CN118406711A - A construction method capable of biosynthesizing 1,4-dihydroxy-2-naphthoic acid in Saccharomyces cerevisiae - Google Patents

Abstract

The present invention discloses a construction method capable of biosynthesizing 1,4-dihydroxy-2-naphthoic acid (DHNA) in Saccharomyces cerevisiae. The method aims to construct 6 different plasmids by means of genetic engineering, metabolic engineering and homologous recombination, obtain homologous fragments by polymerase chain reaction, integrate key heterologous genes for DHNA synthesis into Saccharomyces cerevisiae chromosomes by utilizing efficient homologous recombination of Saccharomyces cerevisiae, biosynthesize DHNA, realize the first synthesis of DHNA in Saccharomyces cerevisiae, and provide prerequisite substances for constructing the biosynthesis of MK-7 in Saccharomyces cerevisiae in the future.

Description

A construction method capable of biosynthesizing 1,4-dihydroxy-2-naphthoic acid in Saccharomyces cerevisiae

Technical Field

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 technique

Genetic engineering, also known as gene splicing technology and DNA recombination technology, is based on the theory of molecular genetics and uses modern methods of molecular biology and microbiology to construct hybrid DNA molecules in vitro according to a pre-designed blueprint using genes from different sources. These molecules are then introduced into living cells so that the exogenous genes can be replicated, transcribed, translated and expressed in the recipient cells, thereby changing the original genetic characteristics of the organism, obtaining new varieties and producing 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.

At present, the technology of metabolic engineering using Saccharomyces cerevisiae as a host has made great progress in the production of 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.

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

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

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 (SEQ ID NO.1), connecting a target gene encoding biosynthetic DHNA to pUC19 (SEQ ID NO.1) by seamless cloning to obtain plasmids P1, P2, P3, P4, P6, and P7, obtaining fragments F1, F2, F3, F4, F6, and F7 by polymerase chain reaction, and transferring the six fragments into Saccharomyces cerevisiae BY4742 by conventional yeast transformation methods to obtain an engineered strain capable of biosynthesizing 1,4-dihydroxy-2-naphthoic acid (DHNA). The method for constructing a recombinant engineered strain.

The recombinant engineering strain construction method comprises a P1 plasmid containing a delta site upstream gene fragment delta-UP (SEQ ID NO.9) derived from Saccharomyces cerevisiae BY4742, a promoter Ptdh1 (SEQID NO.13), a terminator Ttef1 (SEQ ID NO.14) and a promoter Ptef1 partial fragment (200 bp); and a gene entC (SEQID NO.2) encoding isochorismate synthase derived from Escherichia coli str. K-12 substr. MG1655.

The recombinant engineering strain construction method comprises a P2 plasmid containing a promoter Ptef1 (SEQ ID NO.15), a terminator Tpgk1 ( SEQ ID NO.16), 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 (SEQ ID NO.3) derived from Escherichia coli str. K-12 substr. MG1655.

The recombinant engineering strain construction method comprises a P3 plasmid containing a promoter Ppgk1 (SEQ ID NO.17), a terminator Tcyc1 (SEQ ID NO.18), a partial fragment of the terminator Tpgk1 (199 bp) and a terminator Ttef (SEQ ID NO.22) derived from Saccharomyces cerevisiae BY4742; a LEU2 (SEQ ID NO.21) gene encoding leucine derived from Saccharomyces cerevisiae S288C; and a 2-butyryl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid synthase menH (SEQ ID NO.4) derived from Escherichia coli str. K-12 substr. MG1655.

The P4 plasmid of the recombinant engineering strain construction method contains the delta site downstream gene fragment delta-DOWN (SEQ ID NO.10) derived from Saccharomyces cerevisiae BY4742, the promoter Ptef1 (SEQ ID NO.15), and the terminator Ttpi1 (SEQ ID NO.19); contains the partial gene (200bp) of LUE2 (SEQ ID NO.21) encoding leucine derived from Saccharomyces cerevisiae S288C, and the promoter Pleu2 (SEQ ID NO.20); contains the o-succinylbenzoate synthase encoding menC (SEQ ID NO.5) derived from Escherichia coli str. K-12 substr. MG1655.

