Method using microorganism method to convert androstenedione so as to produce testosterone

A technology of codon optimization and hydroxysteroids, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of diosgenin consumption, rising saponin price, and low recovery rate, and achieve environmental protection and efficient production of testosterone and raw materials The effect of high utilization

Pending Publication Date: 2016-10-12
JIANGNAN UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0004] Most of the raw materials for the early synthesis of steroidal drugs were directly extracted from animal tissue fluid. Due to the low content, few sources, complex synthetic routes, low recovery rate and high cost of these raw materials, they could not meet the needs of production.
The discovery and application of diosgenin provide rich and cheap raw materials for the industrial production of steroid drugs, but the production process needs to consume a large amount of diosgenin, which makes the price of saponin rise continuously, increases the production cost, and the production process seriously pollute the environment

Method used

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  • Method using microorganism method to convert androstenedione so as to produce testosterone

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1: Construction of recombinant Y.lipolytica Polh bacterial strain

[0034] Using the fully synthesized gene in the laboratory as a template, the gene of the enzyme is obtained by means of PCR, and connected to the cloning vector to achieve a large amount of amplification of the gene. A large number of amplified 17β-hsd3 and gdh gene fragments were purified and connected to pINA1292 and pYLSC vectors respectively. After successful verification, the model strain Y. lipolytica Polh was transformed. Positive transformants were screened on the resistance plate, inoculated in shake flasks for fermentation, and the product TS in the fermentation liquid was detected by HPLC. The product TS was detected, indicating that the recombinant strain with the conversion of AD to TS was successfully constructed. The present invention finally obtains the Y. lipolytica Polh strain with high-efficiency conversion of AD to TS.

Embodiment 2

[0035] Embodiment 2: the enzyme activity assay of recombinant bacterial strain

[0036] Enzyme activity determination method: determined by HPLC method. The specific method is as follows: take 50 mL of the bacterial solution cultured for 24 hours, centrifuge at 4°C, 8,000 r / min for 5 minutes to collect the bacterial cells, wash twice with 50 mL of Tris-HCl buffer with pH 7.0, and resuspend in 5 ml of the buffer. Ultrasonic crushing with 40% power in an ice bath, working for 2 seconds and resting for 5 seconds, and working time for 10 minutes. Centrifuge at 10,000r / min for 30min to obtain the supernatant, which contains the target protein. The enzyme activity assay method is as follows: 1 mL of enzyme activity assay system includes 0.5 mM NADPH, 200 μM AD (dissolved in methanol), and then add an appropriate amount of enzyme solution. Definition of enzyme activity unit: at 37°C, the amount of enzyme that converts 1 μmol AD ​​into TS in 1 min is defined as 1 enzyme activity uni...

Embodiment 3

[0037] Example 3: Detection of whole cell transformation performance of recombinant strains

[0038] The strain was inoculated in 100 mL of BMGY medium with 5% inoculum, cultured for 24 h, then inoculated with 5% inoculum in a 5L fermenter containing 2L of BMMY medium, and continuously added methanol for induction for 4 days. The cells were recovered by centrifugation, washed twice, redissolved with a certain amount of 50mM Tris-HCl pH 7.5 buffer, and transformed by adding substrate AD. The solvent was methylated-β-cyclodextrin (1:1 molar ratio), the biomass was 200g / L, and the substrate concentration was 5g / L. After 3 feeding whole cell transformations, 15g / L androstenedione was converted into 14.3g / L testosterone, which is the first time at home and abroad to use androstenedione transformation and production in Pichia pastoris by constructing a NADPH coenzyme cycle regeneration system Testosterone, and ultimately achieve the purpose of increasing the conversion rate and eff...

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Abstract

The invention relates to a method recombining Yarrowia lipolytica to efficiently and biologically convert androstenedione so as to produce testosterone and belongs to the field of genetic engineering. The method is characterized in that 17beta-hydroxyl steroid dehydrogenase from human and glucose dehydrogenase from saccharomyces cerevisiae are respectively cloned and excessively co-expressed in Y.lipolytica Polh, and the androstenedione is efficiently converted in the Yarrowia lipolytica to produce the testosterone for the first time at home and abroad by building an NADPH coenzyme circulation system. The enzyme activity determination and intracellular coenzyme level researches of the built genetic-engineering strains show that the recombinant strains can continuously and effectively convert the androstenedione to produce the testosterone. When the conversion temperature is 37 DEG C, conversion pH is 7.5, substrate cosolvent is methylated-beta-cyclodextrin, biomass is 200g/L and substrate concentration is 5g/L, 15g/L androstenedione is converted into 14.3g/L testosterone after three material-supplementing whole-cell conversions, the currently-reported highest level of testosterone production using microorganism conversion is achieved, and a foundation is provided for industrial testosterone production using microorganisms.

Description

technical field [0001] The invention discloses a method for biotransforming androstenedione to produce testosterone, which belongs to the fields of genetic engineering and enzyme engineering. Background technique [0002] Steroidal drugs can be obtained through total synthesis or degradation of natural steroidal compounds and transformation of their functional groups. Steroidal drugs have strong pharmacological effects such as anti-infection, just allergy, anti-virus and anti-shock. With the continuous development of the times, steroid drugs have become the second largest class of drugs after antibiotics. In 2000, the sales of steroid drugs in the global drug market have exceeded 20 billion US dollars, accounting for about 66% of the world's total pharmaceutical sales. %. [0003] Classification of steroid hormone drugs: adrenocortical hormones, including hydrocortisone, prednisone, etc. It can treat Addison's disease, anti-inflammation, anti-allergy, anti-shock, etc.; ana...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/81C12N1/19C12P33/00C12R1/645
CPCC12N9/0006C12N15/815C12N2800/102C12P33/00C12Y101/01051C12Y101/9901
Inventor 饶志明邵明龙张显杨套伟徐美娟
Owner JIANGNAN UNIV
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