Integrative Genomic Mining for Enzyme Function to Enable Engineering of a Non-Natural Biosynthetic Pathway

The design of biosynthetic pathways to produce chemicals beyond what is commonly found in Nature requires the ability to discover novel enzyme function. In this study we demonstrated the use of two approaches to discover enzymes that enabled specific production of long chain (C₅-C₈) alcohols from sugar. In the first approach, we combined bioinformatics and molecular modeling to mine sequence databases for enzymes capable of efficiently decarboxylating long chain ketoacids. This integrative genomic mining approach establishes a unique avenue for enzyme function discovery in the rapidly expanding sequence databases. In the second approach we used computational enzyme design to reprogram enzyme specificity.  Both approaches led to enzymes with >100-fold increase in specificity for the targeted reaction. When enzymes from either approach were integrated in vivo, long chain alcohol production levels increased over 10-fold and represented >95% of the total alcohol products produced.