ENGINEERING OF HIGHLY ENANTIOSELECTIVE AND ACTIVE BIO-CATALYSTS FOR EFFICIENT ASYMMETRIC HYDROXYLATION

Abstract

Regio- and stereo-selective biohydroxylation of non-activate carbon atom is a useful reaction for the preparation of valuable pharmaceutical intermediates. However, the practical application of monooxygenases for the hydroxylation of non-natural substrates is often limited, due to the unsatisfied enantioselectivity, inefficient supply of the necessary co-factor, and narrow substrate range. In this thesis, we successfully improved the enantioselectivity of P450pyr monooxygenase of Sphingomonas sp. HXN-200 by directed evolution for the hydroxylation of N-benzylpyrrolidine to (S)-N-benzyl-3-hydroxypyrrolidine, an important pharmaceutical intermediate. Saturation mutagenesis at the key sites selected from the X-ray structure of P450pyr hydroxylase, coupled with a high throughput MS-base screening method, led to the development of a triple mutant I83H/M305Q/A77S that increased the product ee from 53% to 98%. A recombinant E. coli co-expressing P450pyr monooxygenase and glucose dehydrogenase was engineered for the biohydroxylation with improved NADH regeneration and recycling, increasing 2.5 fold of the product concentration. Moreover, the engineered P450pyr variants were found to hydroxylate other substrates to produce useful compounds either with improved activity such as (S)-N-benzyl-4-hydroxypyrrolidine-2-one (>99%ee) or with improved enantioselectivity such as (S)-N-benzyl-4-hydroxypyperidine-2-one (94%ee).