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A Salvia miltiorrhiza p450 mutant used for preparing tanshinone compounds

A technology of hypotanshinone diene and point mutation, which is applied in the direction of introducing foreign genetic material, application, fungi, etc. by using a carrier, can solve the problems of production limitation, reduction of amorphadiene production, and reduction of catalytic activity, and achieve an increase in production , Improving substrate utilization efficiency and optimizing catalytic efficiency

Active Publication Date: 2022-05-17
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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Problems solved by technology

For example, in the synthetic biology of artemisinic acid, there is a problem that the catalytic efficiency of P450 limits the yield of the final product. After the expression of CYP71AV1 and CPR1, the yield of the cultured product of artemisinic acid production engineering yeast Y285 is higher than that of amorphadiene in the previous step. The production of
Paddon et al suggested that the reduced catalytic activity and reduced yield of sesquiterpene products in Y285 may be caused by the inefficiency of cytochrome P450 that catalyzes the oxidation of amorphadiene

Method used

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  • A Salvia miltiorrhiza p450 mutant used for preparing tanshinone compounds
  • A Salvia miltiorrhiza p450 mutant used for preparing tanshinone compounds
  • A Salvia miltiorrhiza p450 mutant used for preparing tanshinone compounds

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Experimental program
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Effect test

Embodiment 1

[0093] Embodiment 1, the acquisition of mutant

[0094] CYP76AH1 and CYP76AH3 proteins are P450 proteins involved in tanshinone biosynthesis. catalytic pathway see figure 1 . figure 1 In , each protein can catalyze the substrate before the arrow, but with different efficiencies.

[0095] The amino acid sequence of CYP76AH1 is shown in sequence 2 of the sequence listing, and its coding gene (CYP76AH1) is shown in sequence 1 of the sequence listing.

[0096] The amino acid sequence of CYP76AH3 is shown in sequence 4 of the sequence listing, and its coding gene (CYP76AH3) is shown in sequence 3 of the sequence listing.

[0097] Sequence analysis, mutation and functional verification of CYP76AH3 revealed four active sites related to the catalytic efficiency of CYP76AH1, which were amino acid residues 301, 306, 395, and 479, respectively. These 4 amino acid sites were mutated in different forms (Table 1), and a total of 15 CYP76AH1 protein mutants were obtained (Table 2).

[0...

Embodiment 2

[0104] Embodiment 2, preparation of recombinant expression vector

[0105] 1. Construction of wild-type recombinant expression vector

[0106] 1. Insert the double-stranded DNA molecule shown in sequence 1 into the BamHI site of the expression vector pESC-His (circular plasmid shown in sequence 5 of the sequence listing) to obtain the recombinant expression vector pESC-His-H1WT (sequencing verified correctly). The DNA molecule shown in sequence 1 encodes the protein shown in sequence 2.

[0107] 2. Insert the double-stranded DNA molecule shown in Sequence 3 between the BamHI sites of the expression vector pESC-His to obtain the recombinant expression vector pESC-His-H3WT (sequence verification is correct). The DNA molecule shown in sequence 3 encodes the protein shown in sequence 4.

[0108] 2. Construction of mutant recombinant expression vector

[0109] 1. Insert the double-stranded DNA molecule 1 between the BamHI sites of the expression vector pESC-His to obtain the re...

Embodiment 3

[0124] Example 3, preparation of recombinant bacteria and extraction of microsomal protein

[0125] 1. The wild-type recombinant expression vector, mutant recombinant expression vector and expression vector pESC-His prepared in Example 2 were respectively introduced into the yeast expression strain BY4741 integrating the Arabidopsis P450 reductase ATR1 to obtain wild-type recombinant bacteria, 15 species Mutant recombinant bacteria and empty vector recombinant bacteria.

[0126] 2. The wild-type recombinant bacteria obtained in step 1 and 15 kinds of mutant recombinant bacteria were inoculated respectively in the defective SD-His liquid medium for yeast transformation (Ubiquino, SD-His liquid medium formula: 8g / LSD-His powder , 20g / L glucose, sterilized for 15min), cultured at 30°C and 200 rpm for 48 hours, centrifuged at 5000 rpm for 5min to collect the bacteria, and used an equal volume of YPL liquid medium (10g / L yeast extract, 20g / L peptone, 20g / L galactose) after resusp...

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Abstract

The invention discloses a salvia miltiorrhiza P450 mutant used for preparing tanshinone compounds. The present invention optimizes its catalytic efficiency by directional modification of CYP76AH3, so that the reaction path that originally requires the synergistic effect of the two proteins CYP76AH1 and CYP76AH3 can be directly catalyzed by the CYP76AH3 mutant in the present invention, and the construction of the CYP76AH3 mutant can shorten the reaction time In the process, the substrate transport path can efficiently complete multi-step reactions in one protein pocket, thereby improving substrate utilization efficiency and increasing the yield of target compounds. In the present invention, the CYP76AH3 mutant with improved conversion efficiency for different products provides substrates and chassis bacteria for the subsequent analysis of tanshinone biosynthesis pathways, and has important guiding significance for the full analysis of tanshinone biosynthesis pathways and the construction of yeast engineering bacteria with high production of tanshinone compounds.

Description

technical field [0001] The invention relates to a salvia miltiorrhiza P450 mutant used for preparing tanshinone compounds. Background technique [0002] In the biocatalysis and synthetic biology research of medicinal natural products, the catalytic efficiency of P450 has always been a difficult problem in the international synthetic biology research. For example, in the synthetic biology of artemisinic acid, there is a problem that the catalytic efficiency of P450 limits the yield of the final product. After the expression of CYP71AV1 and CPR1, the yield of the cultured product of artemisinic acid production engineering yeast Y285 is higher than that of amorphadiene in the previous step. production was significantly reduced. Paddon et al. believe that the reduced catalytic activity and reduced yield of sesquiterpene products in Y285 may be caused by the low efficiency of cytochrome P450 that catalyzes the oxidation of amorphadiene. [0003] There are multiple P450s in the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19C12P15/00
CPCC12N9/0042C12Y106/02004C12N15/81C12P15/00
Inventor 郭娟毛亚平马莹曾雯陈同黄璐琦
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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