The recombinant engineering strain construction method comprises the following steps: the P6 plasmid contains the gene fragment rDNA-DOWN (SEQ ID NO.12) downstream of the rDNA site derived from Saccharomyces cerevisiae BY4742, the promoter Ppgk1 (SEQ ID NO.17), the terminator Tcyc1 (SEQ ID NO.18), and the terminator Ttpi1 (SEQ ID NO.19); contains the His3 gene encoding histidine derived from the pRS413 plasmid; and contains the gene menE (SEQ ID NO.8) encoding o-succinylbenzoate coenzyme A ligase derived from Escherichia coli str. K-12 substr. MG1655.

The recombinant engineering strain construction method comprises a P7 plasmid containing a gene fragment rDNA-UP (SEQ ID NO.11) upstream of the rDNA site , a promoter Ptdh1 (SEQ ID NO.13), a terminator Ttef1 (SEQ ID NO.14), a promoter Ptef1 (SEQ ID NO.15), and a terminator Tpgk1 (SEQ ID NO.16) derived from Saccharomyces cerevisiae BY4742; a partial His3 gene (200 bp) encoding histidine and a promoter Phis3 derived from a pRS413 (SEQ ID NO.23) plasmid; and a gene sll1127 (SEQ ID NO.6) encoding a 1,4-dihydroxy-2-naphthoyl-CoA synthase and a gene slr0204 (SEQ ID NO.7) encoding a 1,4-dihydroxy-2-naphthoyl-CoA hydrolase derived from cyanobacteria Synechocystis sp. PCC 6803.

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.

The recombinant engineering strain construction method obtains fragments F6 and F7 by polymerase chain reaction on P6 and P7 plasmids, and introduces the fragments into the cerevisiae engineering bacteria BD-0 to form a cerevisiae engineering bacteria BD-2.

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.

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-2.

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.

Six recombinant plasmids were constructed using pUC19 (SEQ ID NO.1) as the basic plasmid, and the target genes were connected in sequence by seamless cloning, including:

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

Plasmid P2: pUC19-Ttef1 (partial)-Ptef1-menD-Tpgk1-Ppgk1 (partial)

Plasmid P3: pUC19-Tpgk1 (partial)-Ppgk1-menH-Tcyc1-Ttef-LEU2

Plasmid P4: pUC19-LEU2 (partial)-Pleu2-Ttpi1-menC-Ptef1-delta-DOWN

Plasmid P6: pUC19-His3-Ttpi1-Tcyc1-menE-Ppgk1-rDNA-DOWN

Plasmid P7: pUC19-rDNA-UP-Ptdh1-sll1127-Ttef1-Ptef1-slr0204-Tpgk1-Phis3-His3 (partial)

The fragments obtained by polymerase chain reaction include:

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

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

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

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

Fragment F6: His3-Ttpi1-Tcyc1-menE-Ppgk1-rDNA-DOWN

Fragment F7: rDNA-UP-Ptdh1-sll1127-Ttef1-Ptef1-slr0204-Tpgk1-Phis3-His3 (partial)

Key genes in DHNA biosynthesis pathway:

entC (SEQ ID NO.2): isochorismate synthase gene from Escherichia coli str. K-12 substr. MG1655

menD (SEQ ID NO. 3): 2-butyryl-5-enolpyruvate-6-hydroxy-3-cyclohexene-1-carboxylate synthase gene from Escherichia coli str. K-12 substr. MG1655

menH (SEQ ID NO. 4): 2-butyryl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase gene from Escherichia coli str. K-12 substr. MG1655

menC (SEQ ID NO.5): o-succinylbenzoate synthase gene from Escherichia coli str. K-12 substr. MG1655

sll1127 (SEQ ID NO.6): 1,4-dihydroxy-2-naphthoyl-CoA synthase gene from cyanobacterium Synechocystis sp. PCC 6803

slr0204 (SEQ ID NO.7): 1,4-dihydroxy-2-naphthyl-CoA hydrolase gene from cyanobacterium Synechocystis sp. PCC 6803

menE (SEQ ID NO.8): o-succinylbenzoate-CoA ligase gene from Escherichia coli str. K-12 substr. MG1655

