Computation-driven redesign of an NRPS-like carboxylic acid reductase improves activity and selectivity
Kun Shi, Ju‐Mou Li, Mu-Qiang Wang, Y. J. Zhang, Zhi‐Jun Zhang, Qi Chen, Frank Hollmann, Jian‐He Xu, Hui‐Lei Yu
Abstract
Engineering nonribosomal peptide synthetases (NRPSs) has been a “holy grail” in synthetic biology due to their modular nature and limited understanding of catalytic mechanisms. Here, we reported a computational redesign of the “gate-keeper” adenylation domain of the model NRPS-like enzyme carboxylic acid reductases (CARs) by using approximate mechanism-based geometric criteria and the Rosetta energy score. Notably, Mab CAR3 mutants ACA-1 and ACA-4 displayed a remarkable improvement in catalytic efficiency ( k cat / K M ) for 6-aminocaproic acid, up to 101-fold. Furthermore, G418K exhibited an 86-fold enhancement in substrate specificity for adipic acid compared to 6-aminocaproic acid. Our work provides not only promising biocatalysts for nylon monomer biosynthesis but also a strategy for efficient NRPSs engineering.