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 (C5–C8) 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.
Highlights
The ability to biosynthetically produce chemicals beyond what is commonly found in Nature requires the discovery of novel enzyme function
KIVD belongs to the thiamine pyrophosphate (TPP) enzyme family that is composed of more than 17,000 sequences[12]
Ketoacid decarboxylase GEOs were identified based on sequence similarity to the KIVD used in the current synthetic recursive þ 1 pathway
Summary
The ability to biosynthetically produce chemicals beyond what is commonly found in Nature requires the discovery of novel enzyme function. 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 USDA projects the industrial production of bio-based specialty chemicals to reach B$340 billion USD by 2025, replacing half of our needs for these products from petroleum[2] To accomplish this ambitious goal, non-natural biochemical pathways are needed. In the current synthetic recursive þ 1 pathway, the enzymes LeuA, LeuB, LeuC and LeuD (collectively known as LeuABCD) are recruited to recursively elongate 2-ketobutyrate into longchain 2-ketoacids[3,4] These 2-ketoacids are converted to aldehydes by ketoisovalerate decarboxylase[6] (KIVD) and subsequently to alcohols by alcohol dehydrogenase[3] (ADH; Fig. 1). By replacing KIVD with an enzyme specific for longchain ketoacids, the short-chain substrates were able to re-enter the þ 1 iteration cycle until their conversion to long-chain ketoacids, which are decarboxylated and reduced into longer chain alcohols
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