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the pUC19 plasmid map;

Figure 2 shows the P1 plasmid map, including key components such as promoters and terminators and their names;

Figure 3 shows the P2 plasmid map, including key components such as promoters and terminators and their names;

Figure 4 shows the P3 plasmid map, including key components and names such as promoters and terminators;

Figure 5 shows the P4 plasmid map, including key components and names such as promoters and terminators;

Figure 6 shows the P6 plasmid map, including key components and names such as promoters and terminators;

Figure 7 shows the P7 plasmid map, including key components and names such as promoters and terminators;

FIG8 is a schematic diagram showing the design principle of the DHNA synthesis pathway of the present invention.

Figure 9 shows the DHNA production graph

Detailed ways

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 a plasmid vector for expression and preservation. The present invention is further described below in conjunction with the embodiments.

Example 1: Construction of plasmid

Linear vector: Using pUC19 (SEQ ID NO.1) plasmid as template, primers puc19-F/puc19-R were used to obtain the linear vector puc19. P1 plasmid construction: Using the genome of Saccharomyces cerevisiae BY4742 as template, primers delta-UP-F/delta-UP-R were used to obtain the gene fragment delta-UP (SEQ ID NO.9), wherein primer delta-UP-F contained the 3' end 20bp gene of the linear vector pUC19, and primer delta-UP-R contained the 5' end 20bp gene of the gene fragment Ptdh1 (SEQID NO.13). Using the synthetic plasmid pUC-Ptdh1 as template, primers Ptdh1-F/Ptdh1-R were used to obtain the gene fragment Ptdh1 (SEQ ID NO.13), wherein Ptdh1-R contained the 5' end 20bp gene of the gene fragment entC (SEQ ID NO.2). Using the genome of Escherichia coli str. K-12 substr. MG1655 as a template, primers entC-F/entC-R were used to obtain the gene fragment entC (SEQ ID NO.2), wherein entC-R contained the 5' end 20bp gene of the gene fragment Ttef1-Ptef1 (part). Using the synthetic plasmid pUC-Ttef1-Ptef1 (part) as a template, primers Ttef1-F/Ptef1-bf-R were used to obtain the gene fragment Ttef1-Ptef1 (part), wherein Ptef1-bf-R contained the 3' end 20bp gene of the linear vector pUC19. Using the seamless cloning method, the linear vector pUC19 and the fragment were connected, and the competent Escherichia coli Trans-T1 was transformed. The positive clones were selected for bacterial P and sequencing to obtain the correct plasmid P1.

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 (SEQ ID NO.3). Using Escherichia coli str. K-12 substr. MG1655 genome as template, primers menD-F/menD-R were used to obtain gene fragment menD (SEQ ID NO.3), wherein menD-R contained the 5' end 20 bp gene of gene fragment Tpgk1-Ppgk1 (partial). Using synthetic plasmid pUC- Tpgk1-Ppgk1 (part) as template and primers Tpgk1-F/Ppgk1-bf-R, gene fragment Tpgk1-Ppgk1 (part) was obtained, where 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, transformed into Escherichia coli competent Trans-T1, and positive clones were selected for bacterial P and sequencing to obtain the correct plasmid P2.

