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

ZHANG Kun et al(College of Life Science and Pharmacy,Nanjing University of Technology,Nanjing,Jiangsu 210009)

Metabolic-engineering directed screening;Rhizopus oryzae;Fumaric acid;Integrated mutagenesis;Propylene alcohol

[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.