核黄素工业菌株高通量筛选方法的建立和应用

枯草芽孢杆菌是主要的核黄素工业生产菌之一,高通量筛选技术是选育获得高产核黄素菌株的关键环节。为实现工业菌种选育与高通量筛选技术相结合,对流式细胞分选、液滴微流控分选和96孔板筛选在核黄素工业菌株筛选中的应用进行了研究,并对96孔板筛选方法进行了优化。在流式细胞分析中,来源于同一株工业菌的低产菌株P1与高产菌株R1的细胞荧光与核黄素产量不成正相关。在液滴微流控分析中,P1和R1的发酵液上清荧光与核黄素产量成正相关,然而液滴分选后活细胞的数量很少。在96孔板筛选实验中,振荡培养后P1和R1的发酵液荧光分别为22 264 a.u.、28 647 a.u.;静置培养后荧光分别为7 095 a.u.、10 189 a.u.,核黄素产量与荧光成正相关,且二者荧光差异显著。利用96孔板静置培养的方法对工业菌株S1的突变体库进行筛选,得到的优选菌株核黄素产量为2.53 g/L,相比S1提高了15%。这些结果表明96孔板静置培养-荧光检测筛选可以应用于核黄素工业生产菌产量的提高。

Establishment and Application of High-throughput Screening Method of Riboflavin Industrial Strain

Bacillus subtilis is one of the major riboflavin industrial production bacteria,and high-throughput screening technology is a key step in breeding high-yield riboflavin strains.To achieve the combination of industrial strain breeding and high-throughput screening technology,flow cytometry sorting,droplet microfluidic and 96-well plate screening were applied to screen riboflavin industrial strains,and 96-well plate screening method was optimized.For the flow cytometry analysis,the cell fluorescence of the low-yielding strain P1 and the high-yield strain R1 derived from the same industrial strain were not positively correlated with the riboflavin production.For the droplet microfluidic analysis,the fluorescence of the supernatant of P1 and R1 was positively correlated with the production of riboflavin.However,cell survival rate was extremely low after droplet microfluidic sorting.In the 96-well plate screening experiment,the fermentation liquid fluorescence of P1 and R1 were 22 264 a.u.and 28 647 a.u.after shaking culture respectively;the fluorescence after static culture was 7 095 a.u.and 10 189 a.u.,respectively.The riboflavin production was positively correlated with fluorescence,and the fluorescence difference was significant.The mutant library of the industrial strain S1 was screened by a 96-well plate static culture,and the preferred strain yielded riboflavin 2.53 g/L,which was 15% higher than S1.These results indicate that 96-well plate static culture-fluorescence screening can be applied in increasing riboflavin production by industrial production bacteria.