Breeding And Fermentation Optimization Of Androstene-4-End-3,17-Dione High Producing Mycobacterium Neoaurum

Steroid drugs, containing steroid nucleus structure in their molecule structures, are the second largest drugs next to antibiotic and have been widely used in clinical applications. Chemical synthesis and microbial transformation are two methods commonly used in producing steroid drugs, of which, the microbial transformation method has obvious advantages. Mycobacteria can degrade phytosterols into androst-4-end-3, 17-dione(AD) and androst-1, 4-end-3, 17-dione(ADD), which are important steroid intermediates that can be converted into other steroid drugs. 3-ketosteroid-△1-dehydrogenase(KSDD) is a key enzyme in the process of microbial degradation of phytosterols, which catalyzes the transformation of AD to ADD. Once the enzyme of KSDD is deficient, the yield of AD will be raised.Strain Mycobacterium neoaurum ZAD, efficiently converting phytosterols into AD and ADD, is preserved in our laboratory. In order to further improve the yield and proportion of AD, atmospheric and room temperature plasma technique was used to mutagenize M. neoaurum ZAD and high AD producing mutant was screened. Besides, the proteomics of the mutant was also investigated in this study. The main contents are as follows:(1) Atmospheric and room temperature plasma(ARTP) mutagenesis technique is used to tread the single-cell suspensions of the original strain, M. neoaurum ZAD. Then H+ dependent dehydrogenase deficient screening method is used as the step of primary screening and re-screening by shaking bottles. Finally, a high AD producing mutant was obtained and designated as M. neoaurum ZADF-4. In the mutant M. neoaurum ZADF-4, the yield of AD reaches 6.28±0.11 g/L, the ratio of AD:ADD is 8:1, and the AD molar yield increases from 48.3% to 60.3%.(2) In shake flask scale, optimization of the fermentation medium composition of M. neoaurum ZADF-4 was carried out and the optimal conditions are as follows: glucose, 20 g/L; soy peptone, 10 g/L; K2HPO4, 2g/L; Mg SO4, 0.3 g/L; substrate, 15 g/L; molar ratio of cosolvent to substrate, 3:1; inoculation amount, 10%(v/v); culture temperature, 30 oC. Based on the optimal conditions, the fermentation result of shake flasks shows AD yield is 6.34±0.21 g/L, then the biotransformation was scaled up in a 5 L fermentor and the finally AD yield reached 6.68±0.12 g/L.(3) In mutant of M. neoaurum ZADF-4, the protein expression differences under two conditions adding phytosterols or not is studied by using two-dimensional gel electrophoresis(2-DE)-tandem mass spectrometry(MS/MS) approach, altogether 16 significant different proteins are identified. Among the enzymes related with AD synthesis pathway, the expression of ethanol dehydrogenase was decreased, while the expressions of S-transferase enzyme and α/β hydrolase were increased significantly.(4) At the transcriptional level, quantitative real-time PCR(q PCR) technology is used to validate 2-DE results. q PCR results showed that, in addition to the expression of alcohol dehydrogenase are not correspond with the previous forecast, the other q PCR results consistent with the 2-DE results.Through the above studies, a high AD producing mutant M. neoaurum ZADF-4 was obtained and the proteomics study of ZADF-4 provides a theoretical guidance for its metabolic pathway control.