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A kind of engineering bacterium and its application for preparing testosterone

A technology of testosterone and engineering bacteria, applied in the field of bioengineering, can solve the problems of large environmental pollution, little effect, complex reaction, etc., and achieve the effects of small environmental pollution, short preparation route and wide source of raw materials

Active Publication Date: 2021-03-19
SHENYANG BOTAI PHARM CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above two methods are chemical methods, which have the disadvantages of large environmental pollution and complicated reactions.
At present, the research on the preparation of testosterone by microbial transformation has also begun at home and abroad, but there is still little effect

Method used

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  • A kind of engineering bacterium and its application for preparing testosterone
  • A kind of engineering bacterium and its application for preparing testosterone
  • A kind of engineering bacterium and its application for preparing testosterone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The construction of the expression vector of embodiment 1 testosterone acetate hydrolase gene

[0052] According to the complete sequence analysis of Mycobacterium neoaurum DSM 44074 gene, the primers of testosterone acetate hydrolase gene were F1 and R1;

[0053] Upstream primer F1 (NdeI), as shown in SEQ ID NO:3 (wherein, the bold underline is the restriction site NdeI, and the preceding AAAAA is the protective base), and downstream primer R1 (HindIII) is as shown in SEQ ID NO:4 As shown (wherein, the bold underline is the enzyme cutting site HindIII, and the AAAAA in front is the protective base);

[0054] SEQ ID NO:3 5'-AAAAA AGCGATAAACTCGACCC;

[0055] SEQ ID NO:4 5'-AAAAA ATCCGGCGGTGACGTGGCTC.

[0056] The testosterone acetate hydrolase gene sequence was amplified by PCR using mycobacterium chromosomal DNA as a template and F1 and R1 as primers.

[0057] Testosterone acetate hydrolase gene PCR amplification system: (the size of the testosterone acetate hy...

Embodiment 2

[0068] Embodiment 2 uses engineering bacterium of the present invention to prepare testosterone

[0069] 2.1 Induced expression of transformants

[0070] First, the testosterone acetate hydrolase Escherichia coli BL21 (DE3) transformant obtained as in Example 1 is induced and expressed, and the induced expression method is as follows: first, the bacterial strain is picked from a -80°C refrigerator, and at 37°C, in 3ml of Activate one generation in LBK liquid medium (peptone 10.0g, yeast powder 5.0g, NaCl10.0g, add deionized water to 1000ml, adjust pH to 7.0, steam sterilize. Final concentration of kanamycin is 50μg / ml), take The bacterial solution cultured for 12-16 hours was inoculated into 5 ml of LBK liquid medium with 1% inoculum, and the inducer IPTG was added to a final concentration of 1 mM when the culture reached the logarithmic growth phase. After overnight induction at 22°C, the bacterial cells were collected.

[0071] 2.2 Escherichia coli cell-free lysate tr...

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Abstract

The invention provides special engineering bacteria capable of expressing testosterone acetate hydrolase for producing testosterone. Testosterone acetate hydrolase genes are transferred into host bacteria or inserted into chromosomes of the host bacteria by an expression vector to obtain the special engineering bacteria capable of expressing the testosterone acetate hydrolase. The invention further provides a method for producing the testosterone. Compared with the prior art, the method has the advantages that reaction conditions are mild, synthetic routes are short in the synthetic process, raw material sources are wide, yield is high, and environmental pollution is low. The yield can reach up to 98% or more, and the industrially useful compound-testosterone serving as a medicine and a medical intermediate can be efficiently manufactured.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to an engineering bacterium and the use of the engineering bacterium to prepare testosterone. Background technique [0002] Steroid hormones, also known as steroid hormones, are a class of tetracyclic aliphatic hydrocarbon compounds with cyclopentane polyhydrophenanthrene nuclei and have extremely high medical value. It has a clear role in maintaining life, regulating sexual function, developing the body, regulating immunity, treating skin diseases and birth control. Steroid hormones are endogenous substances discovered during the study of the mammalian endocrine system. Between 1932 and 1939, estrone (Estrone, 1932), estradiol (Estradiol, 1932), testosterone ( Testosterone, 1935) and corticosterone (Corticosterone, 1939) and other pure product crystallization, and then elucidated its chemical structure, since then created a new field of steroid chemistry and steroid drugs...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P33/00C12N15/63C12N1/21C12N1/19C12R1/19C12R1/01C12R1/38
CPCC12N9/18C12P33/00
Inventor 常尊学张鑫隋靓邹雷
Owner SHENYANG BOTAI PHARM CO LTD
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