Abstract: The objective is to further improve the fermentation level of L-glutamic acid while taking high-yield L-glutamic acid-producing Corynebacterium glutamicum GY1 as the research object and using ARTP for global mutagenesis. First,the preparation and regeneration conditions of C. glutamicum GY1 protoplasts were optimized. Then,the optimal ARTP treatment time was selected according to the lethality rate,and then,96-microplate initial screening and shake flask fermentation screening were performed to screen mutants. Finally,the obtained fine mutant strain was subjected to fermentation in a 50 L automated fermenter. Results showed that the optimal conditions for protoplast formation and regeneration were lysozyme concentration 10.0 mg/mL and hydrolysis for 90 min. The optimal treatment time of ARTP was 40 s,and the lethal rate reached 89.6%. After initial screening and shake flask re-screening,the mutant strain YAG117 was obtained,by which the L-glutamic acid content in the shake flask fermentation was 16.3 g/L,which was 13.9% higher than the original strain,and successive 5 generations were genetically stable. Under fed-batch conditions in a 50 L automated fermenter,the concentration of L-glutamic acid was the highest at 36 h,reaching 216.6 g/L,and the glucose conversion rate was 68.87%,which was 12.9% and 10.2% higher than the original strain,respectively. In conclusion,the ARTP mutagenesis of GY1 protoplasts efficiently enriches the types of mutations,accumulates favorable mutations,and increases the ability of C. glutamicum GY1 producing L-glutamic acid. The obtained mutant strain YAG117 shows very promising potential for industrial application.

Key words: L-glutamate, protoplast, ARTP mutagenesis, fermentation