Computation-driven redesign of an NRPS-like carboxylic acid reductase improves activity and selectivity
Kun Shi (East China University of Technology)
Ju Mou Li (East China University of Technology)
Mu Qiang Wang (East China University of Technology)
Yi Ke Zhang (East China University of Technology)
Zhi Jun Zhang (East China University of Technology)
Qi Chen (East China University of Technology)
Frank Hollmann (TU Delft - BT/Biocatalysis)
Jian He Xu (East China University of Technology)
Hui Lei Yu (East China University of Technology)
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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, MabCAR3 mutants ACA-1 and ACA-4 displayed a remarkable improvement in catalytic efficiency (kcat/KM) 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.
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