Efficient breeding method of 4-androstenedione strains

Abstract

The invention provides an efficient breeding method of 4-androstenedione strains. The method includes the five steps of strain culturing, mutagenesis treatment, mutagenic strain culture solution coating and culturing, single colony inoculation and shaking table fermentation, and fermentation broth extraction and thin layer chromatography. By the adoption of the method, miniature fermentation tubes are used, thousands of miniature fermentation tubes can be cultured through one shaking table experiment, in this way, the number of cultured strains is increased per unit time, the culturing time for a preset number of strains is shortened, and then the efficiency of strain screening is improved. Meanwhile, because the miniature fermentation tubes are used, solvent can be directly added into the miniature fermentation tubes for extraction after fermentation is ended; in this way, the extraction steps are simplified, and operation time is shortened. In addition, the bi-phase thin layer chromatography method is adopted for replacing an efficient liquid phase method, an experimenter can detect hundreds of samples each day, in this way, the screening speed of the strains is increased, and the efficiency of strain screening is substantially improved.

Description

technical field [0001] The invention relates to the technical field of biofermentation medicine, in particular to a high-efficiency 4-androstenedione strain breeding method. Background technique [0002] 4-Androstenedione is an important intermediate in the production of steroid hormones. At present, my country's annual output is about 3000-4000 tons. In addition to meeting the needs of domestic pharmaceutical companies, this intermediate also has a certain amount of export. [0003] At present, the production of 4-androstenedione mainly adopts the microbial biotransformation process, that is, the fermentation process, using phytosterols as raw materials, and under the action of mycobacteria, the phytosterols are degraded into 4-androstenediones. Then extract 4-androstenedione from the fermentation broth by organic solvent extraction, and refine it into a qualified intermediate product. [0004] The fermentation level of 4-androstenedione mainly depends on the production c...

AI Technical Summary

Problems solved by technology

[0006] The main difficulty in obtaining high-yield mutant strains is that after the microorganisms are treated with mutagens, the sites of gene mutations are uncertain, resulting in uncertain changes in the physiological and biochemical characteristics of the mutant strains, and the influence on the production of 4-androstenedione The impact is uncertain; and in the mutant strains, the production capacity of 4-androstenedione of the vast majority of mutant strains has no change or decreases, and only a very small number of mutant strains have improved production capacity of 4-androstenedione , the probability of this mutant strain appearing is extremely low, about one in a thousand to one in ten thousand

Therefore, the conventional mutation breeding method needs to use a lot of manual work, a lot of screening work, and it takes months or years to obtain a high-yield strain by chance.

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