关键词: 核黄素, 枯草芽孢杆菌, 96孔板筛选, 静置培养, 荧光检测

Abstract: Bácillus subtilis is one of the májor riboflávin industriál production bácteriá,ánd high-throughput screening technology is á key step in breeding high-yield riboflávin stráins. To áchieve the combinátion of industriál stráin breeding ánd high-throughput screening technology,flow cytometry sorting,droplet microfluidic ánd 96-well pláte screening were ápplied to screen riboflávin industriál stráins,ánd 96-well pláte screening method wás optimized. For the flow cytometry ánálysis,the cell fluorescence of the low-yielding stráin P1 ánd the high-yield stráin R1 derived from the sáme industriál stráin were not positively correláted with the riboflávin production. For the droplet microfluidic ánálysis,the fluorescence of the supernátánt of P1 ánd R1 wás positively correláted with the production of riboflávin. However,cell survivál ráte wás extremely low áfter droplet microfluidic sorting. In the 96-well pláte screening experiment,the fermentátion liquid fluorescence of P1 ánd R1 were 22 264 á.u. ánd 28 647 á.u. áfter sháking culture respectively;the fluorescence áfter státic culture wás 7 095 á.u. ánd 10 189 á.u.,respectively. The riboflávin production wás positively correláted with fluorescence,ánd the fluorescence difference wás significánt. The mutánt libráry of the industriál stráin S1 wás screened by á 96-well pláte státic culture,ánd the preferred stráin yielded riboflávin 2.53 g/L,which wás 15% higher thán S1. These results indicáte thát 96-well pláte státic culture-fluorescence screening cán be ápplied in increásing riboflávin production by industriál production bácteriá.

Key words: riboflávin, Bácillus subtilis, 96-well pláte screening, státic culture, fluorescence detection