耐低pH值纳他霉素生产菌株的诱变选育

Mutagenesis Screening of Natamycin-Producing Strain with Low pH Resistance

采用常压室温等离子体(atmospheric and room-temperature plasma,ARTP)诱变对纳他霉素生产菌株褐黄孢链霉菌(Streptomyces gilvosporeus)TUST01进行诱变处理,结合低pH值平板筛选及24孔深孔板高通量筛选获得耐低pH值菌株S.gilvosporeus Y-10。耐酸菌株Y-10在pH4.0平板上生长良好且在pH4.4和pH7.4摇瓶发酵条件下,纳他霉素产量和生物量较出发菌株都有明显提高。在不控制pH值和控制pH6.3的5 L发酵罐条件下,Y-10生物量较出发菌更高,但纳他霉素产量偏低。高生物量表明Y-10具有较好的耐酸性,而纳他霉素产量低可能是由于在5 L发酵罐发酵过程中菌体异常上浮导致菌体没有充分利用碳源和氮源。对Y-10的耐酸机制进行研究,通过比较Y-10与出发菌的胞内三磷酸腺苷水平发现,Y-10较出发菌株有更高的胞内三磷酸腺苷水平,以保证其在酸性条件下的正常生长。而通过气相色谱-质谱联用技术研究发现Y-10细胞膜脂肪酸的不饱和度更低,可能影响了其纳他霉素的发酵生产。

The natamycin-producing strain Streptomyces gilvosporeus TUST01 was mutagenized by atmospheric and room-temperature plasma(ARTP)method,and low pH plate screening combined with 24-well deep well plate screening was used for high-throughput screening of the strain S.gilvosporeus Y-10 with low pH resistance.The acid-resistant strain Y-10 grew well on the plate of pH4.0,and the yield and biomass of natamycin were significantly increased in shaking flask fermentation at pH4.4 and pH7.4.In the 5 L fermentor without controlling pH value and controlling pH at 6.3,the biomass of strain Y-10 was higher than that of the original strain,but the yield of natamycin was low.The high biomass showed that Y-10 had a good acid resistance,while the low yield of natamycin was possibly because the mycelium was abnormally raised during the fermentation in the 5 L fermentor,resulting in insufficient utilization of carbon and nitrogen sources.In the study on the mechanism of acid resistance of Y-10,by comparing the intracellular adenosine triphosphate(ATP)levels of Y-10 and the original strain,it was found that Y-10 had higher intracellular ATP levels than the starting strain,thus ensuring its normal growth under acidic conditions.It was found that the degree of unsaturation of Y-10 cell membrane fatty acids was lower by gas chromatography-mass spectrometry,which may affect the fermentation production of natamycin of Y-10.