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 (SEQ ID NO.4). Using Escherichia coli str. K-12 substr. MG1655 genome as template, primers menH-F/menH-R were used to obtain gene fragment menH (SEQ ID NO.4), wherein menH-R contained the 5' end 20 bp gene of gene fragment Tcyc1 (SEQ ID NO.18). Using synthetic plasmid pUC-Tcyc1 as template and primers Tcyc1-F/Tcyc1-R, gene fragment Tcyc1 (SEQ ID NO.18) was obtained, wherein Tcyc1-R contained the 5' 20 bp gene of gene fragment Ttef (SEQ ID NO.22). Using synthetic plasmid pUC-Ttef as template and primers Ttef-F/Ttef-R, gene fragment Ttef (SEQ ID NO.22) was obtained, wherein Ttef-R contained the 5' 20 bp gene of gene fragment LEU2 (SEQ ID NO.21). Using synthetic plasmid pUC-LEU2 as template and primers LEU2-F/LEU2-R, gene fragment LEU2 (SEQ ID NO.21) was obtained, wherein LEU2-R contained the 3' 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 Trans-T1, and the positive clones were selected for bacterial P and sequencing to obtain the correct plasmid P3.

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 (SEQ ID NO.19). Using synthetic plasmid pUC-Ttpi1 as template, primers Ttpi1-F/Ttpi1-R were used to obtain gene fragment Ttpi1 (SEQ ID NO.19), wherein Ttpi1-R contained the 5' end 20 bp gene of gene fragment menC (SEQ ID NO.5). Using the genome of Escherichia coli str. K-12 substr. MG1655 as a template, primers menC-F/menC-R were used to obtain gene fragment menC (SEQ ID NO.5), wherein menC-R contained the 5' end 20 bp gene of gene fragment Ptef1 (SEQ ID NO.15). Using synthetic plasmid pUC-Ptef1 as a template, primers Ptef1-F/Ptef1-R were used to obtain gene fragment Ptef1 (SEQ ID NO.15), wherein Ptef1-R contained the 5' end 20 bp gene of gene fragment delta-DOWN (SEQ ID NO.10). Using the genome of Saccharomyces cerevisiae BY4742 as a template, the primers delta-DOWN-F/delta-DOWN-R were used to obtain the gene fragment delta-DOWN (SEQ ID NO.10), wherein delta-DOWN-R contained the 5' 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 P4.

Construction of P6 plasmid: Using plasmid pRS413 (SEQ ID NO.23) as template, using primers His3-F/His3-R, fragment His3 was obtained, wherein primer His3-F contained the 3' end 20 bp gene of the linear vector pUC19, and primer His3-R contained the 5' end 20 bp gene of gene fragment Ttpi1 (SEQ ID NO.19). Using synthetic plasmid pUC-Ttpi1 as template, using primers Ttpi1-F/Ttpi1-R2, gene fragment Ttpi1 (SEQ ID NO.19)-2 was obtained, wherein Ttpi1-R2 contained the 5' end 20 bp gene of gene fragment Tcyc1 (SEQ ID NO.18). Using synthetic plasmid pUC-Tcyc1 as template and primers Tcyc1-F/Tcyc1-R2, gene fragment Tcyc1 (SEQ ID NO.18)-2 was obtained, wherein Tcyc1-R2 contained the 5' end 20 bp gene of gene fragment menE (SEQ ID NO.8). Using Escherichia coli str. K-12 substr. MG1655 genome as template and primers menE-F/menE-R, gene fragment menE (SEQ ID NO.8) was obtained, wherein menE-R contained the 5' end 20 bp gene of gene fragment Ppgk1 (SEQ ID NO.17). Using synthetic plasmid pUC-Ppgk1 as template, primers Ppgk1-F/ Ppgk1-R2 were used to obtain gene fragment Ppgk1 (SEQ ID NO.17)-2, wherein Ppgk1-R2 contained the 5' end 20bp gene of gene fragment rDNA-DOWN (SEQ ID NO.12). Using the genome of Saccharomyces cerevisiae BY4742 as template, primers rDNA-DOWN-F/rDNA-DOWN-R were used to obtain gene fragment rDNA-DOWN (SEQID NO.12), wherein rDNA-DOWN-R contained the 5' end 20bp gene of linear vector pUC19. Using seamless cloning method, linear vector pUC19 and fragment were connected, transformed into competent Escherichia coli Trans-T1, positive clones were selected for bacterial P and sequencing, and correct plasmid P6 was obtained.

