CN113122512B - Salvia miltiorrhiza P450 mutant for preparing tanshinone compounds - Google Patents

Salvia miltiorrhiza P450 mutant for preparing tanshinone compounds Download PDF

Info

Publication number
CN113122512B
CN113122512B CN202010041299.0A CN202010041299A CN113122512B CN 113122512 B CN113122512 B CN 113122512B CN 202010041299 A CN202010041299 A CN 202010041299A CN 113122512 B CN113122512 B CN 113122512B
Authority
CN
China
Prior art keywords
protein
mutated
sequence
cyp76ah3
tanshinone
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202010041299.0A
Other languages
Chinese (zh)
Other versions
CN113122512A (en
Inventor
郭娟
毛亚平
马莹
曾雯
陈同
黄璐琦
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Materia Medica of CAMS
Original Assignee
Institute of Materia Medica of CAMS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Materia Medica of CAMS filed Critical Institute of Materia Medica of CAMS
Priority to CN202010041299.0A priority Critical patent/CN113122512B/en
Publication of CN113122512A publication Critical patent/CN113122512A/en
Application granted granted Critical
Publication of CN113122512B publication Critical patent/CN113122512B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004Oxidoreductases (1.)
    • C12N9/0012Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C12N9/0038Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12N9/0042NADPH-cytochrome P450 reductase (1.6.2.4)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P15/00Preparation of compounds containing at least three condensed carbocyclic rings
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y106/00Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/02Oxidoreductases acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12Y106/02004NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Mycology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a salvia miltiorrhiza P450 mutant for preparing tanshinone compounds. The invention optimizes the catalytic efficiency by directionally transforming CYP76AH3, so that a reaction path which needs to be completed by the synergistic action of the CYP76AH1 and CYP76AH3 can be directly catalyzed by the CYP76AH3 mutant in the invention, the construction of the CYP76AH3 mutant can shorten a substrate transportation path in the reaction process, and multiple reactions can be efficiently completed in one protein pocket, thereby improving the utilization efficiency of the substrate and the yield of the target compound. The invention provides a substrate and Chassis bacteria for the subsequent analysis of the tanshinone biosynthesis pathway aiming at the CYP76AH3 mutant with improved conversion efficiency of different products, and has important guiding significance for the full analysis of the tanshinone biosynthesis pathway and the construction of yeast engineering bacteria for high-yield tanshinone compounds.

