for 3 minutes to separate the two phases. The extracted tetraene-menadione was recovered from the organic phase. The HPLC chromatographic conditions were as follows: An Aminex HPX-87H Ion Exclusion Column (300 mm × 7.8 mm, Bio-Rad, USA) was used for the organic acid; the UV detection wavelength was 248 nm; the column oven temperature was 40 °C; the injection volume was 10 μL; the flow rate was 1.2 mL / min; and the mobile phase was methanol:dichloromethane (9:1, v / v). The supernatant was used for sample analysis. Standard solutions of different concentrations (1-100 mg / L) were prepared using tetraene-naphthoquinone standard. A standard curve was plotted based on the peak area and concentration of the standard solutions to calculate the product yield.

[0146] After fermentation, the yield of tetraenmenaquinone from recombinant Yersinia lipolytica was 2.23 g / L, as determined by HPLC. This means that 2.23 g of tetraenmenaquinone could be obtained per liter of fermentation broth. Currently reported yields of MK-4 synthesized via microbial methods are all in the milligram range. For example, the yield from the Japanese method based on *Flavobacterium mutagenesis* was 280 mg / L (Journal of Fermentation and Bioengineering, 1989, 67(2):102-106); and the yield from the genetically engineered *Bacillus subtilis* method from Jiangnan University in my country was 145 ± 2.8 mg / L (Enzyme and Microbial Technology, 2020, 141:109652). The method for biosynthesizing tetraenmenaquinone using *Yersinia lipolytica* described in this invention not only has a safe strain and a short biochemical reaction process, but also a high yield, making it a promising method for industrial application.

[0147] This invention provides a method for biosynthesizing tetraene-menaquinone using *Yarrowia lipolytica*. 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 recombinant lipophilic yeast, characterized in that, The recombinant *Yersinia lipolytica* expressed genes related to the mevalonate pathway, genes related to the biosynthesis of gerany-gerany pyrophosphate, and an aromatic isopentenyltransferase encoding gene; wherein the aromatic isopentenyltransferase encoding gene is derived from *Homo sapiens*. Homo sapiens Its nucleotide sequence is shown in SEQ ID No. 7; the N-terminus of the aromatic isopentenyltransferase encoding gene is linked to a solubilization tag; the solubilization tag is a small molecule ubiquitin-related modified protein, the nucleotide sequence of which is shown in SEQ ID No. 10; The mevalonate pathway-related genes mentioned include the gene encoding acetyl-CoA thiolase. ERG10 Isopentenyl diphosphate isomerase encoding gene IDI and the gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase HMGR Combinations in truncated genes; The aforementioned gerany-gerany pyrophosphate biosynthesis-related genes include the gerany-gerany pyrophosphate synthase encoding gene. GGPPs and farnesyl pyrophosphate synthase encoding gene FPS The combination; The aforementioned ERG10 and IDI All were derived from Yersinia lipolytica. Yarrowia lipolytica The aforementioned HMGR Derived from Yersinia lipolytica Yarrowia lipolytica The aforementioned GGPPs Derived from Prunus pedunculata Phomopsis amygdali The FPS mentioned is derived from domestic chickens. Gallus gallus ; The aforementioned ERG10 The NCBI RefSeq is NC_090774.1; the aforementioned IDI The NCBI RefSeq is NC_090775.1; the aforementioned HMGR The truncated gene is the one whose NCBI RefSeq value is XP_503558.

3. HMGR The sequence was obtained by truncating 500 amino acid residues from the N-terminus of the sequence; derived from Prunus persicae. Phomopsis amygdali of GGPPs The nucleotide sequence of the FPS is shown in SEQ ID No. 3; the nucleotide sequence of the FPS is shown in SEQ ID No.

6.

2. The method for constructing recombinant lipophilic yeast according to claim 1, characterized in that, Gene expression cassettes encoding mevalonic acid pathway-related genes, gerany-gerany pyrophosphate biosynthesis-related genes, and aromatic isopentenyltransferase genes were inserted into the genome of Yersinia lipolytica.

3. The application of the recombinant Yersinia lipolytica according to claim 1 in the biosynthesis of tetraene-menaquinone.

4. The application according to claim 3, characterized in that, The recombinant Yersinia lipolyticis was inoculated into the fermentation medium and fermented at 28-30℃ for 24-36 h. Then, menaquinone was added and the culture was continued for 24-90 h.

5. The application according to claim 4, characterized in that, The concentration of the added menaquinone is 2-15 g / L.

Images