Discovery of New Carbonyl Reductases Using Functional Metagenomics and Applications in Biocatalysis

Enzyme discovery for use in the manufacture of chemicals, requiring high stereoselectivities, continues to be an important avenue of research. Here, a sequence directed metagenomics approach is described to identify short chain carbonyl reductases. PCR from a metagenomic template generated 37 enzymes, with an average 25% sequence identity, twelve of which showed interesting activities in initial screens. Six of the most productive enzymes were then tested against a panel of 21 substrates, including bulkier substrates that have been noted as challenging in biocatalytic reductions. Two enzymes were selected for further studies with the Wieland Miescher ketone. Notably, enzyme SDR-17, when co-expressed with a co-factor recycling system produced the anti-(4aR,5S) isomer in excellent isolated yields of 89% and 99% e.e. These results demonstrate the viability of a sequence directed metagenomics approach for the identification of multiple homologous sequences with low similarity, that can yield highly stereoselective enzymes with applicability in industrial biocatalysis. (Figure presented.)

Conversion of Alcohols to Phosphorothiolates Using a Thioiminium Salt as Coupling Agent

We report a method for the direct and rapid conversion of primary and secondary alcohols to the corresponding phosphorothiolates in yields ranging from 64% to 97%, using as a coupling agent the iminium salt prepared from N,N-dimethylthioformamide and Meerwein’s salt. Selective reaction of primary alcohols in the presence of secondary alcohols is possible. The reaction of secondary alcohols proceeds stereospecifically with inversion of configuration