Description

Salvia miltiorrhiza P450 mutant for preparing tanshinone compounds
Technical Field
The invention relates to a salvia miltiorrhiza P450 mutant for preparing tanshinone compounds.
Background
In the biological catalysis and synthetic biology research of medicinal natural products, the catalytic efficiency of P450 is always a difficult problem in the international synthetic biology research. For example, the synthetic biology of artemisinic acid has the problem that the catalytic efficiency of P450 limits the yield of the final product, and after CYP71AV1 and CPR1 are expressed, the yield of the culture product of the artemisinic acid production engineering yeast Y285 is obviously reduced compared with the yield of the amorphadiene in the last step. Paddon et al believe that the reduced catalytic activity and reduced yield of sesquiterpene products in Y285 may be caused by the inefficient catalytic oxidation of amorphadiene cytochrome P450.
The biosynthetic pathway of the tanshinone compound contains a plurality of P450, and the functions of P450 enzymes CYP76AH1 and CYP76AH3 are cloned and verified by combining omics technology, bioinformatics and synthetic biology means in a laboratory where the applicant belongs (figure 1). Due to the heterozygosity of the catalytic function of P450, the biosynthesis pathway of the tanshinone compounds is divided into two branches, and two compounds which are modified at the 7-position and the 11-position on the basis of the iron rust alcohol and the cryptomerin are generated. In experiments of obtaining tanshinone precursor compounds by direct catalysis of P450 enzymes and yeast engineering strains containing wild-type CYP76AH1 and CYP76AH3 genes, the catalytic efficiency of P450 is an important difficult problem to be solved urgently, and the two P450 s, namely CYP76AH1 and CYP76AH3, are key components and speed-limiting steps in the biosynthesis research of the tanshinone compounds.
Disclosure of Invention
The invention aims to provide a salvia miltiorrhiza P450 mutant for preparing tanshinone compounds.
In the first aspect, the present invention is a protected protein obtained by mutating at least one of the 301 th amino acid residue, 306 th amino acid residue, 395 th amino acid residue and 479 th amino acid residue of a protein represented by sequence 4 in a sequence table.
The protein is any one of the following (a1) - (a 15):
(a1) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 301 th amino acid residue is mutated from E to D, and the 306 th amino acid residue is mutated from E to S;
(a2) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 301 th amino acid residue is mutated from E to D, and the 479 th amino acid residue is mutated from F to V;
(a3) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 301 th amino acid residue is mutated from E to D, and the 395 th amino acid residue is mutated from M to I;
(a4) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 395 th amino acid residue is mutated from M to I;
(a5) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 306 th amino acid residue is mutated from E to S, and the 395 th amino acid residue is mutated from M to I;
(a6) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 306 th amino acid residue is mutated from E to S;
(a7) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 301 th amino acid residue is mutated from E to D;
(a8) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 301 th amino acid residue is mutated from E to D, the 306 th amino acid residue is mutated from E to S, and the 395 th amino acid residue is mutated from M to I;
(a9) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 395 th amino acid residue is mutated from M to I, and the 479 th amino acid residue is mutated from F to V;
(a10) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 301 th amino acid residue is mutated from E to D, the 395 th amino acid residue is mutated from M to I, and the 479 th amino acid residue is mutated from F to V;
(a11) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 306 th amino acid residue is mutated from E to S, the 395 th amino acid residue is mutated from M to I, and the 479 th amino acid residue is mutated from F to V;
(a12) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 301 th amino acid residue is mutated from E to D, the 306 th amino acid residue is mutated from E to S, the 395 th amino acid residue is mutated from M to I, and the 479 th amino acid residue is mutated from F to V;
(a13) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 301 th amino acid residue is mutated from E to D, the 306 th amino acid residue is mutated from E to S, and the 479 th amino acid residue is mutated from F to V;
(a14) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 306 th amino acid residue is mutated from E to S, and the 479 th amino acid residue is mutated from F to V;
(a15) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 479 th amino acid residue is mutated from F to V.
The invention also protects a gene encoding any of the proteins described above.
The gene may specifically be any one of the following (a1) - (a 15):
(A1) mutating the 903 th g to c, the 916 th g to t and the 917 th a to c from the 5' end of the sequence 3 to obtain DNA molecules;
(A2) mutating 903 th g to c, 1435 th t to g and 1437 th c to g of the sequence 3 from the 5' end to obtain a DNA molecule;
(A3) mutating the 903 th g to c and the 1185 th g to a from the 5' end of the sequence 3 to obtain a DNA molecule;
(A4) mutating the 1185 th g of the 5' end of the sequence 3 into a DNA molecule obtained by a;
(A5) mutating the 916 th g to t, the 917 th a to c and the 1185 th g to a from the 5' end of the sequence 3 to obtain a DNA molecule;
(A6) mutating the 916 th g of the sequence 3 from the 5' end to t, and mutating the 917 th a to c to obtain a DNA molecule;
(A7) mutating the 903 th g of the 5' end of the sequence 3 into a DNA molecule obtained by c;
(A8) mutating the 903 th g to c, the 916 th g to t, the 917 th a to c and the 1185 th g to a from the 5' end of the sequence 3 to obtain DNA molecules;
(A9) mutating the 1185 th g to a, the 1435 th t to g and the 1437 th c to g of the sequence 3 from the 5' end to obtain a DNA molecule;
(A10) mutating the 903 th g to c, the 1185 th g to a, the 1435 th t to g and the 1437 th c to g of the sequence 3 from the 5' end to obtain a DNA molecule;
(A11) mutating 916 th g to t, 917 th a to c, 1185 th g to a, 1435 th t to g, and 1437 th c to g from 5' end of the sequence 3 to obtain a DNA molecule;
(A12) mutating the 903 th g to c, the 916 th g to t, the 917 th a to c, the 1185 th g to a, the 1435 th t to g and the 1437 th c to g from the 5' end of the sequence 3 to obtain a DNA molecule;
(A13) mutating the 903 th g to c, the 916 th g to t, the 917 th a to c, the 1435 th t to g and the 1437 th c to g of the sequence 3 from the 5' end to obtain a DNA molecule;
(A14) mutating 916 th g to t, 917 th a to c, 1435 th t to g, and 1437 th c to g from 5' end of the sequence 3 to obtain a DNA molecule;
(A15) and (3) mutating t from 1435 th site of the 5' end of the sequence 3 into g, and mutating c from 1437 th site into g.
The invention also protects a recombinant expression vector, an expression cassette or a recombinant bacterium containing the gene.
The recombinant expression vector may specifically be any one of the following (1) to (15):
(1) inserting the DNA molecule of (A1) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(2) inserting the DNA molecule of (A2) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(3) inserting the DNA molecule of (A3) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(4) inserting the DNA molecule of (A4) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(5) inserting the DNA molecule of (A5) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(6) inserting the DNA molecule of (A6) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(7) inserting the DNA molecule of (A7) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(8) inserting the DNA molecule of (A8) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(9) inserting the DNA molecule of (A9) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(10) inserting the DNA molecule of (A10) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(11) inserting the DNA molecule of (A11) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(12) inserting the DNA molecule of (A12) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(13) inserting the DNA molecule of (A13) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(14) inserting the DNA molecule of (A14) into a BamH I site of an expression vector pESC-His to obtain a recombinant expression vector;
(15) and (D) inserting the DNA molecule described in (A15) into the BamH I site of expression vector pESC-His to obtain a recombinant expression vector.
The recombinant strain was obtained BY introducing the recombinant expression vector described above into the yeast expression strain BY4741 containing the Arabidopsis thaliana P450 reductase ATR 1.
In a second aspect, the invention provides a use of any one of the above proteins or genes encoding the proteins, which is at least one of the following (b1) - (b 5):
(b1) preparing tanshinone compounds;
(b2) using sub-tanshinone diene as a substrate to catalyze and generate the rust alcohol;
(b3) catalyzing and generating cryptotanshinone by using sub-tanshinone diene as a substrate;
(b4) using sub-tanshinone diene as a substrate to catalyze and generate 11-hydroxyl rust alcohol;
(b5) using hypotanshinone diene as substrate to catalyze and generate 11-hydroxy cedrol.
The invention also protects the application of any one of the recombinant expression vector, the expression cassette or the recombinant bacterium, which is at least one of the following (b1) - (b 6):
(b1) preparing tanshinone compounds;
(b2) using sub-tanshinone diene as a substrate to catalyze and generate the rust alcohol;
(b3) catalyzing and generating cryptotanshinone by using sub-tanshinone diene as a substrate;
(b4) using sub-tanshinone diene as a substrate to catalyze and generate 11-hydroxyl rust alcohol;
(b5) catalyzing to generate 11-hydroxy cedrol by using sub-tanshinone diene as a substrate;
