Genetically engineered bacterium and application thereof in preparation of 22-hydroxy-23, 24-bisnorchol-4-ene-3-one

A technology of genetically engineered bacteria and hydroxyl, applied in the field of genetic engineering, can solve problems such as being unsuitable for large-scale industrial production, and achieve the effects of easy separation and purification, reducing pollution and reducing production costs

Active Publication Date: 2020-12-04
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

[0009] Xu et al. reported that the mycobacteria obtained by genetic modification used a large number of resting cells to transform phytosterols. The substrate feeding concentration was 40 g / l, and the transformation took 144 hours. The substrate conversion rate was only 60%, and the main product obtained was It is 22-hydroxyl-23,24-dinorchol-4-en-3-one, the molar yield is 47%, and other by-products are included in the reaction, which is not suitable for large-scale industrial production (CN201480078679.4; 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)

Method used

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  • Genetically engineered bacterium and application thereof in preparation of 22-hydroxy-23, 24-bisnorchol-4-ene-3-one
  • Genetically engineered bacterium and application thereof in preparation of 22-hydroxy-23, 24-bisnorchol-4-ene-3-one
  • Genetically engineered bacterium and application thereof in preparation of 22-hydroxy-23, 24-bisnorchol-4-ene-3-one

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Construction of gene knockout plasmid

[0026] 3-sterone-∆ 1 -Dehydrogenase encoding gene ( kstd ) as shown in SEQ ID NO: 1, 17-hydroxy-3-oxo-4-pregnene-20-carboxy-CoA aldolase encoding gene ( ltp2 ) such as SEQ ID NO: 2, according to the following methods to construct the knockout kstd and knockout ltp2 of plasmids.

[0027] Starting strain Mycobacterium neoaurum (Strain No.: Mycobacterium neoaurum NRRLB-3805) genome was used as template for PCR amplification.

[0028] PCR system:

[0029] 5×Phusion GC Buffer 10 μl

[0030] 2mM dNTPs 5 μl

[0031] Primer F 1 μl

[0032] Primer R 1 μl

[0033] Template DNA 50-100ng

[0034] DMSO 1.5 μl

[0035] Phusion 0.5 μl

[0036] wxya 2 O to 50 μl

[0037] 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:

[0038] for kstd :

[0039] Upstream fragment primers:

[0040] ...

Embodiment 2

[0060] Example 2: Screening of knockout strains

[0061] Electrotransform the constructed gene knockout plasmid into the starting strain Mycobacterium neoaurum (Strain No.: Mycobacterium neoaurum NRRL B-3805) Competent cells, coated with Kan-resistant LB plates (tryptone: 10g / L, yeast extract: 5g / L, sodium chloride: 10g / L, Kan: 50μg / ml, agar: 1.5% ) and add IPTG and X-gal for the first screening. Pick blue single colonies to sucrose plate (tryptone: 10g / L, yeast extract: 5g / L, sucrose: 10g / L, agar: 1.5%) (add IPTG and X-gal) from it, carry out the second time filter. Pick the white colony on the sucrose plate to the liquid LB medium, culture at 30°C for about 36 hours, extract the genome, and use the Up-F and Down-R primers of the target gene for PCR verification. If the gene knockout is successful, the PCR product should be a single fragment of about 1900 bp. figure 2 shows that the successful kstd Knockout mycobacterial strains. right kstd The knockout strains w...

Embodiment 3

[0062] Embodiment 3: Construction of gene expression bacterial strain

[0063] 3.1 Expression plasmid construction

[0064] 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.

[0065] PCR system:

[0066] 5×Phusion GC Buffer 10 μl

[0067] 2mM dNTPs 5 μl

[0068] Primer F 1 μl

[0069] Primer R 1 μl

[0070] Template DNA 50-100ng

[0071] DMSO 1.5 μl

[0072] Phusion 0.5 μl

[0073] wxya 2O to 50 μl

[0074] 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 use...

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Abstract

The invention relates to the technical field of gene engineering, in particular to mycobacterium genetically engineered bacteria and application thereof in preparation of 22-hydroxy-23, 24-bisnorchol-4-ene-3-one. According to the genetically engineered strain provided by the invention, the generation of other products such as 4-AD and ADD is greatly reduced, 22-hydroxy-23, 24-bisnorchol-4-ene-3-one is selectively generated, the production efficiency and the product quality are improved, and the effect is very remarkable.

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 22-hydroxyl-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, cardio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/60C12N9/10C07K14/35C12P33/00C12R1/32
CPCC07K14/35C12N9/0008C12N9/1029C12N9/88C12P33/00C12Y102/01003C12Y203/01009
Inventor 马延和吴洽庆冯进辉朱敦明李雪梅王玉张瑞刘祥涛
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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