Xinqi Xu,Fahui Xu,Jingjing Chen,Shumin Wang,Xialian Wang,Bingmei Su,Lian Xu,Juan Lin
Baeyer-Villiger monooxygenases (BVMOs) can catalyze the asymmetric sulfoxidation to form pharmaceutical prazoles in environmentally friendly approach. In this work, the thermostable BVMO named PockeMO had high sulfoxidation activity towards rabeprazole sulfide to form ( R )-rabeprazole but demonstrated significant overoxidation activity to form undesired sulfone by-product. To address this issue, the enzyme was engineered based on the computer assisted comparison for the substrate binding conformations. A mutant S482Y/L532Q (MU2) was obtained with much alleviated overoxidation activity and enhanced sulfoxidation activity towards rabeprazole sulfide. The catalytic efficiency for ( R )-rabeprazole oxidation decreased 35 folds and increased 40 folds for the sulfoxidation of lansoprazole sulfide. The structural mechanism for the selectivity improvement was illuminated to find a selectivity decision pocket that was conservatively present in BVMOs composed of 4 loops. In upscaled reaction system, the substrate loading for MU2 increased from 40 mM to 100 mM for the synthesis of enantiopure ( R )-rabeprazole. The sulfone content decreased from 16.6 % to 1.2 % compared to PockeMO. Lansoprazole sulfide could also be fully converted into enantiopure ( R )-lansoprazole at 50 mM in 4 h by MU2 while PockeMO almost did not have activity. This work indicated the synthetic applicability of MU2 for active pharmaceutical ( R )-prazoles.
Xinqi Xu,Jinchang Zhong,Bingmei Su,Lian Xu,Xiaokun Hong,Juan Lin
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/Δ 1 -dehydrogenation from inexpensive androstenedione using an engineered polycyclic ketone monooxygenase (PockeMO) and an appropriate 3-ketosteroid-Δ 1 -dehydrogenase ( Re KstD). 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 ( k cat / K m) 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, Re KstD 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.
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