(b6) the protein described hereinbefore was prepared.
In a third aspect, the present invention also provides a method for preparing the protein, comprising the steps of: the recombinant bacteria described above are cultured to obtain the protein from the recombinant bacteria.
In the method, the method for obtaining the protein from the recombinant bacteria specifically comprises the following steps: (a) inoculating the recombinant strain into a defective SD-His liquid culture medium for yeast transformation, culturing, collecting the strain, suspending the strain by using an equal volume of YPL liquid culture medium, and performing induction culture at 30 ℃ and 200 r/min; (b) after the step (a) is finished, centrifugally collecting thalli from a culture system, and resuspending cells by using TEK; (c) after the step (b) is finished, centrifugally collecting the thalli, and resuspending the thalli by ice-bath TESB; (d) after the step (c) is finished, adding glass beads to the surface of the contact solution, vigorously shaking the yeast cells at low temperature until the yeast cells are completely crushed, adding TESB into the crushed cells, recovering the supernatant, centrifuging, collecting the supernatant, adding a precipitator of 0.225M NaCl and 0.15g/mL PEG4000, and centrifuging at high speed after ice bath to obtain a precipitate; (e) after completion of step (d), TEG is added to dissolve the precipitate to obtain the protein.
In a fourth aspect, the invention also protects a method of making rust alcohol comprising the steps of: using sub-tanshinone diene as a substrate, and carrying out catalytic reaction by adopting the protein to obtain the rust alcohol.
The invention also protects any one of the following methods;
(A) the method for preparing the cryptomerin comprises the following steps: using sub-tanshinone diene as a substrate, and carrying out catalytic reaction by adopting the protein to obtain cryptomerin;
(B) a method of preparing 11-hydroxyrust alcohol comprising the steps of: using sub-tanshinone diene as a substrate, and carrying out catalytic reaction by adopting the protein to obtain 11-hydroxyl rust alcohol;
(C) the method for preparing 11-hydroxycedrol comprises the following steps: using sub-tanshinone diene as a substrate, and carrying out catalytic reaction by adopting the protein to obtain 11-hydroxy cedrol;
(D) the method for preparing the tanshinone compound comprises the following steps: using sub-tanshinone diene as a substrate, and carrying out catalytic reaction by adopting the protein to obtain tanshinone compounds; the tanshinone compound is any one or combination of more of rustic alcohol, cryptomerin, 11-hydroxyl rustic alcohol and 11-hydroxyl cryptomerin.
In the above (A), the protein is the protein of (a1), (a2), (a3), (a7), (a8), (a5), (a6), (a4), (a9), (a10), (a11), (a13), or (a 14);
in the above (B), the protein is the protein described in the above (a4), the protein described in (a5), the protein described in (a6), the protein described in (a7), the protein described in (a1), the protein described in (a3), the protein described in (a2), the protein described in (a9), the protein described in (a8), or the protein described in (a 11);
in the above (C), the protein is the protein described in the above (a4), the protein described in (a5), the protein described in (a6), the protein described in (a3), the protein described in (a7), the protein described in (a1), the protein described in (a2), the protein described in (a9), or the protein described in (a 8).
In the method of the fourth aspect, the reaction system of the catalytic reaction may specifically be: 100mM Tris-HCl, 1mM NADPH, 5. mu.M FAD, 5. mu.M FMN, 4mM glucose-6-phosphate, 1U glucose-6-phosphate dehydrogenase, 2. mu.M DTT, 500. mu.g of the protein, 100. mu.M of the substrate, tanshinone diene. The reaction conditions may be specifically 30 ℃ and 200rpm for 2 hours.
The tanshinone compound can be Rusitol, salutaol, 11-hydroxyrust alcohol or 11-hydroxysalutaol.
The invention optimizes the catalytic efficiency by directionally modifying CYP76AH 3. The targeted mutation generates the function-integrated P450 mutant with the highest CYP76AH3 efficiency, so that a reaction path which needs to be completed by the synergistic action of the CYP76AH1 and the CYP76AH3 can be directly catalyzed and completed by the CYP76AH3 mutant, the construction of the CYP76AH3 mutant can shorten a substrate transportation path in the reaction process, and multiple reactions can be efficiently completed in one protein pocket, thereby improving the utilization efficiency of substrates and improving the yield of target compounds.
Mutants with different catalytic activities of CYP76AH3 on the substrate sub-tanshinone diene are constructed by different mutation site combinations, and screening is carried out according to the yield of the target product to obtain a mutant library of a single tanshinone compound with high catalytic activity. The invention provides a substrate and Chassis bacteria for the subsequent analysis of the tanshinone biosynthesis pathway aiming at the CYP76AH3 mutant with improved conversion efficiency of different products, and has important guiding significance for the full analysis of the tanshinone biosynthesis pathway and the construction of yeast engineering bacteria for high-yield tanshinone compounds.
Drawings
Figure 1 is the CYP76AH1 and CYP76AH3 catalytic pathways.
FIG. 2 shows the statistical results of rust alcohol production.
FIG. 3 shows the statistical results of the production of 11-hydroxyrust alcohol.
FIG. 4 shows statistical results of the yield of cryptomerin.
FIG. 5 shows the statistical results of 11-hydroxycedrol production.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Expression strain BY4741 Saccharomyces cerevisiae strain: purchased from bio-technology ltd, warrior, beijing, inc, cat #: NRR 1030.
Glucose-6-phosphate dehydrogenase: purchased from bio-engineering (shanghai) gmbh, cat #: A003997. definition of enzyme activity: the amount of enzyme required to consume one micromole of NAD per micromole of reaction using glucose-6-phosphate as a substrate at 37 ℃ and pH7.8 was 1 unit.
Example 1 obtaining of mutants
The CYP76AH1 and CYP76AH3 proteins are P450 proteins involved in tanshinone biosynthesis. The catalytic pathway is shown in FIG. 1. In FIG. 1, each protein catalyzes the substrate in front of the arrow, but the efficiency of production is different.
The amino acid sequence of CYP76AH1 is shown as sequence 2 in the sequence table, and the coding gene (CYP76AH1) is shown as sequence 1 in the sequence table.
The amino acid sequence of CYP76AH3 is shown as sequence 4 in the sequence table, and the coding gene (CYP76AH3) is shown as sequence 3 in the sequence table.
Sequence analysis, mutation and functional verification of CYP76AH3 revealed four active sites related to CYP76AH1 catalytic efficiency, namely amino acid residues 301, 306, 395 and 479. These 4 amino acid positions were subjected to different forms of mutagenesis (Table 1), resulting in a total of 15 mutants of the CYP76AH1 protein (Table 2).
TABLE 14 amino acid sites and their mutated forms
Figure GDA0003527812730000071
TABLE 2 mutant information
Figure GDA0003527812730000072
Figure GDA0003527812730000081
In table 2, the expression pattern of the mutant forms is: pre-mutation-digit-post mutation.
Example 2 preparation of recombinant expression vector
Construction of wild type recombinant expression vector
1. The double-stranded DNA molecule shown in the sequence 1 is inserted between BamH I sites of an expression vector pESC-His (circular plasmid shown in the sequence 5 of the sequence table) to obtain a recombinant expression vector pESC-His-H1WT (the sequencing verification is correct). The DNA molecule shown in sequence 1 encodes the protein shown in sequence 2.
2. And inserting the double-stranded DNA molecule shown in the sequence 3 between BamH I sites of an expression vector pESC-His to obtain a recombinant expression vector pESC-His-H3WT (the sequencing verification is correct). The DNA molecule shown in sequence 3 encodes the protein shown in sequence 4.
Second, construction of mutant recombinant expression vector
1. Double-stranded DNA molecule 1 was inserted between BamH I sites of expression vector pESC-His to obtain recombinant expression vector pESC-His-E301D (correct sequencing). The double-stranded DNA molecule 1 is obtained by point mutation of the wild-type CYP76AH3 gene. Compared with the wild-type CYP76AH3 gene, the double-stranded DNA molecule 1 differs only in that: the 903 nd g of the 5' end of the sequence 3 is mutated into c. DNA molecule 1 encodes the protein AH 3-E301D. Compared with the wild-type CYP76AH3 protein, the protein AH3-E301D differs only in that: the 301 st E of the sequence 4 is mutated to D.
2. Double-stranded DNA molecule 2 was inserted between BamH I sites of expression vector pESC-His to obtain recombinant expression vector pESC-His-E306S (correct sequencing). The double-stranded DNA molecule 2 is obtained by point mutation of the wild-type CYP76AH3 gene. The double-stranded DNA molecule 2 differs from the wild-type CYP76AH3 gene only in that: the 916 th g of the sequence 3 from the 5' end is mutated into t, and the 917 th a is mutated into c. DNA molecule 2 encodes the protein AH 3-E306S. Compared with the wild-type CYP76AH3 protein, the protein AH3-E306S differs only in that: e at position 306 shown in the sequence 4 is mutated into S.
3. Double-stranded DNA molecule 3 was inserted between BamHI sites of expression vector pESC-His to give recombinant expression vector pESC-His-M395I (correct sequencing). The double-stranded DNA molecule 3 is obtained by point mutation of the wild-type CYP76AH3 gene. The double-stranded DNA molecule 3 differs from the wild-type CYP76AH3 gene only in that: the 1185 th position g of the sequence 3 from the 5' end is mutated into a. DNA molecule 3 encodes the protein AH 3-M395I. Compared with the wild-type CYP76AH3 protein, the protein AH3-M395I differs only in that: m at 395 th site shown in the sequence 4 is mutated into I.
