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3-sterone-delta[1]-dehydrogenase gene, relevant carriers, engineering strains and applications thereof

A technology of genetic engineering and dehydrogenase, applied in the field of molecular biology, 3-sterone-△1-dehydrogenase, can solve the problems of high toxicity, low conversion efficiency, steroid nucleus degradation, serious environmental pollution, etc.

Active Publication Date: 2009-10-28
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Steroidal compounds can also be dehydrogenated at C1 and 2 positions by chemical methods. Selenium dioxide method is generally used. This method often makes the product contain a small amount of selenium that is difficult to remove and is harmful to human body. Steroid 1 The dehydrogenation yield of - position is low, the toxicity is high, and the environment is seriously polluted. The microbial conversion method is an ideal choice, but it still has some defects, such as low conversion efficiency or side reactions such as steroid nuclear degradation during the conversion process. Wait

Method used

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  • 3-sterone-delta[1]-dehydrogenase gene, relevant carriers, engineering strains and applications thereof
  • 3-sterone-delta[1]-dehydrogenase gene, relevant carriers, engineering strains and applications thereof
  • 3-sterone-delta[1]-dehydrogenase gene, relevant carriers, engineering strains and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Transformation of steroidal compounds by Mycobacterium neoaurum NwIB-01 (preservation number CCTCC M 209094)

[0061] Using phytosterols as the only carbon source, a sterol-degrading microorganism was isolated from activated sludge. After chemical and physical mutations such as nitrosoguanidine and ultraviolet light, the inventor screened a strain with good production capacity. A mutant strain of traits, which can degrade and transform sterols to produce AD ​​and ADD. After identification of the strain, the microbial strain was identified as Mycobacterium neoaurum, named Mycobacterium neoaurum NwIB-01, and the strain was preserved on April 28, 2009. In Wuhan China Center for Type Culture Collection (Accession No. CCTCC M 209094).

[0062] Use 1%-10% surfactants, polymers or organic solvents (such as Tween80, ethanol, silicone oil, soybean oil, etc.) to help dissolve the steroids. The final transformation medium is inoculated with 1%-50% seeds by using two...

Embodiment 2

[0065] Example 2 3-sterone-△ 1 - Obtainment, molecular identification and heterologous expression of dehydrogenase gene in E. coli

[0066] This example only uses the PCR method as an example to clone 3-sterone-△ from mycobacterium Mycobacterium neoaurum NwIB-01 1 - the sequence of SEQ ID NO: 1 of the dehydrogenase.

[0067] 2.1, 3-sterone-△ 1 - Acquisition of the conserved sequence of the dehydrogenase gene (see Figure 4 )

[0068] By the known mycobacterial 3-sterone-△ 1-Analysis of dehydrogenase genes, 3-sterone-△ of three strains of mycobacteria were selected 1 - Gene homologous comparison of dehydrogenase gene (data from NCBI, see the sequence number below):

[0069] Mycobacterium avium 104 (gb|CP000479.1|);

[0070] Mycobacterium vanbaalenii PYR-1 (gb|CP000511.1|);

[0071] Mycobacterium smegmatis str. MC2 155 (gb|CP000480.1|);

[0072] 3-sterone-△ 1 -After the relevant information of the conserved sequence of dehydrogenase, the software Oligo 6.0 and Prim...

Embodiment 3

[0098] Example 3 3-sterone-△ 1 -Construction method of mycobacterium genetically engineered strain with dehydrogenase gene deletion

[0099] Construction of mycobacterial gene deletion plasmids, which are electrotransformed into mycobacteria. Kanapenicillin and hygromycin were used for double-antibody screening, and then sucrose plate was used for re-screening to obtain gene knockout clones. The above clones were verified by PCR method. Gene deletion double crossover as Figure 6 As shown, the final gene deletion strain was named NwIB-201. It should be noted here that the NwIB-201 obtained here is the NwIB-909 strain 3-sterone-△ 1 - Obtained by deletion of the dehydrogenase gene, NwIB-909 is a derivative strain of NwIB-01, which lacks the activity of 3-sterone-9α-hydroxylase, the key enzyme for steroid nuclear degradation, and thus cannot degrade steroid parent nuclear.

[0100] Specific steps are as follows:

[0101] 1. Knockout plasmid construction: design knockou...

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Abstract

The invention provides a 3-sterone-Delta[1]-dehydrogenase. By constructing various expression vectors and converting relevant strains, the invention obtains various engineering strains such as non-3-sterone-Delta[1]-dehydrogenase activity strains or high 3-sterone-Delta[1]-dehydrogenase activity strains; the engineering strains can be adopted to selectively prepare androst-4-alkene-3, 17-diketone and 9Alpha-androst-4-alkene-3, 17-diketone or androst-1, 4-diene-3, 17-diketone and 3-ketone-1, 4-dienes steroid compounds. The engineering strains can greatly improve the production efficiency and product quality of the steroid medical production system, are beneficial for reducing the energy consumption of steroid drugs during the production process, improving the utilization ratio of the prodrugs and simplifying the production steps, reduce the production cost, have moderate reaction conditions and friendly environment, are suitable for wide generalization and application and have higher economic benefits and social benefits.

Description

technical field [0001] The present invention relates to the technical field of molecular biology, more specifically, relates to 3-sterone-△ 1 -Dehydrogenase technical field, especially a kind of 3-sterone-△ 1 -Dehydrogenase gene, related vectors and engineering bacteria and application. Background technique [0002] Steroids, also known as steroids, are a class of compounds with cyclopentane polyhydrophenanthrene as the core and similar structures. Its basic structure is shown in the figure below. Due to the difference in substituents, double bond positions or stereo configurations on the steroid core, a series of compounds with unique physiological functions have been formed. In animals, steroids are the main endogenous sex hormones, secreted by sex organs and adrenal cortex, and closely related to reproduction, bone and brain development, regulation of biological effects, and maintenance of homeostasis. Steroid drugs mostly refer to steroid hormone drugs, such as adrena...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/63C12N1/21C12P33/02C12N1/20C12R1/19C12R1/32C12R1/465
Inventor 王风清魏东芝魏巍范书鵫
Owner EAST CHINA UNIV OF SCI & TECH
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