Mining and engineering of pyrroline-5-carboxylate reductase for biocatalytic production of l-pipecolic acid with self-sufficient cofactor recycling

l-pipecolic acid (l-PA) is an essential chiral intermediate for local anesthetics and macrolide antibiotics. To achieve more stable and cost-effective biosynthesis of l-PA from l-lysine (l-Lys), a cascade enzymatic pathway with self-sufficient cofactor recycling was developed, incorporating lysine-6-dehydrogenase (LysDH) and pyrroline-5-carboxylate reductase (P5CR). To overcome bottlenecks in the pathway, Ec-P5CR from Enterococcus casseliflavus was identified as a promising biocatalyst for enhancing l-PA production. For further improvement of l-PA yield, protein engineering was performed on Ec-P5CR. The resulting variant K261W, combined with Rp-LysDH from Rhodobacter pomeroyi DSS-3, achieved significantly enhanced yield of 93 % at 100 mM l-Lys, as well as an impressive yield of 83 % at 500 mM l-Lys. MD simulations revealed that improved hydride transfer efficiency was mainly responsible for the enhanced performance of K261W, leading to shorter distances between catalytic residues and substrates. This work paves the way for efficient and sustainable l-PA synthesis, showcasing the potential of enzyme optimization in industrial applications.