4. The double-stranded DNA molecule 4 is inserted between BamH I sites of the expression vector pESC-His to obtain a recombinant expression vector pESC-His-F479V (correct sequencing verification). The double-stranded DNA molecule 4 is obtained by point mutation of the wild-type CYP76AH3 gene. Compared with the wild-type CYP76AH3 gene, the double-stranded DNA molecule 4 differs only in that: and (3) mutating t from 1435 th position of the 5' end of the sequence 3 into g, and mutating c from 1437 th position into g. DNA molecule 4 encodes the protein AH 3-F479V. Compared with the wild-type CYP76AH3 protein, the protein AH3-F479V differs only in that: f at position 479 shown in the sequence 4 is mutated into V.
5. Double-stranded DNA molecule 5 was inserted between BamH I sites of expression vector pESC-His to give recombinant expression vector pESC-His-E301D, M395I (correct sequencing). The double-stranded DNA molecule 5 is obtained by point mutation of the wild-type CYP76AH3 gene. The double-stranded DNA molecule 5 differs from the wild-type CYP76AH3 gene only in that: c is mutated from 903 g at the 5' end of the sequence 3, and a g at the 1185 position is mutated into a. DNA molecule 5 encodes the proteins AH3-E301D, M395I. Compared with the wild-type CYP76AH3 protein, the proteins AH3-E301D, M395I differ only in that: the 301 th E of the sequence 4 is mutated into D, and the 395 th M is mutated into I.
6. The double-stranded DNA molecule 6 is inserted between BamH I sites of the expression vector pESC-His to obtain a recombinant expression vector pESC-His-E301D, F479V (correct sequencing). The double-stranded DNA molecule 6 is obtained by point mutation of the wild-type CYP76AH3 gene. Compared with the wild-type CYP76AH3 gene, the double-stranded DNA molecule 6 differs only in that: and (3) mutating the 903 th position g to c, the 1435 th position t to g and the 1437 th position c to g from the 5' end of the sequence. DNA molecule 6 encodes the proteins AH3-E301D, F479V. The proteins AH3-E301D, F479V differ from the wild-type CYP76AH3 protein only in that: e at the 301 th position shown in the sequence 4 is mutated into D, and F at the 479 th position is mutated into V.
7. Double-stranded DNA molecule 7 was inserted between BamH I sites of expression vector pESC-His to give recombinant expression vector pESC-His-E306S, M395I (correct sequencing). The double-stranded DNA molecule 7 is obtained by point mutation of the wild-type CYP76AH3 gene. The double-stranded DNA molecule 7 differs from the wild-type CYP76AH3 gene only in that: the 916 th g of the sequence 3 from the 5' end is mutated into t, the 917 th a is mutated into c, and the 1185 th g is mutated into a. DNA molecule 7 encodes the proteins AH3-E306S, M395I. Compared with the wild-type CYP76AH3 protein, the proteins AH3-E306S, M395I differ only in that: e at position 306 shown in the sequence 4 is mutated into S, and M at position 395 is mutated into I.
8. The double-stranded DNA molecule 8 is inserted between BamH I sites of the expression vector pESC-His to obtain recombinant expression vectors pESC-His-E301D, E306S (correct sequencing verification). The double-stranded DNA molecule 8 is obtained by point mutation of the wild-type CYP76AH3 gene. The double-stranded DNA molecule 8 differs from the wild-type CYP76AH3 gene only in that: c is mutated from 903 g at the 5' end of the sequence 3, t is mutated from 916 g, and c is mutated from 917 a. The DNA molecule 8 encodes the proteins AH3-E301D, E306S. The proteins AH3-E301D, E306S differ from the wild-type CYP76AH3 protein only in that: e at the 301 th site shown in the sequence 4 is mutated into D, and E at the 306 th site is mutated into S.
9. Double-stranded DNA molecule 9 was inserted between BamH I sites of expression vector pESC-His to give recombinant expression vector pESC-His-E306S, F479V (correct sequencing). The double-stranded DNA molecule 9 is obtained by point mutation of the wild-type CYP76AH3 gene. The double-stranded DNA molecule 9 differs from the wild-type CYP76AH3 gene only in that: the 916 th g of the sequence 3 from the 5' end is mutated into t, the 917 th a is mutated into c, the 1435 th t is mutated into g, and the 1437 th c is mutated into g. DNA molecule 9 encodes the proteins AH3-E306S, F479V. The proteins AH3-E306S, F479V differ from the wild-type CYP76AH3 protein only in that: e at position 306 shown in the sequence 4 is mutated into S, and F at position 479 is mutated into V.
10. The double-stranded DNA molecule 10 was inserted between BamH I sites of the expression vector pESC-His to give the recombinant expression vector pESC-His-M395I, F479V (correct sequencing). The double-stranded DNA molecule 10 is obtained by point mutation of the wild-type CYP76AH3 gene. The double-stranded DNA molecule 10 differs from the wild-type CYP76AH3 gene only in that: the sequence 3 is mutated from 1185 th position of 5' end to a, t 1435 th position to g and c 1437 th position to g. The DNA molecule 10 encodes the proteins AH3-M395I, F479V. Compared with the wild-type CYP76AH3 protein, the proteins AH3-M395I, F479V differ only in that: m at 395 th site shown in the sequence 4 is mutated into I, and F at 479 th site is mutated into V.
11. The double-stranded DNA molecule 11 was inserted between BamH I sites of the expression vector pESC-His to give recombinant expression vectors pESC-His-E301D, E306S, M395I (sequencing confirmed to be correct). The double-stranded DNA molecule 11 is obtained by point mutation of the wild-type CYP76AH3 gene. The double-stranded DNA molecule 11 differs from the wild-type CYP76AH3 gene only in that: c is mutated from 903 g to 916 g, t is mutated from 917 a to c, and a is mutated from 1185 g to a from 5' end of the sequence 3. The DNA molecule 11 encodes the proteins AH3-E301D, E306S, M395I. The proteins AH3-E301D, E306S, M395I differ from the wild-type CYP76AH3 protein only in that: e at the 301 th site shown in the sequence 4 is mutated into D, E at the 306 th site is mutated into S, and M at the 395 th site is mutated into I.
12. The double-stranded DNA molecule 12 was inserted between BamH I sites of the expression vector pESC-His to give recombinant expression vectors pESC-His-E301D, E306S, F479V (correct sequencing). The double-stranded DNA molecule 12 is obtained by point mutation of the wild-type CYP76AH3 gene. The double-stranded DNA molecule 12 differs from the wild-type CYP76AH3 gene only in that: c is mutated from 903 g to 916 g, t is mutated from 917 a to c, t is mutated from 1435 to g, and c is mutated from 1437 to g from 5' end of the sequence 3. The DNA molecule 12 encodes the proteins AH3-E301D, E306S, F479V. The proteins AH3-E301D, E306S, F479V differ from the wild-type CYP76AH3 protein only in that: and (3) mutating the 301 th E shown in the sequence 4 into D, the 306 th E into S, and the 479 th F into V.
13. The double-stranded DNA molecule 13 was inserted between BamH I sites of the expression vector pESC-His to give recombinant expression vectors pESC-His-E301D, M395I, F479V (correct sequencing). The double-stranded DNA molecule 13 is obtained by point mutation of the wild-type CYP76AH3 gene. The double-stranded DNA molecule 13 differs from the wild-type CYP76AH3 gene only in that: c is mutated from 903 g to 1185 g, g to a is mutated from 1435 t to g, and c to g is mutated from 1437 c of the sequence 3 from the 5' end. The DNA molecule 13 encodes the proteins AH3-E301D, M395I, F479V. The proteins AH3-E301D, M395I, F479V differ from the wild-type CYP76AH3 protein only in that: e at the 301 th site shown in the sequence 4 is mutated into D, M at the 395 th site is mutated into I, and F at the 479 th site is mutated into V.
14. The double-stranded DNA molecule 14 was inserted between BamH I sites of the expression vector pESC-His to give recombinant expression vectors pESC-His-E306S, M395I, F479V (correct sequencing). The double-stranded DNA molecule 14 is obtained by point mutation of the wild-type CYP76AH3 gene. The double-stranded DNA molecule 14 differs from the wild-type CYP76AH3 gene only in that: the 916 th g of the sequence 3 from the 5' end is mutated into t, the 917 th a is mutated into c, the 1185 th g is mutated into a, the 1435 th t is mutated into g, and the 1437 th c is mutated into g. The DNA molecule 14 encodes the proteins AH3-E306S, M395I, F479V. Compared with the wild-type CYP76AH3 protein, the proteins AH3-E306S, M395I and F479V only differ: e at the 306 th site shown in the sequence 4 is mutated into S, M at the 395 th site is mutated into I, and F at the 479 th site is mutated into V.
15. The double-stranded DNA molecule 15 was inserted between BamH I sites of the expression vector pESC-His to give recombinant expression vectors pESC-His-E301D, E306S, M395I, F479V (correct sequencing). The double-stranded DNA molecule 15 is obtained by point mutation of the wild-type CYP76AH3 gene. The double-stranded DNA molecule 15 differs from the wild-type CYP76AH3 gene only in that: c is mutated from 903 g to 916 g, t is mutated from 917 a to c, g is mutated from 1185 to a, t is mutated from 1435 to g, and c is mutated from 1437 to g. The DNA molecule 15 encodes the proteins AH3-E301D, E306S, M395I, F479V. Compared with the wild-type CYP76AH3 protein, the proteins AH3-E301D, E306S, M395I, F479V differ only in that: and (3) mutating the 301 th E shown in the sequence 4 into D, the 306 th E into S, the 395 th M into I and the 479 th F into V.
Example 3 recombinant bacteria preparation and microsomal protein extraction
1. The wild-type recombinant expression vector, the mutant recombinant expression vector and the expression vector pESC-His prepared in example 2 were introduced into the yeast expression strain BY4741 into which the Arabidopsis P450 reductase ATR1 was integrated, to obtain wild-type recombinant bacteria, 15 mutant recombinant bacteria and empty-vector recombinant bacteria.
2. Respectively inoculating the wild recombinant bacteria and 15 mutant recombinant bacteria obtained in the step 1 into a defective SD-His liquid culture medium (pantono, SD-His liquid culture medium formula: 8g/LSD-His powder, 20g/L glucose, sterilization for 15min) for yeast transformation, culturing for 48 hours at 30 ℃ at 200 r/min, centrifuging at 5000 r/min for 5min to collect thalli, resuspending the thalli by using an equal volume of YPL liquid culture medium (10g/L yeast extract, 20g/L peptone, 20g/L galactose), and then carrying out induction culture at 30 ℃ at 200 r/min in a shaking flask.