P7 plasmid construction: Using the genome of Saccharomyces cerevisiae BY4742 as a template, using primers rDNA-UP-F/ rDNA-UP-R, the gene fragment rDNA-UP (SEQ ID NO.11) was obtained, wherein the primer rDNA-UP-F contained the 3' end 20bp gene of the linear vector pUC19, and the primer rDNA-UP-R contained the 5' end 20bp gene of the gene fragment Ptdh1 (SEQ ID NO.13). Using the synthetic plasmid pUC-Ptdh1 as a template, using primers Ptdh1-F/Ptdh1-R2, the gene fragment Ptdh1 (SEQ ID NO.13)-2 was obtained, wherein Ptdh1-R2 contained the 5' end 20bp gene of the gene fragment sll1127. Using pUC-sll1127 as a template and primers sll1127-F/sll1127-R, gene fragment sll1127 was obtained, wherein sll1127-R contained the 5' end 20 bp gene of gene fragment Ttef1 (SEQ ID NO.14). Using synthetic plasmid pUC-Ttef1 as a template and primers Ttef1-F/Ttef1-R, gene fragment Ttef1 (SEQ ID NO.14) was obtained, wherein Ttef1-R contained the 5' end 20 bp gene of gene fragment Ptef1 (SEQ ID NO.15). Using synthetic plasmid pUC-Ptef1 as a template and primers Ptef1-F/Ptef1-R2, gene fragment Ptef1 (SEQ ID NO.15)-2 was obtained, wherein Ptef1-R2 contained the 5' end 20 bp gene of gene fragment slr0204. Using pUC-slr0204 as a template and primers slr0204-F/slr0204-R, gene fragment slr0204 was obtained, wherein slr0204-R contained the 5' end 20 bp gene of gene fragment Ppgk1 (SEQ ID NO.17). Using synthetic plasmid pUC-Ppgk1 as a template and primers Ppgk1-F/Ppgk1-R, gene fragment Ppgk1 (SEQ ID NO.17) was obtained, wherein Ppgk1-R contained the 5' end 20 bp gene of gene fragment Phis3-His3 (part). Using plasmid pRS413 (SEQ ID NO.23) as a template and primers Phis3-F/His3-bf-R, fragment Phis3-His3 (part) was obtained, wherein His3-bf-R contained the 3' 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 Trans-T1, and the positive clones were selected for bacterial P and sequencing to obtain the correct plasmid P7.

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

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

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

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

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

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

Target fragment F7: Using plasmid P7 as template and primers F7-F/F7-R, fragment F7 was obtained.

The fragments F1, F2, F3, and F4 obtained above were introduced into Saccharomyces cerevisiae BY4742 by conventional yeast transformation method to obtain BD-0. Using BD-0 as the base bacteria, the fragments F6 and F7 were transferred by conventional yeast transformation method to obtain the Saccharomyces cerevisiae engineered bacteria BD-2. After transformation, SC (-Leu2, -His3) plates (22g/L glucose, 6.7 g/L amino-free yeast nitrogen source, 2 g/L drop-out, 0.02 g/L Ura, 0.02 g/L Trp, 20g/L agar) were used for screening. Single colonies with good growth were picked in the clean bench, suspended in a 96-well plate containing 20 µL ddH2O, set the program in the PCR instrument, and boiled at 99℃ for 10 min. Prepare the PCR system and load 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 solution. Take the boiled bacterial solution and add it to a system. Prepare the gel for DNA electrophoresis and wait for it to solidify. After the PCR is completed, add DNA loading buffer, apply 10 μL to each well, and run electrophoresis for 8-12 minutes. Observe whether there is the target band and record the corresponding number.

