高产琥珀酸菌株的通量筛选

Throughput screening of a high succinic acid-producing strain

摘以琥珀酸放线杆菌Actinobacillus succinogenes F3-21为出发菌株,分别用吖啶黄、紫外线、紫外线-硫酸二乙酯和亚硝基胍进行诱变,产生突变菌库。用"96孔板培养-HPLC浓缩检测-厌氧瓶复筛"的模式筛选高产突变株。从1056株突变株中,筛选到一株高产菌株Ⅵ-10-C。连续传代10次,产酸水平不变。在5 L发酵罐中补料分批发酵72 h,Ⅵ-10-C产琥珀酸87.6 g/L,生产强度1.22 g/(L·h),糖酸转化率0.66g/g;琥珀酸产量比出发菌提高了30%。代谢通量与关键酶活性分析表明:相比于F3-21,Ⅵ-10-C发酵过程中从磷酸烯醇式丙酮酸节点处流向草酰乙酸的代谢流量增加了28.9%,相对应的磷酸烯醇式丙酮酸羧化激酶(PEPCK)酶活提高了23.5%。结果表明用"96孔板培养-HPLC浓缩检测-厌氧瓶复筛"的模式能快速有效筛选高产琥珀酸菌株。

Screening of high succinie acid-producing strain using the procedure of ＂cultured in 96-well plates, concentrated detection by HPLC and fermentation in anaerobic bottle＂ was studied. The mutant libraries derived from Actinobacillus succinogenes 173-21 were obtained by mutagenesis of acridine yellow, ultraviolet, UV-EMS and NTG treatments respectively. A high succinic acid-producing mutant VI-10-C was selected from 1056 mutants and its yield was not decreased after 10 times of passage. In a 5 L fermentor with fed-batch fermentation by the mutant VI-10-C, 87.6 g/ L succinic acid was obtained at 72 h , which was 30 % higher than that of strain F3-21. And the productivity and yield were 1.22 g/（L. h） and 66 g/g correspondingly. Furthermore comparing with F3-21, the metabolic flux from phosphoenolpyruvate to oxaloacetate in VI-10-C was increased by 28. 9% and the activity of phosphoenolpyruvate carboxykinase increased by 23.5 % correspondingly. The results showed that the throughput screening method, which was ＂cultured in 96-well plates, concentrated detection by HPLC and fermentation in anaerobic bottle＂, was successfully applied in breeding high succinic acid-producing strain.