3. After completing step 2, the culture system was centrifuged to collect the cells, and the cells were resuspended with TEK.
4. After completion of step 3, the cells were collected by centrifugation and resuspended in TESB.
5. After completion of step 4, glass beads (Sigma-Aldrich, cat # G4649) were added to just the surface of the contact solution, the yeast cells were vigorously shaken until the cells were completely disrupted, and the supernatant was recovered by high-speed centrifugation.
6. After completion of step 5, the supernatant was taken, 0.225M NaCl and 0.15g/mL PEG4000 were added, ice-cooled for 15min, followed by high-speed centrifugation and discarding of the supernatant.
7. After step 6 is completed, TEG is added to dissolve the precipitate to obtain microsomal protein for later use.
Example 4 enzymatic reaction and detection of reaction product
Protein to be tested: wild-type microsomal proteins, 15 mutant microsomal proteins, and empty carrier microsomal proteins were prepared as in example 3.
Enzymatic reaction
Preparing a reaction system: 100mM Tris-HCl, 1mM NADPH, 5. mu.M FAD, 5. mu.M FMN, 4mM glucose-6-phosphate, 1U glucose-6-phosphate dehydrogenase, 2. mu.M DTT, 500. mu.g microsomal protein, 100. mu.M substrate tanshinone diene.
The reaction system is reacted for 2 hours or more at 30 ℃.
The tanshinone diene is obtained by fermentation and extraction of the yeast engineering strain in the experiment, and can be extracted by the method disclosed in the literature CYP76AH1 catalysts turn over of milliradine in biochemical and energetic heterologous production of ferruginol in yeases, PNAS,110(2013),12108-13.
And II, after the step I is finished, adding equal volume of ethyl acetate into the reaction system, and performing vortex oscillation and uniform mixing to extract a product for UPLC detection.
The UPLC chromatographic detection method comprises the following steps: the company Water UPLC, column model BEH C18 column (50X2.1mm,1.7 μm). Mobile phase A is acetonitrile, mobile phase B is: 0.1% formic acid-water, flow rate 0.5mL/min, gradient elution.
Part of the standard samples are purchased from Beijing Congxin Dekoku technologies, Inc., and the specific information is as follows: rust alcohol: the goods number is: 514-62-5; hydroxyl saligenin: the goods number is: 88664-08-8; and 5, cryptomeriol: the goods number is: 511-05-7.
The hydroxyl rust alcohol is obtained by fermenting and extracting the engineering yeast strain in the experiment, and can be obtained by specifically referring to the following references: cytochrome P450 pathological leads to a biofunctional biochemical pathway for tanshinones, New phytologists, 210(2016),525-34.
The content of the rust alcohol catalyzing the sub-tanshinone diene by CYP76AH1 and the content of the cryptomeril, 11-hydroxyrust alcohol and 11-hydroxycryptomeril catalyzing the sub-tanshinone diene by CYP76AH3 are 1, and the four products catalyzed by the mutant are normalized.
The results are shown in FIGS. 2 to 5.
The comparative data are shown in Table 1.
TABLE 1 comparison of amounts of the CYP76AH3 mutant protein and the wild-type CYP76AH3 protein catalyzing the production of tanshinone
Figure GDA0003527812730000131
The above results indicate that the mutant CYP76AH3E301D,E306SAnd CYP76AH3E301D,F479VThe yield of the rust alcohol generated by isocatalysis of the sub-tanshinone diene is obviously improved by 3.36 and 4.79 times compared with the efficiency of the wild type CYP76AH1 protein, and the mutant CYP76AH3M395IAnd CYP76AH3E306S,M395IThe yield of the 11-hydroxyferruginol generated by catalyzing the sub-tanshinone diene is obviously improved by 3.64 and 2.01 times compared with the efficiency of the wild type CYP76AH3 protein, and the mutant CYP76AH3E301D、CYP76AH3E301D,E306S、CYP76AH3E301D,M395IAnd CYP76AH3E301D,F479VIsocatalytic production of tanshinone dienesThe yield of the raw cryptomerin is obviously improved by 10.77, 19.98, 11.78 and 15.27 times compared with the efficiency of the wild-type CYP76AH3 protein, and the mutant CYP76AH3M395IAnd CYP76AH3E306S,M395IThe efficiency of catalyzing the hypotanshinone diene to generate the 11-hydroxy cedrol is obviously improved by 2.78 and 2.39 times compared with the efficiency of the wild type CYP76AH3 protein.
Sequence listing
<110> institute of traditional Chinese medicine of Chinese academy of traditional Chinese medicine
<120> a salvia miltiorrhiza P450 mutant for preparing tanshinone compounds
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1488
<212> DNA
<213> Salvia miltiorrhiza Bunge (Salvia militirhiza Bunge)
<400> 1
atggattctt ttcctctcct cgccgcgctc ttcttcatcg ccgcgacgat aacattcctc 60
tccttccggc gccggaggaa cctccctccg gggccgttcc cctacccgat cgtcggaaac 120
atgctgcaac tcggcgccaa ccctcaccag gtgttcgcga agctctcaaa gagatacggc 180
ccgctgatgt cgatccatct cggcagcctc tacaccgtga tcgtctcctc cccggagatg 240
gcgaaggaga tcctccacag gcacgggcag gtcttctccg gccgcaccat cgcgcaggcg 300
gtgcacgcgt gcgaccacga caagatctcc atggggttcc tccccgtggc cagcgagtgg 360
cgcgacatgc gcaagatatg caaggagcag atgttctcca accagagcat ggaggccagc 420
cagggcctcc gccgccagaa gctgcagcag ctcctcgacc acgtccagaa atgctccgac 480
agcggccgcg cggtcgacat ccgcgaggcc gccttcatca ccacgctcaa cctcatgtcg 540
gccacgctct tctcctcgca ggccaccgag ttcgactcca aggccaccat ggagttcaag 600
gagatcatcg agggcgtcgc caccatcgtc ggcgtgccca acttcgccga ctacttcccc 660
atcctccgcc ccttcgaccc gcagggtgtc aagcgcaggg cggatgtttt cttcggcaaa 720
ttgctcgcca aaatcgaagg gtatctcaat gagaggctcg aatcgaagag ggccaatccc 780
aatgcgccaa agaaggatga ctttttggag atagtggttg atattattca ggccaacgag 840
ttcaagttga agacgcatca cttcacccat ctcatgctgg acttgttcgt gggaggatcg 900
gacacgaaca cgacctcgat cgagtgggcc atgtcggagc tagtgatgaa cccggacaag 960
atggctcgac tgaaggcgga gctgaagagc gtggccggag acgagaagat cgtggatgag 1020
tcggcaatgc cgaagctgcc gtacctgcaa gccgtgataa aggaggtgat gcggatccac 1080
ccgccgggcc cactgctcct ccctcgcaaa gcggagagcg atcaggaggt gaacggctat 1140
ctcatcccca agggaactca gatactcatc aacgcgtatg cgatagggag ggatccgagt 1200
atctggaccg acccggagac gttcgacccg gaacgcttcc tcgataacaa gatagacttc 1260
aagggccagg attacgagct ccttccgttc gggtcgggta gacgggtctg ccccggcatg 1320
ccgctcgcga cccggatcct gcacatggct actgcgactc tggttcataa cttcgactgg 1380
aaactggaag atgactccac ggcggccgcc gatcacgccg gcgagttgtt tggggtggcc 1440
gtgcgcaggg cagttccgct taggattatc ccaatagtta agtcttag 1488
<210> 2
<211> 495
<212> PRT
<213> Salvia miltiorrhiza Bunge (Salvia militirhiza Bunge)
<400> 2
Met Asp Ser Phe Pro Leu Leu Ala Ala Leu Phe Phe Ile Ala Ala Thr
1 5 10 15
Ile Thr Phe Leu Ser Phe Arg Arg Arg Arg Asn Leu Pro Pro Gly Pro
20 25 30
Phe Pro Tyr Pro Ile Val Gly Asn Met Leu Gln Leu Gly Ala Asn Pro
35 40 45
His Gln Val Phe Ala Lys Leu Ser Lys Arg Tyr Gly Pro Leu Met Ser
50 55 60
Ile His Leu Gly Ser Leu Tyr Thr Val Ile Val Ser Ser Pro Glu Met
65 70 75 80
Ala Lys Glu Ile Leu His Arg His Gly Gln Val Phe Ser Gly Arg Thr
85 90 95
Ile Ala Gln Ala Val His Ala Cys Asp His Asp Lys Ile Ser Met Gly
100 105 110
Phe Leu Pro Val Ala Ser Glu Trp Arg Asp Met Arg Lys Ile Cys Lys
115 120 125
Glu Gln Met Phe Ser Asn Gln Ser Met Glu Ala Ser Gln Gly Leu Arg
130 135 140
Arg Gln Lys Leu Gln Gln Leu Leu Asp His Val Gln Lys Cys Ser Asp
145 150 155 160
Ser Gly Arg Ala Val Asp Ile Arg Glu Ala Ala Phe Ile Thr Thr Leu
165 170 175
Asn Leu Met Ser Ala Thr Leu Phe Ser Ser Gln Ala Thr Glu Phe Asp
180 185 190
Ser Lys Ala Thr Met Glu Phe Lys Glu Ile Ile Glu Gly Val Ala Thr
195 200 205
Ile Val Gly Val Pro Asn Phe Ala Asp Tyr Phe Pro Ile Leu Arg Pro
210 215 220
Phe Asp Pro Gln Gly Val Lys Arg Arg Ala Asp Val Phe Phe Gly Lys
225 230 235 240
Leu Leu Ala Lys Ile Glu Gly Tyr Leu Asn Glu Arg Leu Glu Ser Lys
245 250 255
Arg Ala Asn Pro Asn Ala Pro Lys Lys Asp Asp Phe Leu Glu Ile Val
260 265 270
Val Asp Ile Ile Gln Ala Asn Glu Phe Lys Leu Lys Thr His His Phe
275 280 285
Thr His Leu Met Leu Asp Leu Phe Val Gly Gly Ser Asp Thr Asn Thr
290 295 300
Thr Ser Ile Glu Trp Ala Met Ser Glu Leu Val Met Asn Pro Asp Lys
305 310 315 320
Met Ala Arg Leu Lys Ala Glu Leu Lys Ser Val Ala Gly Asp Glu Lys
325 330 335
Ile Val Asp Glu Ser Ala Met Pro Lys Leu Pro Tyr Leu Gln Ala Val
340 345 350
Ile Lys Glu Val Met Arg Ile His Pro Pro Gly Pro Leu Leu Leu Pro
355 360 365
Arg Lys Ala Glu Ser Asp Gln Glu Val Asn Gly Tyr Leu Ile Pro Lys
370 375 380
Gly Thr Gln Ile Leu Ile Asn Ala Tyr Ala Ile Gly Arg Asp Pro Ser
385 390 395 400
Ile Trp Thr Asp Pro Glu Thr Phe Asp Pro Glu Arg Phe Leu Asp Asn
405 410 415
Lys Ile Asp Phe Lys Gly Gln Asp Tyr Glu Leu Leu Pro Phe Gly Ser
420 425 430
Gly Arg Arg Val Cys Pro Gly Met Pro Leu Ala Thr Arg Ile Leu His
435 440 445
Met Ala Thr Ala Thr Leu Val His Asn Phe Asp Trp Lys Leu Glu Asp
450 455 460
Asp Ser Thr Ala Ala Ala Asp His Ala Gly Glu Leu Phe Gly Val Ala
465 470 475 480
Val Arg Arg Ala Val Pro Leu Arg Ile Ile Pro Ile Val Lys Ser
485 490 495
<210> 3
<211> 1485
<212> DNA
<213> Salvia miltiorrhiza Bunge (Salvia militirhiza Bunge)
<400> 3
atggattctt tctctcttct ggctgcactc tttttcatca gcgccgcaac atggtttatc 60
tcctcccggc ggcggaggaa cctcccaccg gggccattcc cgtatccgat cgtcggaaac 120
atgctgcagc tgggggccca accccacgag acattcgcca aactgtcaaa gaaatacggc 180
ccgctgatgt cggtccatct cggcagcctg tacaccgtga tcgtgtcctc gccggagatg 240
gcgaaagaga tcatgctgaa atacgggacg gtgttctccg gcagaacggt ggcgcaggcg 300
gtgcacgcgt gcgaccacga caagatctcg atggggttcc tcccgatcgg ggcggagtgg 360
cgcgacatgc gcaagatatg caaagagcag atgttctcgc accagagcat ggaagacagc 420
cagggcctcc gcaagcagaa gctgcagcag ctgctcgacc acgcccacag atgctccgag 480
cagggccgcg ccatcgacat ccgcgaggcc gccttcatca ccaccctcaa cctcatgtcc 540
gccaccctct tctccatgca ggccaccgag ttcgactcca aggtcaccat ggagttcaag 600
gagatcatcg agggcgtcgc cagcatcgtc ggcgtcccca acttcgccga ctacttcccc 660
atcctccgcc ccttcgaccc gcagggggtc aagcgcaggg ccgacgtcta cttcggcaga 720
ctgctggctc taatcgaggg ctatctcaac gacagaatcc aatccagaaa ggccaacccc 780
gacgccccca agaaggatga cttcctcgag acgctcgtcg atattctcaa ctccaacgac 840
aacaagctca agaccgatca cctcctgcat ctcatgctcg acctcttcgt cgggggttcg 900
gagacgagca ccaccgagat cgagtggatc atggaggagc tcgtggcgca cccggacaag 960
