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Preparation of boldenone by sequential conversion of Arthrobacter simplex and genetically-engineered yeast strain

A technology of genetically engineered strains and Arthrobacter simplex is applied in the directions of genetic engineering, plant genetic improvement, biochemical equipment and methods, etc. It can solve the problems such as the lack of Arthrobacter simplex and yeast strains, and achieve good application value and promotion prospects. Improve coenzyme utilization, easy to achieve effect

Active Publication Date: 2019-07-05
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant research on the joint fermentation of BD produced by Arthrobacter simplex and yeast strains

Method used

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  • Preparation of boldenone by sequential conversion of Arthrobacter simplex and genetically-engineered yeast strain
  • Preparation of boldenone by sequential conversion of Arthrobacter simplex and genetically-engineered yeast strain
  • Preparation of boldenone by sequential conversion of Arthrobacter simplex and genetically-engineered yeast strain

Examples

Experimental program
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Effect test

Embodiment 117

[0064] Example 1 Acquisition of 17β carbonyl reductase gene and construction of Pichia pastoris genetic engineering strain

[0065] (1) Acquisition of 17β carbonyl reductase gene ayr1

[0066] Using the Saccharomyces cerevisiae W303 genome as a template, amplify the 17β carbonyl reductase (GenBank number: KZV10489.1) encoding gene ayr1 (GeneID: 854682), design the primer pair as follows, and recover the PCR product with GelExtraction Kit (Omega, USA) , the recovered fragment was connected to the vector pPIC3.5K, the recombinant plasmid pPIC3.5K-ayr1 was constructed, and transformed into E.coli JM109 to obtain a genetically engineered strain containing the target gene. The bacteria solution PCR verification and double enzyme digestion verification showed that the recombinant plasmid pPIC3.5K-ayr1 was successfully constructed (such as figure 1 and figure 2 shown). By sequencing the above clones and comparing them with the obtained ayr1 sequence, the sequencing results were c...

Embodiment 2

[0086] The establishment of embodiment 2 double bacterium sequence catalytic system

[0087] In the following examples, the effects of Pichia genetically engineered strains on AD or ADD, the simultaneous transformation and sequential transformation of Arthrobacter simplex and Pichia genetically engineered strains on the synthesis of BD, and the effects of simple Arthrobacter reaction systems were investigated respectively in the following examples. Effect of inactivation on BD synthesis.

Embodiment 2-1

[0089] Pick Pichia pastoris genetic engineering strain P.pastoris GS115AYR1 S.c Single clone, inoculated into 30mL BMGY, 30°C, 200r / min, shake to OD 600 =5-6; Centrifuge at 1500g-3000g at room temperature for 5min, collect the cells, remove the supernatant, resuspend the cells with 30mL BMMY, and induce expression; every 24h, add methanol to a final concentration of 0.5% to continue induction for 4 days; after induction for 4 days Add 5g / L of substrate AD (or ADD) and HP-β-CD with a molar ratio of 1:1 to the fermented broth, 30°C, 200r / min to continue conversion for 4 days, sample 1mL, and equal volume of acetic acid Ethyl ester ultrasonic extraction, HPLC analysis. Such as Figure 5 As shown, Pichia pastoris genetic engineering strain P.pastoris GS115AYR1 S.c After the growing cells converted the substrates AD and ADD, the yields of the corresponding products TS (testosterone) and BD were 59% and 69%, respectively. This indicated that ADD was more suitable as a substrate ...

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Abstract

The invention relates to a production method of steroidal drugs, in particular to a method of preparing a boldenone steroidal compound by sequential fermentation of Arthrobacter simplex and a genetically-engineered yeast strain. The method comprises: providing a Pichia pastoris genetically-engineered strain capable of efficiently expressing 17beta carbonyl reductase; providing a process of fermenting the compound AD by microbial sequential conversion to prepare a compound BD, wherein the method refers to fermenting the AD via Arthrobacter simplex, deactivating the reaction system, and preparing the compound boldenone through sequential fermentation of the Pichia pastoris genetically-engineered strain.

Description

[0001] Technical field: [0002] The invention relates to a production method of steroidal drugs, in particular to a method for sequentially fermenting and preparing boldenone steroidal compounds using Arthrobacter simplex and genetically engineered yeast strains. [0003] Background technique: [0004] Boldenone (BD), also known as Boldenone, is a white or off-white crystalline powder with a molecular formula of C 19 h 26 o 2 . Boldenone is a derivative of testosterone, so it inherits most of the properties of testosterone, and has male hormone ability and protein synthesis ability. Boldenone can keep the nitrogen balance of muscle fiber cells positive at any time, accelerate the synthesis of protein in muscle fiber cells, and expand the muscle fiber cells, which has a great effect on strengthening muscles and endurance of physical exercisers. For a long time, there are not many raw material manufacturers of Boldenone in China, and most of them are synthesized by chemical ...

Claims

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Application Information

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IPC IPC(8): C12P33/00C12N15/53C12R1/84C12R1/06
CPCC12P33/00C12N9/0006C12Y101/01184
Inventor 王敏申雁冰汤睿夏梦雷骆健美屠琳娜耿宇菡
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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