Directed Evolution Of Adenylosuccinate Synthetase And Its Application In Metabolic Engineering

Adenylosuccinate(sAMP) synthetase, whihch is encoded by purA in Bacillus subtilis, catalyzes the first step of the conversion of IMP to AMP, is generally considered as one of the most important enzymes involved in the de novo biosynthesis of purine metabolites. In previous report, inaction of purA could enhance the metabolic flow through purine pathway and consequently increase the production of inosine, guanosine and riboflavin. However, degeneration of purA gene drastically decreased the growth rate and cause a negative effect on fermentation.In this study, sAMP synthetase evolution was accomplished by targeted mutations using site-saturation mutagenesis(SSM). Furthermore, the optimal leaky mutation was introduced into inosine-producing B. subtilis to investigated the influence of purA leaky mutation on inosine production.Firstly, we established a host strain denoted as SS2 which its comK gene was overexpressed and pur A gene was knocked-out. Then, the template plasmid pSSN-purA for SSM was constructed. we selected two candidate saturation mutagenesis sites(Thr238 and Pro242) were selected and the SSM library was established. Optimal leaky mutation(P242N) was selected according to the growth condition and riboflavin production. The growth condition was in accord with the wild type, while the mutated sAMP synthetase activity showed a significantly reduced(53.6%) than that of wild type. Therefore, the blockage of IMP to AMP conversion was partly relieved due to the leaky mutation of purA.In order to test whether purAP242 N mutation could be a possible approach to enhance inosine production in B. subtilis, several gene-targeted mutations including knock-out of purR, overexpress purF were introduced into B. subtilis 168 step by step. Then deoD and pupG which encoding to purine nucleoside phosphorylase catalyzed inosine converse to hypoxanthine were knocked out and the mutant was donated as I12. Finally, I15 was constructed when purAP242 N mutation was introduced into I12 genome. The fermentation experiment showed that I15 led to approximately 4-fold enhancement in inosine production than that of I12.