atggccaagg tgaaggcgga gctgaagagc gtgatggggg atgagaaggt ggtggacgag 1020
tcgctcatgc ccaggctgcc gtatctgcaa gcggtcgtca aggaatccat gcggctgcac 1080
ccgccgggcc cactccttct tcctcgcaag gcggagagcg atcaagtcgt caacggctac 1140
ctcatcccga aagggactca ggtgctcatc aacgcgtggg ccatgggcag agactccacc 1200
atctggaaca atccagacgc cttccaaccc gaacgcttcc tcgataacaa gatcgacttc 1260
aaaggccaag attacgagct cattcccttc gggtcgggtc ggcgggtctg ccccggtatg 1320
cccctcgcca accgcatgct gcacaccgtc accgccacgc tcgttcacaa cttcgattgg 1380
aagctcgaac gccccgatgc gcccctcgcc gagcaccagg gcgtgttgtt tggcttcgcc 1440
gtgcgcaggg ctgtgccgct caggatcgtt ccgtataagg catga 1485
<210> 4
<211> 494
<212> PRT
<213> Salvia miltiorrhiza Bunge (Salvia militirhiza Bunge)
<400> 4
Met Asp Ser Phe Ser Leu Leu Ala Ala Leu Phe Phe Ile Ser Ala Ala
1 5 10 15
Thr Trp Phe Ile Ser Ser Arg Arg Arg Arg Asn Leu Pro Pro Gly Pro
20 25 30
Phe Pro Tyr Pro Ile Val Gly Asn Met Leu Gln Leu Gly Ala Gln Pro
35 40 45
His Glu Thr Phe Ala Lys Leu Ser Lys Lys Tyr Gly Pro Leu Met Ser
50 55 60
Val His Leu Gly Ser Leu Tyr Thr Val Ile Val Ser Ser Pro Glu Met
65 70 75 80
Ala Lys Glu Ile Met Leu Lys Tyr Gly Thr Val Phe Ser Gly Arg Thr
85 90 95
Val Ala Gln Ala Val His Ala Cys Asp His Asp Lys Ile Ser Met Gly
100 105 110
Phe Leu Pro Ile Gly Ala Glu Trp Arg Asp Met Arg Lys Ile Cys Lys
115 120 125
Glu Gln Met Phe Ser His Gln Ser Met Glu Asp Ser Gln Gly Leu Arg
130 135 140
Lys Gln Lys Leu Gln Gln Leu Leu Asp His Ala His Arg Cys Ser Glu
145 150 155 160
Gln Gly Arg Ala Ile Asp Ile Arg Glu Ala Ala Phe Ile Thr Thr Leu
165 170 175
Asn Leu Met Ser Ala Thr Leu Phe Ser Met Gln Ala Thr Glu Phe Asp
180 185 190
Ser Lys Val Thr Met Glu Phe Lys Glu Ile Ile Glu Gly Val Ala Ser
195 200 205
Ile Val Gly Val Pro Asn Phe Ala Asp Tyr Phe Pro Ile Leu Arg Pro
210 215 220
Phe Asp Pro Gln Gly Val Lys Arg Arg Ala Asp Val Tyr Phe Gly Arg
225 230 235 240
Leu Leu Ala Leu Ile Glu Gly Tyr Leu Asn Asp Arg Ile Gln Ser Arg
245 250 255
Lys Ala Asn Pro Asp Ala Pro Lys Lys Asp Asp Phe Leu Glu Thr Leu
260 265 270
Val Asp Ile Leu Asn Ser Asn Asp Asn Lys Leu Lys Thr Asp His Leu
275 280 285
Leu His Leu Met Leu Asp Leu Phe Val Gly Gly Ser Glu Thr Ser Thr
290 295 300
Thr Glu Ile Glu Trp Ile Met Glu Glu Leu Val Ala His Pro Asp Lys
305 310 315 320
Met Ala Lys Val Lys Ala Glu Leu Lys Ser Val Met Gly Asp Glu Lys
325 330 335
Val Val Asp Glu Ser Leu Met Pro Arg Leu Pro Tyr Leu Gln Ala Val
340 345 350
Val Lys Glu Ser Met Arg Leu His Pro Pro Gly Pro Leu Leu Leu Pro
355 360 365
Arg Lys Ala Glu Ser Asp Gln Val Val Asn Gly Tyr Leu Ile Pro Lys
370 375 380
Gly Thr Gln Val Leu Ile Asn Ala Trp Ala Met Gly Arg Asp Ser Thr
385 390 395 400
Ile Trp Asn Asn Pro Asp Ala Phe Gln Pro Glu Arg Phe Leu Asp Asn
405 410 415
Lys Ile Asp Phe Lys Gly Gln Asp Tyr Glu Leu Ile Pro Phe Gly Ser
420 425 430
Gly Arg Arg Val Cys Pro Gly Met Pro Leu Ala Asn Arg Met Leu His
435 440 445
Thr Val Thr Ala Thr Leu Val His Asn Phe Asp Trp Lys Leu Glu Arg
450 455 460
Pro Asp Ala Pro Leu Ala Glu His Gln Gly Val Leu Phe Gly Phe Ala
465 470 475 480
Val Arg Arg Ala Val Pro Leu Arg Ile Val Pro Tyr Lys Ala
485 490
<210> 5
<211> 6706
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240
gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300
ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360
tttttttttt cccctagcgg atgactcttt ttttttctta gcgattggca ttatcacata 420
atgaattata cattatataa agtaatgtga tttcttcgaa gaatatacta aaaaatgagc 480
aggcaagata aacgaaggca aagatgacag agcagaaagc cctagtaaag cgtattacaa 540
atgaaaccaa gattcagatt gcgatctctt taaagggtgg tcccctagcg atagagcact 600
cgatcttccc agaaaaagag gcagaagcag tagcagaaca ggccacacaa tcgcaagtga 660
ttaacgtcca cacaggtata gggtttctgg accatatgat acatgctctg gccaagcatt 720
ccggctggtc gctaatcgtt gagtgcattg gtgacttaca catagacgac catcacacca 780
ctgaagactg cgggattgct ctcggtcaag cttttaaaga ggccctactg gcgcgtggag 840
taaaaaggtt tggatcagga tttgcgcctt tggatgaggc actttccaga gcggtggtag 900
atctttcgaa caggccgtac gcagttgtcg aacttggttt gcaaagggag aaagtaggag 960
atctctcttg cgagatgatc ccgcattttc ttgaaagctt tgcagaggct agcagaatta 1020
ccctccacgt tgattgtctg cgaggcaaga atgatcatca ccgtagtgag agtgcgttca 1080
aggctcttgc ggttgccata agagaagcca cctcgcccaa tggtaccaac gatgttccct 1140
ccaccaaagg tgttcttatg tagtgacacc gattatttaa agctgcagca tacgatatat 1200
atacatgtgt atatatgtat acctatgaat gtcagtaagt atgtatacga acagtatgat 1260
actgaagatg acaaggtaat gcatcattct atacgtgtca ttctgaacga ggcgcgcttt 1320
ccttttttct ttttgctttt tctttttttt tctcttgaac tcgacggatc tatgcggtgt 1380
gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata 1440
ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg 1500
aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc 1560
cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa 1620
ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt 1680
cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac 1740
ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta 1800
gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 1860
cgccgctaca gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc 1920
gatcggtgcg ggcctcttcg ctattacgcc agctgaattg gagcgacctc atgctatacc 1980
tgagaaagca acctgaccta caggaaagag ttactcaaga ataagaattt tcgttttaaa 2040
acctaagagt cactttaaaa tttgtataca cttatttttt ttataactta tttaataata 2100
aaaatcataa atcataagaa attcgcttat ttagaagtgt caacaacgta tctaccaacg 2160
atttgaccct tttccatctt ttcgtaaatt tctggcaagg tagacaagcc gacaaccttg 2220
attggagact tgaccaaacc tctggcgaag aattgttaat taagagctca gatcttatcg 2280
tcgtcatcct tgtaatccat cgatactagt gcggccgccc tttagtgagg gttgaattcg 2340
aattttcaaa aattcttact ttttttttgg atggacgcaa agaagtttaa taatcatatt 2400
acatggcatt accaccatat acatatccat atacatatcc atatctaatc ttacttatat 2460
gttgtggaaa tgtaaagagc cccattatct tagcctaaaa aaaccttctc tttggaactt 2520
tcagtaatac gcttaactgc tcattgctat attgaagtac ggattagaag ccgccgagcg 2580
ggtgacagcc ctccgaagga agactctcct ccgtgcgtcc tcgtcttcac cggtcgcgtt 2640
cctgaaacgc agatgtgcct cgcgccgcac tgctccgaac aataaagatt ctacaatact 2700
agcttttatg gttatgaaga ggaaaaattg gcagtaacct ggccccacaa accttcaaat 2760
gaacgaatca aattaacaac cataggatga taatgcgatt agttttttag ccttatttct 2820
ggggtaatta atcagcgaag cgatgatttt tgatctatta acagatatat aaatgcaaaa 2880
actgcataac cactttaact aatactttca acattttcgg tttgtattac ttcttattca 2940
aatgtaataa aagtatcaac aaaaaattgt taatatacct ctatacttta acgtcaagga 3000
gaaaaaaccc cggatccgta atacgactca ctatagggcc cgggcgtcga catggaacag 3060
aagttgattt ccgaagaaga cctcgagtaa gcttggtacc gcggctagct aagatccgct 3120
ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata 3180
gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga 3240
cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg 3300
aagatccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 3360
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 3420
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 3480
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 3540
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 3600
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 3660
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 3720
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 3780
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 3840
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 3900
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 3960
tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 4020
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4080
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 4140
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4200
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 4260
tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 4320
agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 4380
gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 4440
ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 4500
gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 4560
cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 4620
acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 4680
cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 4740
cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 4800
ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 4860
tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 4920
atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 4980
tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 5040
actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 5100
aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 5160
ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 5220
ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 5280
cgaaaagtgc cacctgaacg aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc 5340
gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa 5400
cgcgaaagcg ctattttacc aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc 5460
aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa 5520
tgcaacgcga gagcgctatt ttaccaacaa agaatctata cttctttttt gttctacaaa 5580
aatgcatccc gagagcgcta tttttctaac aaagcatctt agattacttt ttttctcctt 5640
tgtgcgctct ataatgcagt ctcttgataa ctttttgcac tgtaggtccg ttaaggttag 5700
aagaaggcta ctttggtgtc tattttctct tccataaaaa aagcctgact ccacttcccg 5760
cgtttactga ttactagcga agctgcgggt gcattttttc aagataaagg catccccgat 5820
tatattctat accgatgtgg attgcgcata ctttgtgaac agaaagtgat agcgttgatg 5880
attcttcatt ggtcagaaaa ttatgaacgg tttcttctat tttgtctcta tatactacgt 5940
ataggaaatg tttacatttt cgtattgttt tcgattcact ctatgaatag ttcttactac 6000
aatttttttg tctaaagagt aatactagag ataaacataa aaaatgtaga ggtcgagttt 6060
agatgcaagt tcaaggagcg aaaggtggat gggtaggtta tatagggata tagcacagag 6120
atatatagca aagagatact tttgagcaat gtttgtggaa gcggtattcg caatatttta 6180
gtagctcgtt acagtccggt gcgtttttgg ttttttgaaa gtgcgtcttc agagcgcttt 6240
tggttttcaa aagcgctctg aagttcctat actttctaga gaataggaac ttcggaatag 6300
gaacttcaaa gcgtttccga aaacgagcgc ttccgaaaat gcaacgcgag ctgcgcacat 6360
acagctcact gttcacgtcg cacctatatc tgcgtgttgc ctgtatatat atatacatga 6420
gaagaacggc atagtgcgtg tttatgctta aatgcgtact tatatgcgtc tatttatgta 6480
ggatgaaagg tagtctagta cctcctgtga tattatccca ttccatgcgg ggtatcgtat 6540
gcttccttca gcactaccct ttagctgttc tatatgctgc cactcctcaa ttggattagt 6600
ctcatccttc aatgctatca tttcctttga tattggatca tctaagaaac cattattatc 6660
atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 6706

