复合诱变筛选丙烯醇抗性的富马酸高产米根霉菌株

Integrated Mutagenesis to Screen High Fumaric Acid Producing Mutant of Rhizopus oryzae with Propylene Alcohol Resistance

[目的]筛选具有丙烯醇抗性的富马酸高产米根霉菌株。[方法]以溴甲酚绿与丙烯醇平板作为双初筛标准,建立代谢调控选育米根霉富马酸高产菌株的方法。[结果]利用紫外诱变对米根霉孢子的最佳诱变条件分别为紫外灯30 W,垂直照射距离30 cm,诱变时间1 min;化学诱变剂亚硝酸浓度0.1 mol/L,诱变时间10 min。优化条件下,通过紫外和化学的复合诱变获得了1株产量高、稳定性好的米根霉丙烯醇抗性突变株ME-F12,富马酸产量达到50.39 g/L,与原始菌株相比提高23%,连续传代5次后,产量无明显降低,具有较高稳定性,副产物乙醇浓度只有1.87 g/L,明显低于原始菌株。[结论]为微生物发酵法生产富马酸的工业化奠定了扎实的基础。

[ Objective] The research aimed to screen high fumaric acid producing mutant of Rhizopus oryzae. [ Method] Using metabolic engineering method, a two-standard screening approach was devised to select the mutant by bromocresol green and propylene alcohol methods, The enhancement of fumaric acid production from R. oryzae ME-F10 was attempted by ultraviolet （UV） and nitrous acid mutagenesis. [ Result] After a series screenings, it was found that the optimal condition for UV mutagenesis was a one-minute period, the optimal conditions for nitrous acid mutagenesis were a 10-minute period and nirous acid concentration of 0.1 mol/L. A selected mutant by nitrous acid was subjected to an integrated mutagenesis by UV and nitrous acid. After two steps of inducement, one strain named ME-F12 was selected as an ideal one since it exhibited high fumaric acid production of 50.39 g/L with an increase of 23 % than the original strain, It showed high stability with little regression after five generations and it produced little byproduct of 1.87 g/L ethanol,indicating propylene alcohol resistance. [ Conclusion] The study provided stable foundation for the industrial production of fumaric acid with microbiological fermentation.