Construction method of saccharomyces cerevisiae strain for biosynthesis of 1, 4-dihydroxy-2-naphthoic acid

Abstract

The invention discloses a construction method of a saccharomyces cerevisiae strain capable of biologically synthesizing 1, 4-dihydroxy-2-naphthoic acid (DHNA). Six different plasmids are constructed by means of genetic engineering, metabolic engineering, homologous recombination and the like, homologous fragments are obtained through polymerase chain reaction, key heterologous genes synthesized by DHNA are integrated into chromosomes of saccharomyces cerevisiae by means of efficient homologous recombination of the saccharomyces cerevisiae, the DHNA is generated through biosynthesis, first synthesis of the DHNA in the saccharomyces cerevisiae is achieved, and the yield of the DHNA in the saccharomyces cerevisiae is increased. And a prerequisite substance is provided for constructing biosynthesis of MK-7 in the saccharomyces cerevisiae in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering and biological metabolism, and more specifically, designs homologous fragments and introduces them into brewer's yeast Saccharomyces cerevisiae BY4742 to transform the metabolic pathway of brewer's yeast so that it can biosynthesize 1,4-dihydroxy-4-naphthoic acid (DHNA). Background Art

[0002] Genetic engineering, also known as gene splicing technology and DNA recombination technology, is based on molecular genetics and uses modern methods of molecular biology and microbiology to construct hybrid DNA molecules in vitro according to pre-designed blueprints with genes from different sources, and then introduce them into living cells so that the exogenous genes can be replicated, transcribed, translated and expressed in the recipient cells, in order to change the original genetic characteristics of the organism, obtain new varieties and produce new products. Among many model microorganisms, Saccharomyces cerevisiae is widely used in the biotechnology industry because of its powerful genetic tools and the GRAS (generally regarded as safe) status of the strain, which is suitable for large-scale operations. Unlike prokaryotes, Saccharomyces cerevisiae, as a model eukaryote, has multiple organelles that can provide different environments and compartments for biosynthesis, and can express enzymes that prokaryotes cannot express, such as cytochrome P450 enzymes. In addition, Saccharomyces cerevisiae also shows high tolerance to harsh industrial conditions. Therefore, Saccharomyces cerevisiae has been developed as a platform microorganism for metabolic engineering to produce natural products from bacteria, fungi and plant species.

[0003] At present, the technology of metabolic engineering using Saccharomyces cerevisiae as a host has been used to produce specific fuels, chemicals and drugs, such as free fatty acids, fatty acid ethyl esters, fatty alcohols and alkanes and other fatty acids and their derivatives; terpenoids with important commercial application value, including monoterpenoids (such as geraniol, linalool, menthol, lavandinol, etc.) widely used as perfume ingredients, food flavors, medicines and pesticides; sesquiterpenoids (such as nerolidol, farnesol, patchouli alcohol used as flavors, antimalarial drug artemisinin, antifungal agent capsaicin, farnesene and bisabolene as biofuels, etc.); diterpenoids (such as claryol and carnosic acid used in the cosmetics industry, and anticancer drug taxol, etc.); triterpenoids (such as squalene, hydrocortisone, etc.); tetraterpenoids (such as β-carotene, lycopene, etc.); butanol isomers and pharmaceutical proteins have made great progress.

[0004] Vitamin K is collectively referred to as a group of fat-soluble compounds containing a 2-methyl-1,4-naphthoquinone moiety, but with a different isoprenoid side chain structure at position 3. Vitamin K exists naturally in two forms, phylloquinone and a series of menaquinones (including MK-4, MK-7, etc.), both of which play an important role in hemostasis. 1,4-Dihydroxy-4-naphthoic acid is a precursor of MK-7, which is biosynthesized using Saccharomyces cerevisiae, providing important implications for the future biosynthesis of MK-7 in Saccharomyces cerevisiae. Summary of the invention

[0005] In order to solve the problems of the prior art, the present invention proposes the following technical solutions:

[0006] A method for constructing a 1,4-dihydroxy-2-naphthoic acid biosynthetic yeast strain, characterized by comprising the following steps: constructing homology arm fragments on a basic plasmid pUC19, connecting a target gene encoding biosynthetic DHNA to pUC19 by seamless cloning to obtain plasmids P1, P2, P3, P4, P5, and P6, obtaining fragments F1, F2, F3, F4, F5, and F6 by polymerase chain reaction, and transferring the six fragments into Saccharomyces cerevisiae BY4742 by a yeast transformation method to obtain an engineered strain capable of biosynthesizing 1,4-dihydroxy-2-naphthoic acid (DHNA). The method for constructing a recombinant engineered strain.