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

The 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 Xs (1 kb requires 30-60 s, so the time must be sufficient), 35 cycles, and finally extension at 72°C for 5 min and insulation at 16°C.

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

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, invert the mixture several times to mix well, then place it at room temperature for use, and invert it again to mix well before use.

Example 3: Shake flask fermentation of Saccharomyces cerevisiae BY4742 and engineered bacteria BD-2 in shake flask fermentation medium (aerobic)

In a sterile clean bench, BD-2 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.

Pick a single colony that grows well on the solid plate and inoculate it 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 Ura, 0.02 g/L Trp, 0.1 g/L Leu, 0.02 g/L His), then place the centrifuge tube in a shaker at 30°C and 220 rpm for about 20 h to reach the logarithmic growth phase, at which time the OD600 is approximately 0.5-1.

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 in a certain proportion for fermentation culture, so that the OD600 at the beginning of the fermentation remained consistent at 0.1.

The culture was carried out 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.

Example 4: DHNA extraction and detection

Take 1 ml of the fermentation broth and centrifuge it at 12,000 rpm for 5 min, remove the supernatant and keep the bacteria.

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 at room temperature for 15 min.

The supernatant was drawn up with a disposable syringe, filtered with a 0.2 µm organic phase filter membrane, and added to a sample bottle. After marking, the sample was tested by HPLC.

HPLC detection conditions: Shim-pack GIST C18 column (250 mm×4.6 mm, 5 μm); detector: Shimadu SPD-20A, UV detection wavelength (nm): 254; column temperature (℃): 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.

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.

The beneficial effects of the present invention are:

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-2 biosynthesized DHNA, with the highest yield at 72h, reaching 3.16±1.08 mg/L. It can be seen that:

(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-2.

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

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 (10)

1. A construction method capable of biosynthesizing 1, 4-dihydroxyl-2-naphthoic acid in saccharomyces cerevisiae is characterized by comprising the following steps: constructing a homologous arm fragment on a basic plasmid pUC19 (SEQ ID NO. 1), connecting a target gene encoding biosynthesis DHNA to pUC19 (SEQ ID NO. 1) in a seamless cloning mode to obtain plasmids P1, P2, P3, P4, P6 and P7, obtaining fragments F1, F2, F3, F4, F6 and F7 through polymerase chain reaction, and transferring 6 fragments into Saccharomyces cerevisiae Saccharomyces cerevisiae BY and 4742 by a yeast transformation method to obtain an engineering strain capable of biosynthesizing 1, 4-dihydroxy-2-naphthoic acid (DHNA).

2. The engineering strain construction method according to claim 1, wherein: the P1 plasmid contains delta-UP (SEQ ID NO. 9), promoter Ptdh1 (SEQ ID NO. 13), terminator Ttef1 (SEQ ID NO. 14) and promoter Ptef1 partial fragment (200 bp) which are the delta site upstream gene fragment derived from Saccharomyces cerevisiae Saccharomyces cerevisiae BY 4742; contains gene entC (SEQ ID NO. 2) encoding isochorismate synthase derived from Escherichia coli ESCHERICHIA COLI str. K-12 substr MG 1655.

3. The engineering strain construction method according to claim 1, wherein: the P2 plasmid contains a promoter Ptef1 (SEQ ID NO. 15), a terminator Tpgk1 (SEQ ID NO. 16) and a partial fragment of terminator Ttef1 (202 bp) and a partial fragment of promoter Ppgk1 (200 bp) derived from Saccharomyces cerevisiae Saccharomyces cerevisiae BY 4742; contains a gene menD (SEQ ID NO. 3) encoding 2-butyryl-5-enolpyruvate-6-hydroxy-3-cyclohexene-1-carboxylic acid synthase derived from E.coli ESCHERICHIA COLI str. K-12 substr. MG 1655.

