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Genetically engineered bacterium and application thereof to preparation of 9alpha,22-dihydroxy-23,24-bisnorcholest-4-ene-3-ketone

A technology of genetically engineered bacteria and steroids, applied in the field of genetic engineering, can solve the problems of difficulty in separation and purification and high production costs, and achieve the effects of reducing pollution, improving production efficiency and product quality, and reducing production costs.

Active Publication Date: 2020-12-18
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on the preparation of 9α, 22-dihydroxy-23, 24-bisnorcholest-4-en-3-one by biological methods. The research of Wang Fengqing et al. (patent: CN201910510202.3, reference "Xu L Q , Liu Y J, Yao K, et al. Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism, Scientific Repots, 2016, 6: 21928"), Knock out the ester acyl Coenzyme A thiolase gene, which can be used to produce 9ɑ,22-dihydroxy-23,24-dinorcholesta-4-dien-3-one, using a large number of resting cells to convert phytosterols, the substrate feeding concentration is 40 g / l, conversion 144 h, the substrate conversion rate is only 50%, the main product obtained is 9ɑ,22-dihydroxy-23,24-bisnorcholesta-4-dien-3-one, and the molar yield is 30%, the reaction also contains other by-products, resulting in difficulties in separation and purification and high production costs

Method used

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  • Genetically engineered bacterium and application thereof to preparation of 9alpha,22-dihydroxy-23,24-bisnorcholest-4-ene-3-ketone
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  • Genetically engineered bacterium and application thereof to preparation of 9alpha,22-dihydroxy-23,24-bisnorcholest-4-ene-3-ketone

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: Construction of gene inactivation strain

[0022] 3-sterone-∆ 1 -Dehydrogenase encoding gene ( kstd1, kstd2, kstd3 ) as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, to start the strain Mycobacteriu. smegmatis mc 2 The genome of 155 (strain number: ATCC 700084) was used as a template for PCR amplification, and the inactivation kstd1, kstd2 and kstd3 strains.

[0023] This study uses CRISPR-Cas12a-assisted recombineering (references: Yan MY, Yan HQ, Ren GX, Zhao JP, Guo XP, Sun YC. CRISPR-Cas12a-assisted recombineering in bacteria[J]. Appl Environ Microbiol , 2017, 83(17): e00947-17) to construct a knockout strain. first in kstd1 The 31-base sequence targeting the lagging strand was selected as the gene-specific crRNA, and the pCR-Hyg plasmid was used as a template to amplify with two pairs of primers, KstD1-F / R and pCR-Hyg-F / R, to obtain two The plasmid fragments were ligated and transformed and sent for sequencing, and the correct extract...

Embodiment 2

[0053] Embodiment 2: Construction of gene expression strain

[0054] 1. Expression plasmid construction

[0055] Primers cxgAB-F and cxgAB-R were used to amplify from Mycobacterium Acetyl-CoA acetyltransferase / thiolase gene (cxgA) and DNA binding protein gene (cxgB) in the sp. NRRL B-3805 genome, after obtaining the cxgAB fragment with a 15bp homology arm with the plasmid pMV261, and expressing Plasmid pMV261 was digested and purified by EcoRI and HindIII to obtain a single-fragment connection, and the recombinant expression plasmid 261-cxgAB was obtained.

[0056] PCR system:

[0057] 5×Phusion GC Buffer 10 μl

[0058] 2mM dNTPs 5 μl

[0059] Primer F 1 μl

[0060] Primer R 1 μl

[0061] Template DNA 50-100ng

[0062] DMSO 1.5 μl

[0063] Phusion 0.5 μl

[0064] wxya 2 O to 50 μl

[0065] PCR program: 3 min at 98°C; denaturation at 98°C for 10 s, annealing at 58°C for 20 s, extension at 72°C for 30 s, 30 cycles; 10 min at 72°C. The primer sequences used are:

...

Embodiment 3

[0071] Example 3: Fermentative production of 9α, 22-dihydroxy-23, 24-bisnorcholest-4-en-3-one

[0072]Seed medium: glucose 6 g / L, yeast powder 15 g / L, NaNO 3 5.4 g / L, glycerol 2 g / L, NH 4 h 2 PO 4 0.6g / L, kanamycin 0.1 mg / L, pH 7.5, sterilized at 115°C for 30 minutes.

[0073] Fermentation medium: defatted soybean powder 10 g / L, corn steep liquor 5 g / l, diammonium hydrogen phosphate 3 g / L, soybean oil 150 g / L, kanamycin 0.1 mg / L, pH 7.5, 121°C Sterilize for 30 minutes.

[0074] Fermentation culture steps:

[0075] 1. Cultivate LB plates (tryptone: 10g / L, yeast extract: 5g / L, sodium chloride: 10g / L, agar: 15 g / L) at 37°C for 72 hours to activate the strains;

[0076] 2. Inoculate from the activated plate into the seed medium, cultivate at 180 rpm and 37°C for 3 days; sample the seed liquid under aseptic conditions for sampling and microscopic examination, and inoculate without any bacteria in the microscopic examination;

[0077] 3. Inoculate 10% of the inoculum into 3...

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Abstract

The invention relates to the technical field of gene engineering, in particular to a mycobacterium genetically engineered bacterium and application thereof to preparation of 9alpha,22-dihydroxy-23,24-bisnorcholest-4-ene-3-ketone. The genetically engineered bacterium is constructed by inactivating three genes kstd 1, kstd 2 and kstd 3 of 3-sterone-delta1-dehydrogenase in mycobacteria and overexpressing genes encoding acetyl coenzyme A acetyltransferase / thiolase and DNA binding protein, and can selectively generate 9alpha,22-dihydroxy-23,24-bisnorcholest-4-ene-3-ketone, thereby reducing byproduct 9-OH-AD. The production efficiency and the product quality are greatly improved, the product is easy to separate and purify, the production cost is reduced, and meanwhile, the pollution to the environment is reduced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a mycobacterium genetically engineered bacterium and its application in the preparation of 9α, 22-dihydroxy-23, 24-bisnorcholest-4-en-3-one. Background technique [0002] Steroids are a class of compounds with a cyclopentane polyhydrophenanthrene ring structure, usually with methyl groups at C-10 and C-13 and alkyl side chains at C-17. Steroids, as a component of cell membranes, play an important role in living organisms. Some steroids also act as hormones and signaling molecules. Since the discovery of steroid drugs in the 1950s, more than 300 steroid drugs have been identified so far. Steroidal drugs have strong pharmacological effects such as anti-infection, anti-allergy, anti-virus and anti-shock. In recent years, the application range of steroid drugs in the medical field has been continuously expanded, and they are widely used in the treatment of rheumatism, ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P33/02C12R1/32
CPCC07K14/35C12N9/1029C12P33/02C12Y103/99004C12Y203/01009
Inventor 马延和李雪梅吴洽庆冯进辉朱敦明张瑞王玉徐自祥卜丹丹
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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