[0007] The recombinant engineering strain construction method comprises a P1 plasmid containing a delta site upstream gene fragment delta-UP, a promoter Ptdh1, a terminator Ttef1 and a promoter Ptef1 partial fragment (200 bp) derived from Saccharomyces cerevisiae BY4742; and a gene entC encoding isochorismate synthase derived from Escherichia colistr.K-12substr.MG1655.

[0008] The recombinant engineering strain construction method comprises a P2 plasmid containing a promoter Ptef1, a terminator Tpgk1, a partial fragment of the terminator Ttef1 (202 bp) and a partial fragment of the promoter Ppgk1 (200 bp) derived from Saccharomyces cerevisiae BY4742; and a plasmid encoding a 2-butyryl-5-enolpyruvate-6-hydroxy-3-cyclohexene-1-carboxylic acid synthase menD derived from Escherichia colistr.K-12substr.MG1655.

[0009] The recombinant engineering strain construction method comprises a P3 plasmid containing a promoter Ppgk1, a terminator Tcyc1, a partial fragment of the terminator Tpgk1 (199 bp) and a terminator Ttef derived from Saccharomyces cerevisiae BY4742; a LEU2 gene encoding leucine derived from Saccharomyces cerevisiae S288C; and a gene encoding 2-butyryl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid synthase menH derived from Escherichia colistr.K-12substr.MG1655.

[0010] The recombinant engineering strain construction method comprises a P4 plasmid containing a delta site downstream gene fragment delta-DOWN, a promoter Ptef1, and a terminator Ttpi1 derived from Saccharomyces cerevisiae BY4742; a LUE2 partial gene (200 bp) encoding leucine, and a promoter Pleu2 derived from Saccharomyces cerevisiae S288C; and a gene encoding o-succinylbenzoate synthase menC derived from Escherichia colistr.K-12substr.MG1655.

[0011] The recombinant engineering strain construction method comprises a P5 plasmid containing a gene fragment rDNA-UP upstream of the rDNA site, a promoter Ptdh1, and a terminator Ttef1, which are derived from Saccharomyces cerevisiae BY4742; a His3 partial gene (200 bp) encoding histidine and a promoter Phis3, which are derived from a pRS413 plasmid; and a gene menB encoding a 1,4-dihydroxy-2-naphthoyl-CoA synthase and a gene menI encoding a 1,4-dihydroxy-2-naphthoyl-CoA hydrolase, which are derived from Escherichia coli str.K-12substr.MG1655.

[0012] The method for constructing the recombinant engineering strain comprises: a P6 plasmid containing a gene fragment rDNA-DOWN downstream of the rDNA site derived from Saccharomyces cerevisiae BY4742, a promoter Ppgk1, a terminator Tcyc1, and a terminator Ttpi1; a His3 gene encoding histidine derived from a pRS413 plasmid; and a gene menE encoding o-succinylbenzoate coenzyme A ligase derived from Escherichia coli str.K-12substr.MG1655.

[0013] The recombinant engineering strain construction method is to obtain fragments F1, F2, F3, and F4 by polymerase chain reaction on P1, P2, P3, and P4 plasmids, and introduce them into wild-type brewer's yeast Saccharomyces cerevisiae BY4742 to form a brewer's yeast engineering strain BD-0.

[0014] The recombinant engineering strain construction method obtains fragments F5 and F6 by polymerase chain reaction on P5 and P6 plasmids, and introduces the fragments into the cerevisiae engineering bacteria BD-0 to form a cerevisiae engineering bacteria BD-1.

[0015] The recombinant engineering strain construction method utilizes shaking flask culture, a spectrophotometer to measure OD600, and a high performance liquid chromatography (HPLC) to measure the yield of DHNA.

[0016] In order to biosynthesize 1,4-dihydroxy-2-naphthoic acid (DHNA) in Saccharomyces cerevisiae, the present invention introduces key genes of the DHNA metabolic pathway into Saccharomyces cerevisiae BY4742 through heterologous expression technology combined with genetic engineering and other means to construct an engineered bacterium, thereby obtaining the engineered bacterium BD-1.