4. The engineering strain construction method according to claim 1, wherein: the P3 plasmid contains the promoter Ppgk1 (SEQ ID NO. 17), the terminator Tcyc1 (SEQ ID NO. 18), a partial fragment of terminator Tpgk1 (199 bp) and terminator Ttef (SEQ ID NO. 22) derived from Saccharomyces cerevisiae Saccharomyces cerevisiae BY 4742; contains the LUE2 gene encoding leucine derived from Saccharomyces CEREVISIAE S C; contains a gene menH (SEQ ID NO. 4) encoding 2-butyryl-6-hydroxy 2, 4-cyclohexadiene-1-carboxylic acid synthase derived from Escherichia coli ESCHERICHIA COLI str. K-12 substr. MG 1655.

5. The engineering strain construction method according to claim 1, wherein: the P4 plasmid contains delta-DOWN (SEQ ID NO. 10), promoter Ptef1 (SEQ ID NO. 15), terminator Ttpi1 (SEQ ID NO. 19) which is a gene fragment downstream of the delta site derived from Saccharomyces cerevisiae Saccharomyces cerevisiae BY 4742; a LUE2 part gene (200 bp) containing leucine encoding gene derived from Saccharomyces CEREVISIAE S288C, promoter Pleu; contains gene menC (SEQ ID NO. 5) encoding an o-succinylbenzoic acid synthase derived from E.coli ESCHERICHIA COLI str. K-12 substr. MG 1655.

6. The engineering strain construction method according to claim 1, wherein: the P6 plasmid contains rDNA-DOWN (SEQ ID NO. 12), promoter Ppgk1 (SEQ ID NO. 17), terminator Tcyc1 (SEQ ID NO. 18), terminator Ttpi1 (SEQ ID NO. 19) which is a gene fragment downstream of rDNA locus derived from Saccharomyces cerevisiae Saccharomyces cerevisiae BY 4742; a His3 gene encoding histidine derived from pRS413 (SEQ ID NO. 23) plasmid; contains the gene menE (SEQ ID NO. 8) derived from E.coli ESCHERICHIA COLI str. K-12 substr, MG1655 encoding the o-succinylbenzoic acid coenzyme A ligase.

7. The engineering strain construction method according to claim 1, wherein: the P7 plasmid contains rDNA-UP (SEQ ID NO. 11), promoter Ptdh1 (SEQ ID NO. 13), terminator Ttef1 (SEQ ID NO. 14), promoter Ptef1 (SEQ ID NO. 15) and terminator Tpgk1 (SEQ ID NO. 16) which are gene fragments upstream of rDNA locus derived from Saccharomyces cerevisiae Saccharomyces cerevisiae BY 4742; his3 part gene (200 bp) encoding histidine derived from pRS413 (SEQ ID NO. 23) plasmid, promoter Phis; contains a gene sll1127 (SEQ ID NO. 6) encoding a1, 4-dihydroxy-2-naphthaloyl-CoA synthase and a gene slr0204 (SEQ ID NO. 7) encoding a1, 4-dihydroxy-2-naphthalenyl-CoA hydrolase derived from cyanobacteria Synechocystis sp.PCC 6803.

8. The engineering strain construction method according to claim 1, wherein: the fragments F1, F2, F3 and F4 are obtained by performing polymerase chain reaction on the P1, P2, P3 and P4 plasmids, and are introduced into wild-type saccharomyces cerevisiae Saccharomyces cerevisiaeBY and 4742 to form 1 strain of saccharomyces cerevisiae engineering bacteria BD-0.

9. The engineering strain construction method according to claim 1, wherein: the P6 and P7 plasmids are subjected to polymerase chain reaction to obtain fragments F6 and F7, and the fragments F6 and F7 are introduced into saccharomyces cerevisiae engineering bacteria BD-0 to form 1 strain of saccharomyces cerevisiae engineering bacteria BD-2.

10. The recombinant engineering strain construction method according to claim 1, wherein: the culture was performed in shake flasks using a shaker, the OD600 was measured by a spectrophotometer, and the yield of DHNA was measured by High Performance Liquid Chromatography (HPLC).