高产辅酶Q10类球红细菌的选育及发酵优化

Breeding and fermentation optimization of Rhodobacter sphaeroides with high yield of coenzyme Q10

辅酶Q10(coenzyme Q10,CoQ10)是一种淡黄色的脂溶性醌类,常见于动植物和微生物的细胞内膜中,是天然的抗氧化剂和细胞代谢激活剂。该研究以1株具有CoQ10生产能力的类球红细菌(Rhodobacter sphaeroides)为出发菌株,其在摇瓶水平上初始CoQ10产量为136.47 mg/L,通过常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变和多孔板高通量筛选相结合的方法,确定了ARTP诱变处理的时间为120 s,致死率为90%。在孔板初筛阶段,从5184株菌的突变菌库采用高通量筛选方法获得8株高产菌株,经过复筛得到1株CoQ10的高产突变菌株7p22,其摇瓶水平CoQ10产量达到158.44 mg/L,相比出发菌株提高了16.1%。验证其遗传稳定性后,进一步优化了碳源、前体物质、金属离子等,提高了辅酶Q10的产量。最后,在5 L发酵罐上对突变菌株进行培养条件的优化,CoQ10产量提高至1640.6 mg/L。该研究所使用的结合筛选因子和Craven test检测法的高通量筛选方法可以实现简单、高效地获得高产CoQ10突变菌株。

Coenzyme Q10(CoQ10)is a light yellow and fat soluble quinone commonly existed in the inner cell intima of animals,plants and microorganisms.It is a natural antioxidant and activator of cell metabolism.In this study,a Rhodobacter sphaeroides that can produce CoQ10 was used as the starting strain.Its initial CoQ10 titer was 136.47 mg/L at the shaking flasks.Through the combination of ARTP mutation and porous plate based high-throughput screening,it was determined that the time of ARTP mutation treatment was 120 s and the lethal rate was 90%.In the initial screening stage of the orifice plate,eight high-yielding strains were obtained from the mutant library of 5184 strains by high-throughput screening.After re-screening,a high-yielding mutant strain 7p22 was obtained.The CoQ10 production was 158.44 mg/L,which was 16.1%higher than that of the original strain.In addition,after verifying its genetic stability,the carbon source,precursor substances and metal ions were further optimized to improve the production of coenzyme Q10.Finally,the culture conditions of mutant strains were optimized in 5 L fermenter.The CoQ10 production was further increased to 1640.6 mg/L.The high-throughput screening method combined with the screening factor and the Craven test assay used in this study can achieve simple and synergistic acquisition of high-yielding CoQ10 mutant strains.