[0017] The present invention constructs 6 plasmids, takes Saccharomyces cerevisiae BY4742 as a chassis host bacterium, transfers fragments obtained from the 6 plasmids by polymerase chain reaction into the host bacterium, constructs a DHNA metabolic pathway, enables Saccharomyces cerevisiae to synthesize DHNA, and provides a precursor for the future biosynthesis of MK-7 in Saccharomyces cerevisiae.

[0018] Six recombinant plasmids were constructed based on pUC19, and the target genes were connected in sequence by seamless cloning, including:

[0019] Plasmid P1: pUC19-delta-UP-Ptdh1-entC-Ttef1-Ptef1 (partial)

[0020] Plasmid P2: pUC19-Ttef1 (part)-Ptef1-menD-Tpgk1-Ppgk1 (part)

[0021] Plasmid P3: pUC19-Tpgk1 (partial)-Ppgk1-menH-Tcyc1-Ttef-LEU2 Plasmid P4: pUC19-LEU2 (partial)-Pleu2-Ttpi1-menC-Ptef1-delta-DOWN

[0022] Plasmid P5: pUC19-rDNA-UP-Ptdh1-menB-Ttef1-Ptef1-menI-Tpgk1-Phis3-His3 (partial)

[0023] Plasmid P6: pUC19-His3-Ttpi1-Tcyc1-menE-Ppgk1-rDNA-DOWN fragments were obtained by polymerase chain reaction, including:

[0024] Fragment F1: delta-UP-Ptdh1-entC-Ttef1-Ptef1 (partial)

[0025] Fragment F2: Ttef1 (partial)-Ptef1-menD-Tpgk1-Ppgk1 (partial)

[0026] Fragment F3: Tpgk1 (partial)-Ppgk1-menH-Tcyc1-Ttef-LEU2

[0027] Fragment F4: LEU2 (partial)-Pleu2-Ttpi1-menC-Ptef1-delta-DOWN

[0028] Fragment F5: rDNA-UP-Ptdh1-menB-Ttef1-Ptef1-menI-Tpgk1-Phis3-His3 (partial)

[0029] Fragment F6: His3-Ttpi1-Tcyc1-menE-Ppgk1-rDNA-DOWN

[0030] Key genes in DHNA biosynthesis pathway:

[0031] entC: isochorismate synthase gene from Escherichia coli str.K-12substr.MG1655

[0032] menD: 2-butyryl-5-enolpyruvate-6-hydroxy-3-cyclohexene-1-carboxylate synthase gene from Escherichia colistr.K-12substr.MG1655

[0033] menH: 2-Butyryl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase gene from Escherichia colistr.K-12substr.MG1655

[0034] menC: o-succinylbenzoate synthase gene from Escherichia colistr.K-12substr.MG1655

[0035] menB: 1,4-dihydroxy-2-naphthoyl-CoA synthase gene from Escherichia colistr.K-12substr.MG1655

[0036] menI: 1,4-dihydroxy-2-naphthyl-CoA hydrolase gene from Escherichia coli str.K-12substr.MG1655

[0037] menE: o-succinylbenzoate-CoA ligase gene from Escherichia colistr.K-12substr.MG1655 BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Shown is the pUC19 plasmid map;

[0039] Figure 2 Shown is the P1 plasmid map, including key components such as promoters and terminators and their names;

[0040] Figure 3 Shown is the P2 plasmid map, including key components such as promoters and terminators and their names;

[0041] Figure 4 Shown is the P3 plasmid map, including key components such as promoters and terminators and their names;

[0042] Figure 5 Shown is the P4 plasmid map, including key components such as promoters and terminators and their names;

[0043] Figure 6 Shown is the P5 plasmid map, including key components such as promoters and terminators and their names;

[0044] Figure 7 Shown is the P6 plasmid map, including key components such as promoters and terminators and their names;

[0045] Figure 8 The schematic diagram of the DHNA synthesis pathway design of the present invention is shown as follows

[0046] Fig. 9 Shown is a graph of DHNA production DETAILED DESCRIPTION

[0047] The plasmid designed in the present invention is designed by this laboratory, and the strain can be obtained commercially. In the process of fully synthesizing the exogenous genes required by the present invention, it is necessary to connect them to the plasmid vector for expression and preservation. The present invention is further described below in conjunction with the embodiments.

