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Cytochrome P450 enzyme, construction for electron transfer system engineering bacteria thereof and application thereof

A technology of cytochrome and electron transfer, applied in the field of biotechnology and biotransformation, can solve the problems of weak sedative and hypnotic effects

Active Publication Date: 2017-07-18
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

L-Corydalmine (L-Corydalmine, l-CDL) is the 10-position O-demethylation product of l-THP, its analgesic activity is significantly higher than that of l-THP, and its sedative-hypnotic effect is weak

Method used

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  • Cytochrome P450 enzyme, construction for electron transfer system engineering bacteria thereof and application thereof
  • Cytochrome P450 enzyme, construction for electron transfer system engineering bacteria thereof and application thereof
  • Cytochrome P450 enzyme, construction for electron transfer system engineering bacteria thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1, Construction of CYP105D1 and its electron transfer system co-expression engineering strain

[0061] 1. Acquisition of pET22b-cyp105d1 plasmid

[0062] (1) Using the genomic DNA of Streptomyces griseus ATCC13273 as a template, high-fidelity polymerase Tks Gflex DNA polymerase, and P1 and P2 as primers for PCR amplification. The PCR program was: pre-denaturation at 94°C for 2 minutes, denaturation at 98°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 68°C for 90 seconds, and 30 cycles to obtain a 1239bp fragment containing the cytochrome P450 monooxygenase gene cyp105d1, The nucleotide sequence of this fragment is shown in SEQ ID No.1.

[0063] P1: 5'-GGAATTC CATATG ACGGAATCCACGACGGACC-3'

[0064] (The underline represents the recognition site for Nde I digestion)

[0065] P2: 5'-G GAATTC TCACCAGGCCACGGGCAGGT-3'

[0066] (The underline represents the EcoR I restriction recognition site)

[0067] (2) Nde I and EcoR I double-digest the D...

Embodiment 2

[0107] Example 2. Induced expression of co-expression CYP105D1 and its electron transfer system genetically engineered bacteria

[0108] Strain: recombinant engineered bacteria Escherichia coli BL21(DE3)(pET22b-105d1pDuet-fdx1-fdr1)

[0109] Seed medium (g / L): protein Chen 10; yeast extract 5; NaCl 10; ampicillin 50mg / mL, chloramphenicol 50mg / mL

[0110] Fermentation medium (g / l): tryptone 12; yeast extract 6; sodium chloride 8; D-sorbitol 10; D-glucose 1, the initial pH value was adjusted to 7.5.

[0111] Seed culture: transfer the strains preserved in glycerol tubes to fresh LB liquid medium, and activate at 200 rpm at 37°C for 16 hours; connect 1 ring of activated strains to LB solid medium with corresponding antibiotics, and culture at 37°C for 12 hours; Pick a single colony from the plate into a 250mL Erlenmeyer flask containing 50mL of seed medium, and culture it at 37°C for 12 hours at 200rpm as the primary seed; then transfer the primary seed liquid to another 50mL / ...

Embodiment 3

[0122] Example 3 Biotransformation Synthesis of Corydalis L-Damine by Co-expression of CYP105D1 and Genetic Engineering Bacteria of its Electron Transfer System

[0123] Strain: recombinant engineering bacteria Escherichia coli BL21 (DE3) (pET22b-105d1pDuet-fdx-fdr);

[0124] Seed medium (g / L): protein Chen 10; yeast extract 5; NaCl 10; ampicillin 50mg / mL, chloramphenicol 50mg / mL;

[0125] Fermentation medium (g / l): tryptone 12; yeast extract 6; sodium chloride 8; D-sorbitol 10; D-glucose 1, the initial pH value was adjusted to 7.5.

[0126] Seed culture: transfer the strains preserved in glycerol tubes to fresh LB liquid medium, and activate at 37°C for 16 hours at 200 rpm; connect 1 ring of activated strains to LB solid medium with corresponding antibiotics, and culture at 37°C for 12 hours; Pick a single colony from the plate into a 250mL Erlenmeyer flask containing 50mL of seed medium, and culture it at 37°C for 12 hours at 200rpm as the primary seed; then transfer the pr...

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Abstract

The invention discloses a cytochrome P450 enzyme, a construction for electron transfer system engineering bacteria thereof and an application thereof and belongs to the technical field of biotechnology. The bacterial strain is the engineering strain which utilizes streptomyces griseus (ATCC13273) endogenous ferredoxin and ferredoxin reductase as P450 enzyme CYP105D1 electron transfer systems. The bacterial strain can be utilized to realize the specific oxygen-site demethylation for L-tetrahydropalmatine and supply the feasibility for utilizing genetic engineering strain biotransformation to produce L-corydalmine. Meanwhile, ferredoxin reductases capable of supporting CYP105D1 biological catalysis disclosed by the invention all can promote the biotransformation capacity of CYP105D1 for various natural products, can remove L-tetrahydropalmatine and can realize efficient conversion of natural products, such as, warfarin and 7-ethyoxyl coumarin. An embodiment is provided for efficient biotransformation based on P450.

Description

[0001] Technical field: [0002] The invention belongs to the field of biotechnology and biotransformation, and specifically relates to the construction of an engineering strain co-expressing cytochrome P450 and its electron transfer system, and the method and application of using the engineering strain to biotransform natural products such as L-corydalis damming. [0003] Background technique: [0004] The cytochrome P450 enzyme system (abbreviated as CYP) was discovered as a protein that can combine with CO. In 1958, Klingenberg discovered this pigment protein that can combine with CO in rat liver microsomes. It is also named cytochrome P450 because of its maximum absorption value at 450nm wavelength when its reduced state combines with CO. As the largest superfamily of oxidoreductases, P450 is widely distributed in most organisms, and is currently widely used in drug metabolism research and industrial biotransformation. Because cytochrome P450 has the advantages of reducin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P17/18C12P17/06C12R1/19
CPCC07K14/21C12N9/0071C12N9/0095C12N15/70C12N2800/22C12P17/06C12P17/182C12Y118/01
Inventor 刘吉华沈辰徐涛
Owner CHINA PHARM UNIV
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