title: Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering.
abstract: 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 purA(P242N) 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. PurA(P242N) was introduced into an inosine-producing strain and resulted in an approximately 4.66-fold increase in inosine production, from 0.088±0.009g/L to 0.41±0.051g/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.
authors.affiliation: Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China; Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China.
authors.forename: Xiaoyue
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China; Laboratory of Systems Biology and Biofuels, School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450000, People's Republic of China; Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China.
authors.forename: Guanglu
authors.initials: G
authors.lastname: Wang
authors.affiliation: Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China; Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China.
authors.forename: Xinli
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China; Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China.
authors.forename: Jing
authors.initials: J
authors.lastname: Fu
authors.affiliation: Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China; Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China.
authors.forename: Tao
authors.initials: T
authors.lastname: Chen
authors.affiliation: Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China; Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China. Electronic address: zww@tju.edu.cn.
authors.forename: Zhiwen
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China; Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China.
authors.forename: Xueming
authors.initials: X
authors.lastname: Zhao
mesh_terms: D000265:Adenylosuccinate Synthase / Q000235:genetics* / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D007288:Inosine / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D012256:Riboflavin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007288:Inosine; D000265:Adenylosuccinate Synthase; D012256:Riboflavin
keywords: Adenylosuccinate synthetase; Bacillus subtilis; Directed evolution; Inosine; Riboflavin