[0048] Example 1: Construction of plasmid

[0049] Linear vector: Using pUC19 plasmid as template and primers puc19-F / puc19-R, linear vector puc19 was obtained.

[0050] P1 plasmid construction: Using the genome of Saccharomyces cerevisiae BY4742 as a template, using primers delta-UP-F / delta-UP-R, the gene fragment delta-UP was obtained, wherein primer delta-UP-F contained the 3' end 20 bp gene of the linear vector pUC19, and primer delta-UP-R contained the 5' end 20 bp gene of the gene fragment Ptdh1. Using the synthetic plasmid pUC-Ptdh1 as a template, using primers Ptdh1-F / Ptdh1-R, the gene fragment Ptdh1 was obtained, wherein Ptdh1-R contained the 5' end 20 bp gene of the gene fragment entC. Using the genome of Escherichia coli str.K-12substr.MG1655 as a template, using primers entC-F / entC-R, the gene fragment entC was obtained, wherein entC-R contained the 5' end 20 bp gene of the gene fragment Ttef1-Ptef1 (part). The synthetic plasmid pUC-Ttef1-Ptef1 (partial) was used as a template and primers Ttef1-F / Ptef1-bf-R were used to obtain the gene fragment Ttef1-Ptef1 (partial), wherein Ptef1-bf-R contained the 20bp gene at the 3' end of the linear vector pUC19. The linear vector pUC19 and the fragment were connected by seamless cloning, and transformed into the competent Escherichia coli Trans-T1. The positive clones were selected for bacterial P and sequencing to obtain the correct plasmid P1.

[0051] P2 plasmid construction: Using synthetic plasmid pUC-Ttef1 (partial)-Ptef1 as template, primers Ttef1-bf-F / Ptef1-R were used to obtain gene fragment Ttef1 (partial)-Ptef1, wherein primer Ttef1-bf-F contained the 3' end 20 bp gene of linear vector pUC19, and Ptef1-R contained the 5' end 20 bp gene of gene fragment menD. Using Escherichia coli Escherichia colistr.K-12substr.MG1655 genome as template, primers menD-F / menD-R were used to obtain gene fragment menD, wherein menD-R contained the 5' end 20 bp gene of gene fragment Tpgk1-Ppgk1 (partial). The synthetic plasmid pUC-Tpgk1-Ppgk1 (part) was used as a template and primers Tpgk1-F / Ppgk1-bf-R were used to obtain the gene fragment Tpgk1-Ppgk1 (part), wherein Ptef1-bf-R contained the 3' end 20bp gene of the linear vector pUC19. The linear vector pUC19 and the fragment were connected by seamless cloning, and transformed into the competent Escherichia coli Trans-T1. The positive clones were selected for bacterial P and sequencing to obtain the correct plasmid P2.

[0052] P3 plasmid construction: Using synthetic plasmid pUC-Tpgk1 (partial)-Ppgk1 as template, primers Tpgk1-bf-F / Ppgk1-R were used to obtain gene fragment Tpgk1 (partial)-Ppgk1, wherein primer Tpgk1-bf-F contained the 3' end 20 bp gene of linear vector pUC19, and Ppgk1-R contained the 5' end 20 bp gene of gene fragment menH. Using Escherichia coli Escherichia colistr.K-12substr.MG1655 genome as template, primers menH-F / menH-R were used to obtain gene fragment menH, wherein menH-R contained the 5' end 20 bp gene of gene fragment Tcyc1. Using synthetic plasmid pUC-Tcyc1 as template and primers Tcyc1-F / Tcyc1-R, gene fragment Tcyc1 was obtained, wherein Tcyc1-R contained the 5' end 20bp gene of gene fragment Ttef. Using synthetic plasmid pUC-Ttef as template and primers Ttef-F / Ttef-R, gene fragment Ttef was obtained, wherein Ttef-R contained the 5' end 20bp gene of gene fragment LEU2. Using synthetic plasmid pUC-LEU2 as template and primers LEU2-F / LEU2-R, gene fragment LEU2 was obtained, wherein LEU2-R contained the 3' end 20bp gene of linear vector pUC19. Using seamless cloning method, linear vector pUC19 and fragments were connected, transformed into competent Escherichia coli Trans-T1, positive clones were selected for bacterial P and sequencing, and correct plasmid P3 was obtained.

