Improved Bioproduction of the Nylon 12 Monomer by Combining the Directed Evolution of P450 and Enhancing Heme Synthesis

Abstract

The nylon 12 (PA12) monomer ω-hydroxydodecanoic acid (ω-AmDDA) could be synthesized from lauric acid (DDA) through multi-enzyme cascade transformation using engineered E. coli, with the P450 catalyzing terminal hydroxylation of DDA as a rate-limiting enzyme. Its activity is jointly determined by the heme domain and the reductase domain. To obtain a P450 mutant with higher activity, directed evolution was conducted using a colorimetric high-throughput screening (HTS) system with DDA as the real substrate. After two rounds of directed evolution, a positive double-site mutant (R14R/D629G) with 90.3% higher activity was obtained. Molecular docking analysis, kinetic parameter determination and protein electrophoresis suggested the improved soluble expression of P450 resulting from the synonymous mutation near the N-terminus and the shortened distance of the electron transfer between FMN and FAD caused by D629G mutation as the major reasons for activity improvement. The significantly increased kcat and unchanged Km provided further evidence for the increase in electron transfer efficiency. Considering the important role of heme in P450, its supply was strengthened by the metabolic engineering of the heme synthesis pathway. By combining P450-directed evolution and enhancing heme synthesis, 2.02 ± 0.03 g/L of ω-AmDDA was produced from 10 mM DDA, with a yield of 93.6%.

Engineering Electron Transfer Pathway of Cytochrome P450s

“…Velazquez et al [ 78 ] expressed POR (OMIM:*124015, HNGC:9208) wild type and P228L variant in bacteria, and based on computer and in vitro studies, predicted that the change of proline to leucine might change the rigidity of protein, change the conformation of POR, and lead to a decrease in the rate of electron transfer to cytochrome c and thiazole blue tetrazole (MTT) significantly. Yu et al [ 79 ] used a colorimetric high-throughput screening (HTS) system with DDA as the true substrate to conduct directed evolution and obtained a P450 mutant (R14R/D629G) with higher activity. Molecular docking analysis, kinetic parameter determination, and protein electrophoresis showed that the decrease in electron transfer distance between FMN and FAD caused by the D629G mutation is the main reason for the increased activity.…”