Enhanced thermal and alkaline stability of L-lysine decarboxylase CadA by combining directed evolution and computation-guided virtual screening.

As an important monomer for bio-based nylons PA5X, cadaverine is mainly produced by enzymatic decarboxylation of L-lysine. A key issue with this process is the instability of L-lysine decarboxylase (CadA) during the reaction due to the dissociation of CadA subunits with the accumulation of alkaline cadaverine. In this work, we attempted to improve the thermal and alkaline stability of CadA by combining directed evolution and computation-guided virtual screening. Interestingly, site 477 residue located at the protein surface and not the decamer interface was found as a hotspot in directed evolution. By combinatorial mutagenesis of the positive mutations obtained by directed evolution and virtual screening with the previously reported T88S mutation, K477R/E445Q/T88S/F102V was generated as the best mutant, delivering 37% improvement of cadaverine yield at 50 ºC and pH 8.0. Molecular dynamics simulations suggested the improved rigidity of regional structures, increased number of salt bridges, and enhancement of hydrogen bonds at the multimeric interface as possible origins of the improved stability of the mutant. Using this four-point mutant, 160.7 g/L of cadaverine was produced from 2.0 M Lysine hydrochloride at 50 °C without pH regulation, with a conversion of 78.5%, whereas the wild type produced 143.7 g/L cadaverine, corresponding to 70% conversion. This work shows the combination of directed evolution and virtual screening as an efficient protein engineering strategy.