Rational design on loop regions for precisely regulating flexibility of catalytic center to mitigate overoxidation of prazole sulfides by Baeyer-Villiger monooxygenase.

S-omeprazole and R-rabeprazole are important proton pump inhibitors (PPIs) used for treating peptic disorders. They can be biosynthesized from the corresponding sulfide catalyzed by Baeyer-Villiger monooxygenases (BVMOs). During the development of BVMOs for target sulfoxide preparation, stereoselectivity and overoxidation degree are important factors considered most. In the present study, LnPAMO-Mu15 designed previously and TtPAMO from Thermothelomyces thermophilus showed high (S)- and (R)-configuration stereoselectivity respectively towards thioethers. TtPAMO was found to be capable of oxidating omeprazole sulfide (OPS) and rabeprazole sulfide (RPS) into R-omeprazole and R-rabeprazole respectively. However, the overoxidation issue existed and limited the application of TtPAMO in the biosynthesis of sulfoxides. The structural mechanisms for adverse stereoselectivity between LnPAMO-Mu15 and TtPAMO towards OPS and the overoxidation of OPS by TtPAMO were revealed, based on which, TtPAMO was rationally designed focused on the flexibility of loops near catalytic sites. The variant TtPAMO-S482Y was screened out with lowest overoxidation degree towards OPS and RPS due to the decreased flexibility of catalytic center than TtPAMO. The success in this study not only proved the rationality of the overoxidation mechanism proposed in this study but also provided hints for the development of BVMOs towards thioether substrate for corresponding sulfoxide preparation.

Single-cell enzymatic cascade synthesis of testolactone enabled by engineering of polycyclic ketone monooxygenase and multi-gene expression fine-tuning.

The synthesis of steroids is challenging through multistep steroidal core modifications with high site-selectivity and productivity. In this work, a novel enzymatic cascade system was constructed for synthesis of testolactone by specific C17 lactonization/Δ1-dehydrogenation from inexpensive androstenedione using an engineered polycyclic ketone monooxygenase (PockeMO) and an appropriate 3-ketosteroid-Δ1-dehydrogenase (ReKstD). The focused saturation mutagenesis in the substrate binding pocket was implemented for evolution of PockeMO to eliminate the bottleneck effect. A best mutant MU3 (I225L/L226V/L532Y) was obtained with 20-fold higher specific activity compared to PockeMO. The catalytic efficiency (kcat/Km) of MU3 was 171-fold higher and the substrate scope shifted to polycyclic ketones. Molecular dynamic simulations suggested that the activity was improved by stabilization of the pre-lactonization state and generation of productive orientation of 4-AD mediated by distal L532Y mutation. Based on that, the three genes, MU3, ReKstD and a ketoreductase for NADPH regeneration, were rationally integrated in one cell via expression fine-tuning to form the efficient single cell catalyst E. coli S9. The single whole-cell biocatalytic process was scaled up and could generate 9.0 g/L testolactone with the high space time yield of 1 g/L/h without steroidal by-product, indicating the potential for site-specific and one-pot synthesis of steroid.