[0053] P4 plasmid construction: Using synthetic plasmid pUC-LEU2 (partial)-Pleu2 as template, primers LEU2-bf-F / Pleu2-R were used to obtain gene fragment LEU2 (partial)-Pleu2, wherein primer LEU2-bf-F contained the 3' end 20 bp gene of linear vector pUC19, and Pleu2-R contained the 5' end 20 bp gene of gene fragment Ttpi1. Using synthetic plasmid pUC-Ttpi1 as template, primers Ttpi1-F / Ttpi1-R were used to obtain gene fragment Ttpi1, wherein Ttpi1-R contained the 5' end 20 bp gene of gene fragment menC. Using Escherichia coli str.K-12substr.MG1655 genome as template, primers menC-F / menC-R were used to obtain gene fragment menC, wherein menC-R contained the 5' end 20 bp gene of gene fragment Ptef1. Using synthetic plasmid pUC-Ptef1 as template, primers Ptef1-F / Ptef1-R were used to obtain gene fragment Ptef1, where Ptef1-R contained the 5' end 20bp gene of gene fragment delta-DOWN. Using the genome of Saccharomyces cerevisiae BY4742 as template, primers delta-DOWN-F / delta-DOWN-R were used to obtain gene fragment delta-DOWN, where delta-DOWN-R contained the 5' end 20bp gene of linear vector pUC19. Using seamless cloning method, linear vector pUC19 and fragments were connected, transformed into Escherichia coli competent Trans-T1, positive clones were selected for bacterial P and sequencing, and correct plasmid P4 was obtained.

[0054] P5 plasmid construction: Using the genome of Saccharomyces cerevisiae BY4742 as a template, primers rDNA-UP-F / rDNA-UP-R were used to obtain the gene fragment rDNA-UP, wherein primer rDNA-UP-F contained the 3' end 20bp gene of the linear vector pUC19, and primer rDNA-UP-R contained the 5' end 20bp gene of the gene fragment Ptdh1. Using the synthetic plasmid pUC-Ptdh1 as a template, primers Ptdh1-F / Ptdh1-R2 were used to obtain the gene fragment Ptdh1-2, wherein Ptdh1-R2 contained the 5' end 20bp gene of the gene fragment menB. Using the genome of Escherichia colistr.K-12substr.MG1655 as a template, primers menB-F / menB-R were used to obtain the gene fragment menB, wherein menB-R contained the 5' end 20bp gene of the gene fragment Ttef1. Using synthetic plasmid pUC-Ttef1 as template, primers Ttef1-F / Ttef1-R were used to obtain gene fragment Ttef1, wherein Ttef1-R contained the 5' end 20 bp gene of gene fragment Ptef1. Using synthetic plasmid pUC-Ptef1 as template, primers Ptef1-F / Ptef1-R2 were used to obtain gene fragment Ptef1-2, wherein Ptef1-R2 contained the 5' end 20 bp gene of gene fragment menI. Using Escherichia coli str.K-12substr.MG1655 genome as template, primers menI-F / menI-R were used to obtain gene fragment menI, wherein menI-R contained the 5' end 20 bp gene of gene fragment Ppgk1. Using synthetic plasmid pUC-Ppgk1 as template, primers Ppgk1-F / Ppgk1-R were used to obtain gene fragment Ppgk1, wherein Ppgk1-R contained the 5' end 20bp gene of gene fragment Phis3-His3 (part). Using plasmid pRS413 as template, primers Phis3-F / His3-bf-R were used to obtain fragment Phis3-His3 (part), wherein His3-bf-R contained the 3' end 20bp gene of linear vector pUC19. Using seamless cloning method, linear vector pUC19 and fragment were connected, transformed into competent E. coli Trans-T1, positive clones were selected for bacterial P and sequencing, and correct plasmid P5 was obtained.

