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Method for transforming and producing 9-OH-AD by using whole cells of recombinant corynebacterium glutamicum

A technology of Corynebacterium glutamicum and recombinant bacteria, which is applied in the field of bioengineering and biology, and can solve the problems of reduced production efficiency, long fermentation cycle, low conversion rate, etc.

Active Publication Date: 2018-09-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the role of rate-limiting steps and rate-limiting enzymes in the sterol degradation pathway, at the same time, because 3-sterol-△1-dehydrogenase (Ksdd) in microorganisms can catalyze the conversion of AD into ADD, its existence will also hinder Accumulation of 9-OH-AD, resulting in lower conversion and longer fermentation cycle, greatly reducing production efficiency

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of recombinant bacteria C. glutamicum ATCC13032 / pMJ19-kshA-kshB

[0037] 1. Primer design for 3-sterone 9α-hydroxylase

[0038] According to the kshA gene sequence in the whole genome nucleic acid sequence of Mycobacterium sp.Strain VKM Ac-1817D in NCBI, the PCR primers P1 and P2 of 3-sterone 9α-hydroxylase oxidative subunit KshA were designed.

[0039] P1: 5'-CG GGATCC ATGACGACTGAGCACGCCGG-3' (BamH I)

[0040] P2: 5'- TTTCCTCCCTTTAG TCAGCTTGATTGAGCGGTTTC-3'(rbsR)

[0041] According to the kshB gene sequence in the whole genome nucleic acid sequence of Mycobacterium sp.Strain VKM Ac-1817D in NCBI, the PCR primers P3 and P4 of 3-sterone 9α-hydroxylase reducing subunit KshB were designed.

[0042] P3: 5'- AAAGGAGGGAAATC ATGACTGATGAACCGTTAGGTAG-3'(rbsF)

[0043] P4: 5'-CCC AAGCTT TCACTCGTCGTAGGTCACCTC-3' (Hind III)

[0044] 2. Cloning of 3-sterone 9α-hydroxylase genes kshA and kshB

[0045] Using the synthesized DNA as a template, using ...

Embodiment 2

[0054] Embodiment 2: Recombinant bacteria C.glutamicum ATCC13032 / pMJ19-kshA-kshB whole cell transformation method produces 9-OH-AD

[0055] The recombinant strain C. glutamicum ATCC13032 / pMJ19-kshA-kshB constructed in Example 1 was inoculated in 200mL LBG medium and cultured for 8h, then added IPTG with a final concentration of 0.7mM, induced at 30°C for 8h, collected and obtained cells, and measured by pH 7.0tris-HCl washed twice and resuspended in 200mL substrate buffer (0.3M carbonate buffer, 1g / L AD, 0.5mMMn, pH 7.0), added 1.0g / L substrate AD every hour, 30 After conversion at ℃ for 10 h, the content of AD and 9-OH-AD in the conversion solution was determined by high performance liquid chromatography (HPLC). The results showed that after 10 hours of conversion, the content of 9-OH-AD in the conversion liquid was 9.8g / L, and the molar conversion rate of 9-OH-AD was 99.5%.

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Abstract

The invention discloses a method for transforming and producing 9-OH-AD by using whole cells of recombinant corynebacterium glutamicum, and belongs to the technical field of bioengineering and biology. Through a shuttle plasmid pXMJ19 between escherichia coli and corynebacterium glutamicum, an oxidization subunit KshA and a reducing subunit KshB of 3-ketosteroid 9alpha-hydroxylase from Mycobacterium sp.Strain VKM Ac-1817D are successfully co-expressed in C.glutamicum ATCC13032. A whole-cell thallus of the engineering bacterium is used as a biocatalyst to transform and produce 9alpha-hydroxyl androstane-4-alkene-3,17-diketone; the mole transformation rate of the substrate androstane-4-alkene-3,17-diketone is up to 99.5 percent. The 9alpha-hydroxyl androstane-4-alkene-3,17-diketone producedby the method disclosed by the invention has the advantages of high efficiency, high specifity, low energy consumption and the like.

Description

technical field [0001] The invention relates to a method for transforming and producing 9-OH-AD by whole cells of recombinant Corynebacterium glutamicum, belonging to the fields of bioengineering and biotechnology. Background technique [0002] Steroids are an important class of natural organic compounds that widely exist in biological tissues. Steroids have a variety of physiological functions and exert unique curative effects, so they are widely used in clinical practice. Steroids are the second largest class of drugs next to antibiotics, with an annual growth rate of more than 15%. Steroids usually have a series of unique physiological functions, which are mainly due to the differences in substituents, double bond positions or stereo configurations on the core of steroids. Steroids have similar structures and complex structures and are widely found in animal and plant tissues and some microorganisms. The more common ones are cholesterol, bile acid, sex hormones, adrenal ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12N15/53C12P33/06C12R1/15
CPCC12N9/0071C12N15/77C12P33/06
Inventor 饶志明沙宗焱哈婧雯燕宇刘昭君樊佳慧高梦昕邵明龙张显
Owner JIANGNAN UNIV
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