Asymmetric Cascade Reactions with Hydroxynitrile Lyases

Hydroxynitrile lyases (HNLs) catalyse the asymmetric addition of HCN to carbonyl compounds to give cyanohydrins, a C-C bond forming reaction that has found broad application in academia and industry. Since cyanohydrins are versatile building blocks that can be further transformed into a wide variety of products, multi- enzymatic one-pot reactions (cascades) incorporating HNLs could provide convenient access to various chiral alcohol derivatives. To date, however, this approach is limited largely by the low number of established nitrile- converting enzymes. Recently, nitrile reductases have been identified as a novel class of nitrile-converting enzymes in bacteria. The enzyme QueF catalyses the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1) as a key step in the biosynthesis of the nucleoside queuosine. Within the proposed project, QueF variants obtained by site-directed mutagenesis will be used for the biocatalytic reduction of cyanohydrins. The nitrile reductase will be thoroughly investigated with respect to substrate scope, enantioselectivity and optimal reaction conditions, and it will be co-expressed with a suitable cofactor-regenerating enzyme in a single host. This nitrile-reducing "designer cell" will then be coupled with HNL-catalysed cyanohydrin formation in a multi-enzymatic cascade system, allowing the one-pot conversion of carbonyl compounds into enantiomerically enriched beta-amino alcohols.