[0055] P6 plasmid construction: Using plasmid pRS413 as a template, using primers His3-F / His3-R, the fragment His3 was obtained, wherein primer His3-F contained the 3' end 20bp gene of the linear vector pUC19, and primer His3-R contained the 5' end 20bp gene of the gene fragment Ttpi1. Using synthetic plasmid pUC-Ttpi1 as a template, using primers Ttpi1-F / Ttpi1-R2, the gene fragment Ttpi1-2 was obtained, wherein Ttpi1-R2 contained the 5' end 20bp gene of the gene fragment Tcyc1. Using synthetic plasmid pUC-Tcyc1 as a template, using primers Tcyc1-F / Tcyc1-R2, the gene fragment Tcyc1-2 was obtained, wherein Tcyc1-R2 contained the 5' end 20bp gene of the gene fragment menE. Using the genome of Escherichia colistr.K-12substr.MG1655 as a template, primers menE-F / menE-R were used to obtain gene fragment menE, wherein menE-R contained the 5' end 20 bp gene of gene fragment Ppgk1. Using synthetic plasmid pUC-Ppgk1 as a template, primers Ppgk1-F / Ppgk1-R2 were used to obtain gene fragment Ppgk1-2, wherein Ppgk1-R2 contained the 5' end 20 bp gene of gene fragment rDNA-DOWN. Using the genome of Saccharomyces cerevisiae BY4742 as a template, primers rDNA-DOWN-F / rDNA-DOWN-R were used to obtain gene fragment rDNA-DOWN, wherein rDNA-DOWN-R contained the 5' end 20 bp gene of linear vector pUC19. The linear vector pUC19 and the fragment were connected by seamless cloning method, transformed into Escherichia coli competent cell Trans-T1, and the positive clones were selected for bacterial P and sequencing to obtain the correct plasmid P6.

[0056] Example 2: Obtaining the target fragment and constructing the Saccharomyces cerevisiae engineering strain

[0057] Target fragment F1: Using plasmid P1 as template and primers F1-F / F1-R, fragment F1 was obtained.

[0058] Target fragment F2: Using plasmid P2 as a template and primers F2-F / F2-R, fragment F2 was obtained.

[0059] Target fragment F3: Using plasmid P3 as template and primers F3-F / F3-R, fragment F3 was obtained.

[0060] Target fragment F4: Using plasmid P4 as a template and primers F4-F / F4-R, fragment F4 was obtained.

[0061] Target fragment F5: Using plasmid P5 as template and primers F5-F / F5-R, fragment F5 was obtained.

[0062] Target fragment F6: Using plasmid P6 as template and primers F6-F / F6-R, fragment F6 was obtained.

[0063] The fragments F1, F2, F3, and F4 obtained above were introduced into Saccharomyces cerevisiae BY4742 by conventional yeast transformation method to obtain BD-0. After transformation, SC (-Leu2, -His3) plates (22g / L glucose, 6.7g / L amino-free yeast nitrogen source, 2g / L drop-out, 0.02g / LUra, 0.02g / LTrp, 20g / L agar) were used for screening. Single colonies with good growth were picked up in the clean bench, suspended in a 96-well plate containing 20μL ddH2O, and the program was set in the PCR instrument, and boiled at 99℃ for 10min. Prepare the PCR system and divide it into 96-well plates. You can prepare 2-5 more systems than actually needed to prevent losses caused by the tip of the gun being stained with the solution. Take the boiled bacterial solution and add it to one system. Prepare the gel for DNA electrophoresis and wait for it to solidify. After PCR, add DNA loading buffer, spot 10 μL per well, and run electrophoresis for 8-12 minutes. Observe the presence of the target band and record the corresponding number.

[0064] PCR reaction system configuration: 2 μL each of upstream and downstream primers; 25 μL of 2x Rapid TaqMaster Mix; Template 2-5 μL; add ddH2O to 50 μL.

[0065] PCR conditions were as follows: pre-denaturation at 95°C for 10 min, denaturation at 96°C for 15 s, annealing at 56-66°C for 15 s, extension at 72°C for X s (1 kb requires 30-60 s, so time must be sufficient), 35 cycles, and finally extension at 72°C for 5 min and insulation at 16°C.

[0066] DNA electrophoresis: Large gel (100mL): 100mL 1XTAE buffer, 1.0g agarose, add 8μL nucleic acid dye after appropriate cooling, shake well and pour into the tank with transparent pad and comb. 50mL medium gel, 25mL small gel, add according to the proportion.

[0067] Drop-out amino acid mixture: weigh 0.5 g adenine, 2 g p-aminobenzoic acid, 2 g arginine, 2 g isoleucine, 2 g cysteine, 2 g threonine, 2 g lysine, 2 g aspartic acid, 2 g tryptophan, 2 g methionine, 2 g phenylalanine, 2 g tyrosine, 2 g proline, 2 g valine, 2 g serine, 2 g inositol, 2 g glutamic acid, 2 g glutamate, and 2 g alanine, put them in a container, add two clean and dried marble blocks, mix well by inverting several times, and place at room temperature for use. Mix well by inverting again before use.

