Method for enhancing steroid medicine precursor production by strengthening NADH dehydrogenation

A technology of precursors and steroids, applied in the field of biocatalysis, can solve the problems of low production efficiency, low bacterial activity, and long fermentation period.

Active Publication Date: 2019-05-03
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention will obtain and utilize NDH2 to construct NDH2-enhanced steroid precursor production strains to strengthen the dehydrogenation of NADH in the steroid precursor production strains and solve the fermentation problem caused by excessive accumulati

Method used

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  • Method for enhancing steroid medicine precursor production by strengthening NADH dehydrogenation
  • Method for enhancing steroid medicine precursor production by strengthening NADH dehydrogenation
  • Method for enhancing steroid medicine precursor production by strengthening NADH dehydrogenation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Obtaining of NDH2 gene (ndh)

[0035] The preservation number is mycobacterium fortuitous strain ARL-91 of CGMCC NO.16771 inoculated in the basal medium containing phytosterol (glucose 10g / L, MgSO 4 0.5g / L, K 2 HPO 4 0.5g / L, (NH 4 ) 2 HPO 4 3.5g / L, citric acid 2g / L, ferric ammonium citrate 0.05g / L, phytosterol 5g / L) cultivated for 60 hours to obtain samples for transcriptome sequencing. Align the transcript sequence of the obtained gene to the protein database: nr and KEGG (evalue<0.00001), and obtain the protein with the highest sequence similarity to the transcript of the given gene, so as to obtain the protein corresponding to the transcript of the gene Function annotation information. Searching for NADH dehydrogenase from the annotation information obtained multiple genes annotated as NADH dehydrogenase, and determined that it had the highest homology with Mycobacterium neoaurum VKM Ac-1815D, and identified it as the NDH2 coding gene. Primers ndh...

Embodiment 2

[0039] Example 2 Construction of NDH2-encoding gene self-overexpression genetic engineering expression vector

[0040] Constructing the genetic engineering expression vector for self-overexpression of NDH2 gene, the process includes:

[0041] The NDH2 gene obtained in Example 1 was recovered by double digestion with BamHI and HindIII, and ligated with the pMV261 plasmid that had been digested with the same enzyme at 16°C overnight under the action of T4 DNA ligase, and the ligated product was transformed into Escherichia by chemical transformation. coli DH5α, positive clones were obtained by kanamycin screening. The plasmids in the positive clones were extracted and verified by enzyme digestion ( figure 2 ) and sequencing, the successfully constructed expression vector pMV261-ndh for the overexpression of the NDH2 gene itself was obtained.

Embodiment 3

[0042] Example 3 Construction of NDH2 Gene Enhanced Strain NdhF

[0043] Preparation of Mycobacterium ARL-91 Competent Cells: Inoculate ARL-91 strain into LB medium and culture at 30°C until 0D 600 1.0, according to 10% inoculum amount transferred to the seed medium for secondary seed culture; 24h after adding 2% glycine to continue culturing for 24h. Collect the bacteria by centrifugation, wash the suspended bacteria with 10% pre-cooled glycerin of 1 times, 3 / 4 times, 1 / 2 times and 1 / 4 times the volume of fermentation broth respectively and centrifuge, finally add 1 / 25 times of 10% glycerol Suspend the bacteria and store in sub-packages;

[0044] Electroporation: Take 10 μL of the pMV261-ndh genetic engineering expression vector obtained in Example 2, add it to 100 μL of competent bacteria and let it stand for 30 minutes, then transfer to the electroporation cuvette for clicking. Electroporation conditions were 2kV / cm, 25μF, 720Ω for 3-6ms, placed on ice for 5min, then tran...

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Abstract

The invention belongs to the technical field of biocatalysis, and particularly relates to a method for enhancing the steroid medicine precursor production by strengthening the intracellular NADH dehydrogenation of a steroid medicine production strain. The method introduces NADH dehydrogenase-NDH2 which has no proton-pump function and exists insteroid medicine production strain microorganisms, constructs a NDH2-enhanced steroid medicine precursor production strain to strengthen the dehydrogenation of NADH in the steroid medicine precursor production strain, solves a problems of long fermentation cycle and low production efficiency caused by excessive accumulation of NADH in the process of microbial transformation production of the steroid medicine precursor, and can help reduce energy consumption and save production cost in the production process of the steroid medicine precursor. The method can be effectively used for strengthening the intracellular NADH dehydrogenation of other industrial production strains, and has wide application value. A new method for reducing the production cost of steroid medicine precursor is provided.

Description

Technical field: [0001] The invention belongs to the technical field of biocatalysis, and specifically relates to a method for enhancing the production of steroid drug precursors by strengthening intracellular NADH dehydrogenation of steroid drug production strains. Background technique: [0002] Steroidal drugs are the main marketed products in the pharmaceutical industry and are widely used clinically. Among them, 4-androstene-3,17-dione (AD), 1,4-androstene-3,17-dione (ADD) and 9α-hydroxyandrost-4-ene-3,17- Diketone (9α-hydroxyandrostenedione) is an important precursor for the synthesis of steroids. AD and ADD are the main precursors of synthetic steroid drugs such as progestins, birth control pills and estrogens. 9α-Hydroxyandrostenedione is an important precursor for the production of steroidal drugs with a halogen at the 9α position. Hydrocortisone, 17α-hydroxyprogesterone, eplerenone, and Various important steroid drugs such as dexamethasone, betamethasone, cortiso...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P33/00C12R1/33
Inventor 申雁冰王敏周秀玲张扬夏梦雷骆健美张晓
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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