Abstract
l-asparaginase has high application value in medicine and food industry, but the low yield limits its application. In this study, we designed a synthetic system in Bacillus subtilis to produce l-asparaginase by improving gene expression and optimizing the fermentation agitation speed. Gene expression was improved by respectively increasing transcription levels and translation speeds through screening promoters and RBS sequences. With the optimal promoter, P43, and the synthetic RBS sequence, the yield obtained in a shake flask was 371.87 U/mL, which was 2.09 times that with the original strain. To further enhance production in a 5-L fermenter, a multistage agitation speed control strategy was adopted, involving agitation at 600 rpm for the first 12 h, followed by a gradual increase in speed to 900 rpm, which resulted in the highest yield of l-asparaginase, 5321 U/mL, after 42 h of fermentation.