[0068] Example 3: Shake flask fermentation of Saccharomyces cerevisiae BY4742 and engineered bacteria BD-1 in shake flask fermentation medium (aerobic)

[0069] In a sterile clean bench, BD-1 was streaked on a SC (-Leu2, -His3) solid plate, and BY4742 was streaked on a YPD solid plate, and the plates were inverted and cultured in a 30°C constant temperature incubator for about 2 days.

[0070] A single colony that grew well on the solid plate was picked and inoculated into a 50 mL centrifuge tube with 5 mL SC (22 g / L glucose, 6.7 g / L amino-free yeast nitrogen source, 2 g / L drop-out, 0.02 g / L Ra, 0.02 g / L Trp, 0.1 g / L Leu, 0.02 g / L His), and then the centrifuge tube was placed in a shaker at 30 ° C and 220 rpm for about 20 hours to reach the logarithmic growth phase, at which time OD600 was approximately 0.5-1.

[0071] In a sterile clean bench, the seed solution in the logarithmic phase was transferred to a 250 mL conical flask containing 30 mL of the corresponding fermentation medium according to a certain ratio for fermentation culture, so that the OD600 at the beginning of the fermentation remained consistent at 0.1.

[0072] The culture was placed in a constant temperature shaker according to the corresponding fermentation culture conditions (30°C, 200 rpm), and samples were taken at 72h and 96h to determine the cell mass and the content of each fermentation product.

[0073] Example 4: DHNA extraction and detection

[0074] Take 1 ml of the fermentation liquid and centrifuge it at 12,000 rpm for 5 min, remove the supernatant and keep the bacterial cells.

[0075] Add quartz sand of the same volume as the bacterial cells obtained in the previous step, and add 500 μl of chromatographically pure ethyl acetate, and vortex at 2,400 g for 15 min at room temperature.

[0076] Use a disposable syringe to absorb the supernatant, filter it with a 0.2 μm organic phase filter membrane, add it to the sample bottle, mark it and then perform HPLC detection.

[0077] HPLC detection conditions: Shim-pack GIST C18 chromatographic column (250 mm × 4.6 mm, 5 μm); detector: Shimadu SPD-20A, UV detection wavelength (nm): 254; column temperature (°C): 40; mobile phase: pure acetonitrile: 0.1% formic acid (ammonia water adjusted to pH 4.65) = 3:7, flow rate (mL / min): 1.0, sample volume (μL): 10.

[0078] Quantitative method: Use the same chromatographic conditions to measure the standard solution of standard DHNA, draw a concentration-peak area standard curve, and quantify DHNA.

[0079] The beneficial effects of the present invention are:

[0080] By using heterologous expression technology and combining genetic engineering, six plasmids were constructed to obtain six fragments. The strong homologous recombination ability of Saccharomyces cerevisiae was used to integrate the genes required for DHNA biosynthesis into the genome, reconstruct the metabolic pathway of Saccharomyces cerevisiae, and achieve the first synthesis of DHNA in Saccharomyces cerevisiae. The results showed that the Saccharomyces cerevisiae engineered strain BD-1 biosynthesized DHNA, with the highest yield at 72h, reaching 5.31±1.23mg / L. It can be seen that:

[0081] (1) The present invention utilizes heterologous expression technology and genetic engineering to heterologously introduce key genes for DHNA synthesis into Saccharomyces cerevisiae, thereby enabling strains that were previously unable to synthesize DHNA to synthesize DHNA, thereby obtaining strain BD-1.

[0082] (2) The modified strain of the present invention provides precursor substances for the future biosynthesis of MK-7.

[0083] The technical solutions disclosed and proposed by the present invention can be realized by those skilled in the art by referring to the contents of this article and appropriately changing the conditions, routes and other links. Although the methods and preparation techniques of the present invention have been described through preferred embodiments, relevant technicians can obviously modify or re-combine the methods and technical routes described herein without departing from the content, spirit and scope of the present invention to achieve the final preparation technology. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the spirit, scope and content of the present invention. Matters not covered in the present invention belong to the known technology.

