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A method for constructing Rhizopus niger cpr genetically engineered bacteria by transformation of protoplasts

A technology of protoplast transformation and genetically engineered bacteria, applied in the direction of microorganism-based methods, biochemical equipment and methods, and the use of vectors to introduce foreign genetic materials

Active Publication Date: 2016-04-13
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But there is no report that the protoplast transformation method is applied to Rhizopus niger

Method used

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  • A method for constructing Rhizopus niger cpr genetically engineered bacteria by transformation of protoplasts
  • A method for constructing Rhizopus niger cpr genetically engineered bacteria by transformation of protoplasts

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Embodiment 1

[0032] Embodiment 1: Utilize the protoplast transformation method to carry out the genetic transformation of exogenous gene pCB1004-pgpd-CPR to Rhizopus niger

[0033] Using the method described in the summary of the invention, the exogenous plasmid pCB1004-pgpd-CPR containing the CPR target gene was introduced into Rhizopus niger, and the stably transformed engineering bacteria were obtained through hygromycin screening and applied to the hydroxylation transformation of steroidal compounds . The specific method is as follows:

[0034] 1. Fungal expression plasmid construction

[0035] 1) Design primers based on the Rhizopus niger CPR gene sequence on geneBank:

[0036] CPR-F: 5'- GGATCC ATGACTCGAAACAACTCTCATC-3' (the underline is the BamHI restriction site);

[0037] CPR-R: 5'- AAGCTT TTAACTCCAAACATCTTCTTGG-3' (the underline is the HindIII restriction site)

[0038] The total RNA of Rhizopus oryzae (CICC40467) was extracted according to the instructions of the TakaraRN...

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Abstract

The invention provides a method for exogenous gene genetic transformation of rhizopus nigericans by protoplast transformation technology, which comprises the following steps: treating cell walls of rhizopus nigericans by fungal cell wall degrading enzyme to obtain protoplast, purifying the protoplast, introducing rhizopus oryzae NADPH-cytochrome P450 reductase CYPOR (abbreviated as CPR gene) genes into rhizopus nigericans for the first time by using a protoplast transformation method with hygromycin B as a selective marker, so as to obtain the genetically engineered bacteria. The beneficial effects of the invention are that: the method can be used for molecular biology research of rhizopus nigericans, such as the molecular mechanism of the hydroxylation effect of the P450 enzyme system of the bacteria on steroids, and the like; the method can perform genetic engineering of rhizopus nigericans for genetic improvement breeding so as to increase the conversion rate of hydroxylation of steroid substrates by rhizopus nigericans.

Description

(1) Technical field [0001] The invention relates to a method for constructing Rhizopus niger CPR genetic engineering bacteria by using protoplast transformation. (2) Background technology [0002] Rhizopus nigricans is capable of C11α-hydroxylation on steroids. Hydroxylation biotransformation reaction is usually mediated by P450 enzyme system, cytochrome P450 oxidoreductase (NADPH-cytochromeP450reductase, CPR, E.C.1.6.2.4) can transfer electrons from NADPH to cytochrome P450, cytochrome c, cytochrome b5 and heme proteins such as heme oxidase. [0003] At present, the methods of improving the biotransformation system can be roughly classified into three categories: increasing the solubility of the substrate, releasing the inhibition of the substrate (product), and changing the permeability of the cell membrane (cell wall), which has encountered a bottleneck to a certain extent. With the development of molecular technology, P450 monooxygenase (CYP) and P450 reductase (CPR) g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/80C12N1/15C12N1/14C12R1/845
Inventor 陈小龙顾光志朱廷恒嘉晓勤范永仙沈寅初
Owner ZHEJIANG UNIV OF TECH
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