Integrative genomic mining for enzyme function to enable engineering of a non-natural biosynthetic pathway

TLDR

Biosynthetic production of non‑natural chemicals requires discovery of novel enzyme functions. The study aims to discover enzymes that enable specific production of longer‑chain (C5–C8) alcohols from sugar. They employ a two‑step strategy: first, bioinformatics and molecular modelling mine sequence databases for diverse enzymes catalysing the reaction, and second, computational enzyme design reprograms specificity. The computationally selected enzymes exhibit a median catalytic efficiency 75‑fold higher than naive homologues, and both approaches yield enzymes with >100‑fold specificity, leading to over 10‑fold increases in longer‑chain alcohol production that comprise >95% of total alcohols in vivo.

Abstract

Abstract The ability to biosynthetically produce chemicals beyond what is commonly found in Nature requires the discovery of novel enzyme function. Here we utilize two approaches to discover enzymes that enable specific production of longer-chain (C 5 –C 8 ) alcohols from sugar. The first approach combines bioinformatics and molecular modelling to mine sequence databases, resulting in a diverse panel of enzymes capable of catalysing the targeted reaction. The median catalytic efficiency of the computationally selected enzymes is 75-fold greater than a panel of naively selected homologues. This integrative genomic mining approach establishes a unique avenue for enzyme function discovery in the rapidly expanding sequence databases. The second approach uses computational enzyme design to reprogramme specificity. Both approaches result in enzymes with >100-fold increase in specificity for the targeted reaction. When enzymes from either approach are integrated in vivo , longer-chain alcohol production increases over 10-fold and represents >95% of the total alcohol products.