Claims (12)

1. A protein which is any one of the following (a1) - (a 6):
(a1) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 301 th amino acid residue is mutated from E to D, and the 306 th amino acid residue is mutated from E to S;
(a2) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 301 th amino acid residue is mutated from E to D, and the 479 th amino acid residue is mutated from F to V;
(a3) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 301 th amino acid residue is mutated from E to D, and the 395 th amino acid residue is mutated from M to I;
(a4) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 395 th amino acid residue is mutated from M to I;
(a5) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 306 th amino acid residue is mutated from E to S, and the 395 th amino acid residue is mutated from M to I;
(a6) the protein obtained by performing the following point mutation on the protein shown in the sequence 4 in the sequence table: the 301 th amino acid residue is mutated from E to D.
2. A gene encoding the protein of claim 1.
3. A recombinant expression vector, expression cassette or recombinant bacterium comprising the gene of claim 2.
4. The protein or the coding gene thereof is applied to the catalytic generation of the rust alcohol by taking the sub-tanshinone diene as a substrate; the protein of claim 1, wherein the protein is the protein of (a1), (a2), (a3) or (a 6).
5. The protein or the coding gene thereof is applied to catalyzing and generating the cryptomerin by taking the sub-tanshinone diene as a substrate; the protein of claim 1, which is the protein described in (a1), (a2), (a3), (a4), (a5), or (a 6).
6. The protein or the coding gene thereof is applied to the catalytic generation of 11-hydroxyl rust alcohol by taking the sub-tanshinone diene as a substrate; the protein of claim 1 (a4) or (a 5).
7. The protein or the coding gene thereof is applied to the catalytic generation of 11-hydroxycedrol by using the sub-tanshinone diene as a substrate; the protein of claim 1, wherein the protein is the protein of (a3), (a4) or (a 5).
8. The method for producing the protein according to claim 1, comprising the steps of: culturing the recombinant bacterium according to claim 3 to obtain the protein from the recombinant bacterium.
9. A method of preparing rust alcohol comprising the steps of: using sub-tanshinone diene as a substrate, and carrying out catalytic reaction by using the protein as shown in (a1), (a2), (a3) or (a6) in claim 1 to obtain the iron rust alcohol.
10. The method for preparing the cryptomerin comprises the following steps: carrying out catalytic reaction by using sub-tanshinone diene as a substrate and adopting the protein as shown in (a1), (a2), (a3), (a4), (a5) or (a6) in claim 1 to obtain the cryptomeril.
11. A method of preparing 11-hydroxyrust alcohol comprising the steps of: using sub-tanshinone diene as substrate, adopting the protein of (a4) or (a5) in claim 1 to perform catalytic reaction, and obtaining 11-hydroxyl rust alcohol.
12. The method for preparing 11-hydroxycedrol comprises the following steps: using sub-tanshinone diene as substrate, and adopting the protein described in (a3), (a4) or (a5) in claim 1 to carry out catalytic reaction to obtain 11-hydroxy cedrol.
CN202010041299.0A 2020-01-15 2020-01-15 Salvia miltiorrhiza P450 mutant for preparing tanshinone compounds Active CN113122512B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010041299.0A CN113122512B (en) 2020-01-15 2020-01-15 Salvia miltiorrhiza P450 mutant for preparing tanshinone compounds

