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Method for producing androstadienedione by degrading phytosterol through microorganisms

A technology for androstenedione and androstenedione is applied in the field of microbial degradation of phytosterol to produce androstenedione, which can solve the problems of only 65.7% and the like

Inactive Publication Date: 2020-10-23
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Patent CN106636160A discloses a method for overexpressing Mycobacterium aureus ksdd in Escherichia coli BL21, which can convert AD to ADD, but the feeding concentration is only 0.1% (W / V), and the substrate conversion rate is only 76.5%
Patent CN102703494A discloses a method for transforming AD into ADD by using whole cells of recombinant Bacillus subtilis. The enzyme activity of the genetic engineering strain constructed is significantly higher than that of the control strain, but when 0.1% AD is used as the substrate, the molar conversion of the substrate rate is only 65.7%

Method used

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  • Method for producing androstadienedione by degrading phytosterol through microorganisms
  • Method for producing androstadienedione by degrading phytosterol through microorganisms
  • Method for producing androstadienedione by degrading phytosterol through microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Mycobacterium neoaurium DSM 1381 3-sterone-△ 1 -Acquisition of the dehydrogenase-encoding gene ksdd1

[0067] Using Illumina Miseq sequencing technology to sequence and analyze Mycobacterium aureus DSM 1381, according to the gene annotation information, a complete 3-sterone-△ 1 - dehydrogenase encoding gene ksdd1 sequence, said 3-sterone-△ 1 - The amino acid sequence of the dehydrogenase coding gene ksdd1 is shown in SEQ ID NO: 1, specifically:

[0068] VFYMTAQDYSVFDVVVVGSGAAGMVAALTAAHQGLSTVVVEKAPHYGGSTARSGGGVWIPNNEVLQRDGVKDTAAEARKYLHTIIGDVVPAEKIDTYLDRSPEMLSFVLKNSPLKLCWVPNYSDYYPETPGGKATGRSVEPKPFNAKKLGPDEKGLEPPYGKVPLNMVVLQQDYVRLNQLKRHPRGVLRSIKVGVRSVWANATGKNLVGMGRALIAPLRIGLQKAGVPVLLNTALTDLYLEDGVVRGIYVREAGAPESAEPKLIRARKGVILGSGGFEHNQEMRTKYQRQPITTEWTVGAVANTGDGIVAAEKLGAALELMEDAWWGPTVPLVGAPWFALSERNSPGSIIVNMNGKRFMNESMPYVEACHHMYGGQYGQGAGPGENVPAWMVFDQQYRDRYIFAGLQPGQRIPKKWMESGVIVKADSVAELAEKTGLAPDALKATIDRFNGFARSGVDEDFHRGESAYDRYYGDPTNKPNPNLGEIKNGPFYAAKMVPGDLGTKGGIRTDVHGR...

Embodiment 2

[0076] Example 2: 3-sterone-△ 1 - Construction and transformation of dehydrogenase encoding gene ksdd1 expression vector

[0077] The fragment obtained in Example 1 was double-digested with EcoRI and SalI, and ligated with the integrated expression vector pMV306, which was also double-digested with EcoRI and SalI, and the ligated product was transformed into Escherichia coli DH5α competent cells (purchased from TAKARA company) , and the expression recombinant plasmid was verified by colony PCR screening. The recombinant plasmid pMV-ksdd1 was extracted and sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing analysis. After sequencing, the 3-sterone-△ 1 - the dehydrogenase encoding gene ksdd1 sequence and the nucleotide sequence shown in SEQ ID NO:1.

[0078]Using the plasmid pJES103 (ADDgene: #135410) as a template, design primers to amplify the promoter Hsp60 according to the gene fragment amplification verification method in Example 1. The primer sequences are a...

Embodiment 4

[0086] Embodiment 4 Recombinant bacteria ZFZ-11 transforms phytosterols

[0087] Seed medium (g / L): 6 glucose, 15 corn steep liquor dry powder, 5.4 sodium nitrate, 0.6 diammonium hydrogen phosphate, sterilized at 115°C for 20 minutes.

[0088] Fermentation medium (g / L): glucose 6, corn steep liquor dry powder 15, sodium nitrate 5.4, diammonium hydrogen phosphate 0.6, Tween-801, soybean oil 10% (V / V), phytosterol 5, sterilized at 115°C 20min.

[0089] Inoculate the recombinant strain ZFZ-11 monoclonal into the seed culture medium at 30°C and 160rpm and cultivate it for two days to the late exponential stage, then transfer 10% of the volume of the bacterial liquid to the fermentation medium at 30°C and 160rpm for fermentation until the oil phase is completely emulsified Finally, take an appropriate amount of the oil layer and extract it with an equal amount of ethyl acetate. After the extract is evaporated to dryness, it is dissolved with e.g. methanol, and an appropriate amoun...

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Abstract

The invention relates to the technical field of biology, and provides a method for producing androstadienedione by degrading phytosterol through microorganisms. According to the invention, a new 3-sterone-1-dehydrogenase gene is cloned from Mycobacterium neoaurum DSM 1381 for the first time; through genetic engineering means, recombinant bacteria capable of efficiently expressing 3-ketosterol-1-dehydrogenase are obtained, and when the concentration of substrate phytosterol in a conversion culture medium is as high as 5g / L, the purity of the product androstadienedione in a conversion solution can be 95% or above, so that the residue of the byproduct androstenedione is effectively reduced, and the purity of the product androstadienedione is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for producing androsdienedione by degrading plant sterols by microorganisms. Background technique [0002] Steroid hormone drugs refer to hormone drugs containing steroid structure in their molecular structure. Due to the difference in substituents, double bond positions or stereo configurations on the steroid core, a wide variety of steroid compounds are formed. It has strong anti-infection, anti-allergic, anti-viral and other effects. It is an important class of drugs in clinical practice and plays a very important role in regulating the body. Steroid drugs are mainly divided into mineralocorticoids, glucocorticoids, sex hormones and anabolic hormones. It is widely used in the treatment of rheumatoid arthritis, bronchial asthma, breast cancer, prostate cancer, oral contraceptives, etc. So far, there are about 300 kinds of steroid drugs that have been approved for use, whi...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/74C12N1/21C12P33/02C12R1/32
CPCC12N9/001C12Y103/99004C12N15/74C12P33/02
Inventor 张保国刘相岑袁辰阳曹慧锦杜桂林史吉平
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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