CN113122512B

CN113122512B - Salvia miltiorrhiza P450 mutant for preparing tanshinone compounds

Info

Publication number: CN113122512B
Application number: CN202010041299.0A
Authority: CN
Inventors: 郭娟; 毛亚平; 马莹; 曾雯; 陈同; 黄璐琦
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2020-01-15
Filing date: 2020-01-15
Publication date: 2022-05-17
Anticipated expiration: 2040-01-15
Also published as: CN113122512A

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;

(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

TABLE 2 mutant information

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

The above results indicate that the mutant CYP76AH3_E301D,E306SAnd CYP76AH3_E301D,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 CYP76AH3_M395IAnd CYP76AH3_E306S,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 CYP76AH3_E301D、CYP76AH3_E301D,E306S、CYP76AH3_E301D,M395IAnd CYP76AH3_E301D,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 CYP76AH3_M395IAnd CYP76AH3_E306S,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

1. A protein which is any one of the following (a1) - (a 6):

(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.