Engineering catechol 1, 2-dioxygenase by design for improving the performance of the cis, cis-muconic acid synthetic pathway in Escherichia coli

• Published in: Scientific reports 2015-08, Vol.5 (1), p.13435-13435, Article 13435
• Main Authors: Han, Li, Liu, Pi, Sun, Jixue, Wu, Yuanqing, Zhang, Yuanyuan, Chen, Wujiu, Lin, Jianping, Wang, Qinhong, Ma, Yanhe
• Format: Article
• Language: English
• Subjects: 631/61/318; 631/61/338; Catechol; Catechol 1,2-dioxygenase; Catechol 1,2-Dioxygenase - genetics; Catechol 1,2-Dioxygenase - metabolism; Crystal structure; E coli; Enzymatic activity; Enzymes; Escherichia coli - physiology; Genetic Enhancement - methods; Humanities and Social Sciences; Metabolic Engineering - methods; Muconic acid; multidisciplinary; Mutation; Protein Engineering - methods; Protein structure; Science; Signal Transduction - physiology; Sorbic Acid - analogs & derivatives; Sorbic Acid - isolation & purification; Sorbic Acid - metabolism; Synthetic Biology - methods
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep13435

Regulating and ameliorating enzyme expression and activity greatly affects the performance of a given synthetic pathway. In this study, a new synthetic pathway for cis, cis -muconic acid ( cc MA) production was reconstructed without exogenous induction by regulating the constitutive expression of the important enzyme catechol 1,2-dioxygenase (CatA). Next, new CatAs with significantly improved activities were developed to enhance cc MA production using structure-assisted protein design. Nine mutations were designed, simulated and constructed based on the analysis of the CatA crystal structure. These results showed that mutations at Gly72, Leu73 and/or Pro76 in CatA could improve enzyme activity and the activity of the most effective mutant was 10-fold greater than that of the wild-type CatA from Acinetobacter sp. ADP1. The most productive synthetic pathway with a mutated CatA increased the titer of cc MA by more than 25%. Molecular dynamic simulation results showed that enlarging the entrance of the substrate-binding pocket in the mutants contributed to their increased enzyme activities and thus improved the performance of the synthetic pathway.