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010041299.0A CN113122512B (en) 2020-01-15 2020-01-15 Salvia miltiorrhiza P450 mutant for preparing tanshinone compounds

Publications (2)

Publication Number Publication Date
CN113122512A CN113122512A (en) 2021-07-16
CN113122512B true CN113122512B (en) 2022-05-17

Family

ID=76771325

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010041299.0A Active CN113122512B (en) 2020-01-15 2020-01-15 Salvia miltiorrhiza P450 mutant for preparing tanshinone compounds

Country Status (1)

Country Link
CN (1) CN113122512B (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103695441B (en) * 2013-10-24 2019-07-26 中国中医科学院中药研究所 One CYP450 gene for participating in tanshinone biosynthesis and its coded product and application
CN106148360B (en) * 2015-03-19 2020-03-03 中国中医科学院中药研究所 Key CYP450 gene for catalyzing C20 site methyl hydroxylation in biosynthetic pathway of tanshinone compounds
CN113122513B (en) * 2020-01-15 2022-05-17 中国中医科学院中药研究所 Salvia miltiorrhiza P450 mutant and application thereof

Also Published As

Publication number Publication date
CN113122512A (en) 2021-07-16

Similar Documents

Publication Publication Date Title
CN109370966B (en) Genetically engineered bacterium for producing L-theanine and fermentation method thereof
CN104342411B (en) The Ketoreductase mutant of increased activity, coded sequence and preparation method thereof
CN104342410B (en) Ketone reductase mutant and preparation method thereof
CN104342406B (en) Enhanced formic dehydrogenase mutant of heat stability and preparation method thereof
KR20110063576A (en) Increased heterologous fe-s enzyme actiivty in yeast
CN113584134B (en) Isothermal nucleic acid detection system based on CRISPR-Cas9, and method and application thereof
CN104342412B (en) For producing the Ketoreductase mutant of (S) -4- chloro-3-hydroxyl ethyl butyrate
CN104694452B (en) A kind of recombined bacillus subtilis and its construction method of high yield Pullulanase
CN113122513B (en) Salvia miltiorrhiza P450 mutant and application thereof
CN113302305A (en) Methods of increasing productivity of filamentous fungal cells in the production of polypeptides
CN116745418A (en) Compositions and methods for RNA-encoded DNA replacement of alleles
CN113122512B (en) Salvia miltiorrhiza P450 mutant for preparing tanshinone compounds
CN115161251B (en) Polygene mutant of rhizobium HH103 and application thereof
CN113755518B (en) Method for constructing recombinant yarrowia lipolytica and application thereof
CN113846019B (en) Marine nannochloropsis targeted epigenomic genetic control method
CN111635907B (en) Method for constructing astaxanthin-producing strain
KR20130078265A (en) Infectious cdna clones of foot-and-mouth disease virus of type o and the complete sequences of the clones
CN101492685A (en) Gene sequence of recombinant expression vector and construction method thereof
CN114085868A (en) Targeting vector, recombinant Huh7 cell line, construction method and application
CN110431232B (en) Reagents for extracting and amplifying nucleic acids
CN109182241B (en) Engineering bacterium for expressing epoxide hydrolase and construction method and application thereof
CN108517326A (en) A method of preparing Ribavirin using Permeabilized cells
KR102422842B1 (en) Compositon for regulating translation of RNA using CRISPRi
CN107475279B (en) Construction method and application of expression T vector of Vip gene of Bacillus thuringiensis
RU2761637C1 (en) ESCHERICHIA coli BL21(DE3)/ pET32 v 11-Cre CELL STRAIN PRODUCING SITE-SPECIFIC Cre RECOMBINASE

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant