Abstract: In order to improve the ability to produce ethyl acetate, Nakazawaea ishiwadae GDMCC 60786 was mutagenized by atmospheric and room-temperature plasma (ARTP), ethyl methanesulfonate (EMS) and nitrosoguanidine (NTG). The mutants were sequentially screened by using a plate medium containing tributyrin, shake flask fermentation and addition of substrate. The genetic stability, hemolytic activity and in vitro drug resistance of the selected mutant were evaluated. Mutant N5, which was found to be able to produce a high yield of ethyl acetate, had good genetic stability and in vitro safety. After five successive passages, the average production of ethyl acetate was 764.54 mg/L, and the glucose conversion rate was 38.22%, which were 2.90 times and 25.03% higher than those observed with the original strain, respectively. When ethanol was used as a supplementary carbon source, the yield of ethyl acetate was 1 426.81 mg/L; however, upon the addition of acetic acid, N5 did not grow and lost the ability to produce esters, indicating that the strain was more tolerant to ethanol than acetic acid. Meanwhile, the activities of esterase, acetyl-CoA and alcohol acyltransferase in N5 cells were measured to reach a maximum value after 24 h. In summary, this study successfully constructed a set of suitable mutagenesis system for this strain.

Key words: Nakazawaea ishiwadae GDMCC 60786; ethyl acetate; physicochemical mutagenesis; screening of mutants; enzymatic activity; in vitro safety