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Application of G6PDH gene for improving steroid C11 alpha-hydroxylation ability of rhizopus nigricans and strain

A technology of rhizopus niger and hydroxylation, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve problems such as difficult conversion rate, achieve high application value, high conversion rate and conversion efficiency, and far-reaching theoretical significance

Active Publication Date: 2014-03-12
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After decades of research, Rhizopus niger biotransformation steroid C11α-hydroxylation process and technology has been relatively mature, and the transformation ability of strains has been improved through mutation breeding, so it is difficult to continue to improve on the basis of existing strains and processes Transformation rate, how to use new methods to further improve the production capacity of strains has become a problem of particular concern to personnel in this field

Method used

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  • Application of G6PDH gene for improving steroid C11 alpha-hydroxylation ability of rhizopus nigricans and strain
  • Application of G6PDH gene for improving steroid C11 alpha-hydroxylation ability of rhizopus nigricans and strain
  • Application of G6PDH gene for improving steroid C11 alpha-hydroxylation ability of rhizopus nigricans and strain

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Embodiment 1

[0034] Example 1: Related primer design

[0035] Primer F-g6pdh / R-g6pdh and primer F-hy g were designed according to the known Rhizopus oryzae G6DPH gene sequence (SEQ ID No.1) and E.coli hygromycin resistance gene sequence (SEQ ID No.2) / R-hyg (see Table 1), BamHI and ApaⅠ restriction sites were added to the two ends of primer F-g6pdh / R-g6pdh respectively, and primer F-hyg / R-hyg was used to identify Rhizopus niger transformants by PCR.

[0036] Table 1: Primers involved in the present invention

[0037] Primer name

Embodiment 2

[0038] Embodiment 2: the extraction of Rhizopus oryzae total RNA

[0039] The Rhizopus oryzae (CICC40467) mycelium was ground with liquid nitrogen, and the total RNA was extracted with RNAiso plus Total RNA extraction reagent from Takara Company, and the genomic DNA was removed by DNase I treatment, agarose gel electrophoresis detection and absorbance analysis to ensure the extraction RNA does not degrade and contaminate genomic DNA.

[0040] The total RNA of Rhizopus oryzae extracted by the present invention is analyzed by absorbance: OD 260 / OD 280 =1.85, the RNA concentration is 89μg / ml, the agarose gel electrophoresis picture is as follows figure 1 According to the results of agarose gel electrophoresis, the 28S and 18S RNA bands in the extracted total RNA were clear and not degraded, indicating that the RNA integrity was good, and there was no genomic DNA contamination in the extracted RNA.

Embodiment 3

[0041] Embodiment 3: Rhizopus oryzae G6PDH gene clone

[0042] Using the extracted Rhizopus oryzae RNA as a template and primers F-g6pdh / R-g6pdh (see Table 1) for RT-PCR amplification to obtain the G6PDH gene fragment without introns, the PCR product agarose gel electrophoresis detection results like figure 2 , it can be seen from the figure that the band is between 1200 and 1400bp, which is consistent with the size of the coding sequence of the G6PDH gene (including intron 1439bp and exon 1346bp) reported in the database.

[0043] After purification and recovery, the amplified product was ligated with the PMD19-T Simple vector (purchased from TaKaDa) and then transformed. Picked 5 single colonies from the transformed plate for colony PCR. The results are as follows: image 3 . As can be seen from the figure, the selected 5 transformants were all positive. The No. 2 transformant was selected for expansion culture, the plasmid was extracted and verified by single and double...

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Abstract

The invention provides an application of G6PDH gene for improving steroid C11 alpha-hydroxylation ability of rhizopus nigricans, a method for constructing glucose-6-phosphate dehydrogenase genetically engineered bacteria and a strain screened out after construction. G6PDH is cloned into rhizopus nigricans through a molecular biological method to transform the strain, and the G6PDH catalyzes NADPH regeneration in the transformed rhizopus nigricans cells to provide coenzyme necessary for a catalytic reaction of a cytochrome P450 enzyme system so as to improve the steroid C11 alpha-hydroxylation ability of the rhizopus nigricans. The application provided by the invention has the beneficial effects that the genetically engineered bacteria created by the method provided by the invention are used for converting Wolfowitz oxide C11 alpha-hydroxylation to produce mold oxide; compared with a starting strain, the strain grows faster and is high in raw material utilization rate and high in conversion rate and conversion efficiency, so that the strain has far-reaching theoretical significance and high application value in development and application process of steroid medicine.

Description

(1) Technical field [0001] The present invention relates to the application of G6PDH gene in improving the ability of Rhizopus niger to steroid C11α-hydroxylation, the construction method of glucose-6-phosphate dehydrogenase genetically engineered bacteria, and a strain of steroid C11α- Higher hydroxylation strain - Rhizopus nigericans PIe. (2) Background technology [0002] Products catalyzed by oxidoreductases are widely used in medicine, food, pesticides and other fields. The lack of coenzymes in the enzyme catalysis process is the main factor that limits the biocatalytic synthesis of products by most oxidoreductases. Therefore, the recycling of coenzymes is very important for the enzyme-catalyzed process, and it is of great value in the production of related products such as medicine and food. It has been reported in the literature that the regeneration methods of coenzymes include enzymatic, photochemical, electrochemical and other regeneration methods, among which enz...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N1/15C12N15/53C12R1/845
Inventor 陈小龙范永仙朱廷恒薛海龙张力伟沈寅初
Owner ZHEJIANG UNIV OF TECH
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