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High-expression cytochrome P450 monooxygenase aspergillus ochraceus strain and construction method and application thereof

The technology of monooxygenase Aspergillus ochreus and monooxygenase Aspergillus ochra is applied to the high expression cytochrome P450 monooxygenase Aspergillus ochra strain and its construction and application fields, which can solve the limitation of wide application, the gap of transformation rate, etc. question

Pending Publication Date: 2020-01-21
SHANGHAI INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with the high-yielding ochrax strain, there is still a certain gap in the transformation rate, and the need to add antibiotics during the fermentation also limits its wide application in industrial production to a certain extent.

Method used

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  • High-expression cytochrome P450 monooxygenase aspergillus ochraceus strain and construction method and application thereof
  • High-expression cytochrome P450 monooxygenase aspergillus ochraceus strain and construction method and application thereof
  • High-expression cytochrome P450 monooxygenase aspergillus ochraceus strain and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Construction of recombinant plasmid pB-UTPHD

[0043] According to the genome sequencing results of a strain of Ocheraspermum isolated in our laboratory, the cytochrome P450 monooxygenase coding gene was predicted, and the DNA sequence of 1000 bp upstream and downstream of the gene was determined. Through resistance screening, determine the type of antibiotic and the amount of antibiotic added.

[0044] (1) Construction of upstream and downstream homologous arm recombination plasmids pMD18-Up and pMD18-Down

[0045] Genomic DNA of the original Ochrae sp. strain MF018 was used as a template, primers F-Up and R-Up were used to amplify the upstream homology arm fragment of the cytochrome P450 monooxygenase gene by PCR, and the 1011bp PCR product was carried out on 1% agarose gel Electrophoresis, the size of the fragment was consistent with the expectation; primers F-Down and R-Down were used to perform PCR amplification of the downstream homology arm fragmen...

Embodiment 2

[0119] Example 2: Obtaining of a strain of Ochrae sp. highly expressing cytochrome P450 monooxygenase

[0120] (1) Transformation of recombinant plasmid pB-UTPHD into Aspergillus ochrae MF018

[0121] The recombinant plasmid pB-UTPHD was extracted from Escherichia coli by CaCl 2 / PEG-mediated transfer into Ochrae MF018 protoplasts. After the protoplasts were regenerated and cultured for 6-8 hours, they were spread on a PDA plate containing 125 μg / mL hygromycin resistance, and cultured at 28°C for 5-6 days. Pick a positive single colony for re-screening culture, and the culture conditions are the same as above. The positive transformants obtained after rescreening were verified at the gene level.

[0122] Wherein, the protoplast preparation operation is as follows:

[0123] Pick the original ochrax strain MF018 grown on the PDA plate for 6-8 days in a 50mL centrifuge tube filled with 10mL of normal saline, vortex and mix well, then add the spore liquid to 100mL seed medium,...

Embodiment 3

[0126] Embodiment 3: the identification of Ochrae japonicus recombinant strain

[0127] The positive transformants obtained by re-screening on the hygromycin resistance plate were picked in the seed medium, cultured at 28°C for 18 hours, transferred the seed liquid to the fermentation medium, and cultivated at 28°C for 24 hours. Genomic DNA of positive transformants extracted with DNA extraction kit from OMEGA Company (Catalog No.: D3390-00) was used as a template. Primers F-Screen and R-Screen were designed based on the upstream and downstream DNA sequences of the homology arms of the original strain, and the double crossover verification of recombinant bacteria was carried out by PCR. The PCR product of the original strain MF018 genomic DNA was used as a control. If the fusion gene UTPHD undergoes double exchange at the position of the P450 monooxygenase gene of the original strain, a 6016bp gene fragment (named Sc-UTPHD) (SEQ ID NO: 29) can be amplified; the genomic DNA of...

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Abstract

The invention discloses a high-expression cytochrome P450 monooxygenase aspergillus ochraceus strain, a construction method thereof and application thereof in fermentation production of 11alpha-hydroxy-canrenone. The aspergillus ochraceus strain is currently preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO: M2019616, wherein the recombinant fragment comprises a cytochrome P450 monooxygenase coding gene, upstream and downstream DNA sequences, a promoter, a terminator and an antibiotic resistance gene; and homologous recombination occurs between thegene fragment and the cytochrome P450 monooxygenase gene in the chromosome of an aspergillus ochraceus initial strain MF018. according to the invention, a homologous recombinant aspergillus ochraticastrain is obtained by the double exchange which occurs between the constructed high-expression cytochrome P450 monooxygenase gene and the cytochrome P450 monooxygenase gene in the chromosome of the aspergillus ochraceus initial strain MF018. The invention can realize the effective accumulation of 11alpha-hydroxy-canrenone produced in the fermentation process.

Description

technical field [0001] The invention relates to a high-expression cytochrome P450 monooxygenase ochraetobacter strain MF010 and its construction method and application, belonging to the field of biotechnology. Background technique [0002] Steroid compounds, also known as steroid compounds, are a general term for a class of compounds with cyclopentane polyhydrophenanthrene as the parent nucleus. It is reported that steroids have a wide range of pharmacological activities, and play an extremely important role in anti-inflammatory, anti-tumor, regulation of sexual function and birth control. Studies have shown that natural steroids often have low pharmacological activity, but the introduction of hydroxyl groups on specific sites of steroids can significantly improve their pharmacological and biological activities. Steroidal compounds have complex structures and contain multiple asymmetric centers, and it is difficult to introduce hydroxyl groups at specific positions of the s...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12N15/90C12N15/53C12N9/02C12P33/20C12R1/66
CPCC12N9/0071C12N15/80C12N15/902C12P33/20C12N1/14C12Y114/14
Inventor 李茜茜时丽荣绍丰管世敏张硕蔡宝国
Owner SHANGHAI INSTITUTE OF TECHNOLOGY
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