Claims

1. A method for constructing a 1,4-dihydroxy-2-naphthoic acid biosynthetic yeast strain, characterized in that The method comprises the following steps: constructing homology arm fragments on a basic plasmid pUC19, connecting a target gene encoding biosynthetic DHNA to pUC19 by seamless cloning to obtain plasmids P1, P2, P3, P4, P5, and P6, obtaining fragments F1, F2, F3, F4, F5, and F6 by polymerase chain reaction, and transferring the six fragments into brewer's yeast Saccharomyces cerevisiae BY4742 by a yeast transformation method to obtain an engineering strain capable of biosynthesizing 1,4-dihydroxy-2-naphthoic acid.

2. The method for constructing an engineered strain according to claim 1, characterized in that: The P1 plasmid contains the delta site upstream gene fragment delta-UP, promoter Ptdh1, terminator Ttef1 and a partial fragment of promoter Ptef1 derived from Saccharomyces cerevisiae BY4742; and contains the gene entC encoding isochorismate synthase derived from Escherichia colistr.K-12substr.MG1655.

3. The method for constructing an engineered strain according to claim 1, characterized in that: The P2 plasmid contains the promoter Ptef1, terminator Tpgk1, a partial fragment of the terminator Ttef1 and a partial fragment of the promoter Ppgk1 derived from Saccharomyces cerevisiae BY4742; and contains the gene menD encoding 2-butyryl-5-enolpyruvate-6-hydroxy-3-cyclohexene-1-carboxylate synthase derived from Escherichia colistr.K-12substr.MG1655.

4. The method for constructing an engineered strain according to claim 1, characterized in that: The P3 plasmid contains the promoter Ppgk1, terminator Tcyc1, a partial fragment of the terminator Tpgk1 and the terminator Ttef from Saccharomyces cerevisiae BY4742; the LUE2 gene encoding leucine from Saccharomyces cerevisiae S288C; and the gene menH encoding 2-butyryl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase from Escherichia colistr.K-12substr.MG1655.

5. The method for constructing an engineered strain according to claim 1, characterized in that: The P4 plasmid contains the delta site downstream gene fragment delta-DOWN, promoter Ptef1, and terminator Ttpi1 derived from Saccharomyces cerevisiae BY4742; contains the LUE2 partial gene encoding leucine and promoter Pleu2 derived from Saccharomyces cerevisiae S288C; and contains the gene menC encoding o-succinylbenzoate synthase derived from Escherichia colistr.K-12substr.MG1655.

6. The method for constructing an engineered strain according to claim 1, characterized in that: The P5 plasmid contains the gene fragment rDNA-UP upstream of the rDNA site, promoter Ptdh1, terminator Ttef1, promoter Ptef1, terminator Tpgk1 from Saccharomyces cerevisiae BY4742; it contains the His3 partial gene encoding histidine and promoter Phis3 from the pRS413 plasmid; it contains the gene menB encoding 1,4-dihydroxy-2-naphthoyl-CoA synthase and the gene menI encoding 1,4-dihydroxy-2-naphthoyl-CoA hydrolase from Escherichia coli str.K-12substr.MG1655.

7. The method for constructing an engineered strain according to claim 1, characterized in that: The P6 plasmid contains the gene fragment rDNA-DOWN, promoter Ppgk1, terminator Tcyc1, and terminator Ttpi1 of the rDNA site downstream of Saccharomyces cerevisiae BY4742; the His3 gene encoding histidine derived from the pRS413 plasmid; and the gene menE encoding o-succinylbenzoate coenzyme A ligase derived from Escherichia coli str.K-12substr.MG1655.

8. The method for constructing an engineered strain according to claim 1, characterized in that: The fragments F1, F2, F3, and F4 were obtained by polymerase chain reaction on the P1, P2, P3, and P4 plasmids, and were introduced into the wild-type Saccharomyces cerevisiae BY4742 to form an engineered Saccharomyces cerevisiae strain BD-0.

9. The method for constructing an engineered strain according to claim 1, characterized in that: The fragments F5, F6 and F7 were obtained by polymerase chain reaction on the P5, P6 and P7 plasmids, and introduced into the cerevisiae engineering bacteria BD-0 to form a cerevisiae engineering bacteria BD-1.

10. The method for constructing a recombinant engineering strain according to claim 1, characterized in that: The culture was carried out in a shaker flask, OD600 was measured by a spectrophotometer, and DHNA production was measured by a high performance liquid chromatography.

Images