Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering

• A library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242 of adenylosuccinate synthetase in Bacillus subtilis.
• A leaky mutation purAP242N was found to partially reduce the flux towards AMP derived from IMP and increase concentration of GTP while keeping the requirement of ATP synthesis for cell growth.
• PurAP242N may provide a promising application in purine-based product accumulation, which are directly stemming from purine pathway in B. subtilis.

Adenylosuccinate synthetase (EC. 6.3.4.4) encoded by purA in Bacillus subtilis, catalyzing the first step of the conversion of IMP to AMP, plays an important role in flux distribution in the purine biosynthetic pathway. In this study, we described the use of site saturation mutagenesis to obtain a desired enzyme activity of adenylosuccinate synthetase and its application in flux regulation. Based on sequence alignment and structural modeling, a library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242. Other than purA deletion, the leaky mutation purAP242N partially reduced the flux towards AMP derived from IMP and increased the riboflavin synthesis precursor GTP, while also kept the requirement of ATP synthesis for cell growth. PurAP242N was introduced into an inosine-producing strain and resulted in an approximately 4.66-fold increase in inosine production, from 0.088 ± 0.009 g/L to 0.41 ± 0.051 g/L, in minimal medium without hypoxanthine accumulation. These results underline that the directed evolution of adenylosuccinate synthetase could tailor its activities and adjust metabolic flux. This mutation may provide a promising application in purine-based product accumulation, like inosine, guanosine and folate which are directly stemming from purine pathway in B. subtilis.