화학공학소재연구정보센터
Biotechnology Letters, Vol.42, No.12, 2693-2701, 2020
Computer-aided engineering of adipyl-CoA synthetase for enhancing adipic acid synthesis
Objective To enhance adipic acid production, a computer-aided approach was employed to engineer the adipyl-CoA synthetase fromThermobifida fuscaby combining sequence analysis, protein structure modeling, in silico site-directed mutagenesis, and molecular dynamics simulation. Results Two single mutants ofT. fuscaadipyl-CoA synthetase, E210 beta N and E210 beta Q, achieved a specific enzyme activity of 1.95 and 1.84 U/mg, respectively, which compared favorably with the 1.48 U/mg for the wild-type. The laboratory-level fermentation experiments showed that E210 beta N and E210 beta Q achieved a maximum adipic acid titer of 0.32 and 0.3 g/L. In contrast, the wild-type enzyme yielded a titer of 0.15 g/L under the same conditions. Molecular dynamics (MD) simulations revealed that the mutants (E210 beta N and E210 beta Q) could accelerate the dephosphorylation process in catalysis and enhance enzyme activity. Conclusions The combined computational-experimental approach provides an effective strategy for enhancing enzymatic characteristics, and the mutants may find a useful application for producing adipic acid.