Functional screening and in vitro analysis reveals thioesterases with enhanced substrate specificity profiles that improve short-chain fatty acid production in Escherichia coli

Abstract

Short-chain fatty acid (SCFAs) biosynthesis is pertinent to production of biofuels, industrial compounds, and pharmaceuticals from renewable resources. To expand on Escherichia coli SCFA products, we previously implemented a coenzyme A (CoA)-dependent pathway that condenses acetyl-CoA to a diverse group of short chain fatty acyl-CoAs. To increase product titers and reduce premature pathway termination products, we describe in vivo and in vitro analyses to understand and improve the specificity of the acyl-CoA thioestera enzyme, which releases fatty acids from CoA. A total of 62 putative bacterial thioesterases, including from the cow rumen microbiome, were inserted into a pathway that condenses acetyl-CoA to an acyl-CoAmolecule derived from exogenously provided propionic or isobutyric acid. Functional screening revealed thioesterases that increase production of saturated (valerate), unsaturated (trans-2-pentenoate) and branched (4-methylvalerate) SCFAs compared to overexpression of E. coli thioesterase tesB or native expression of endogenous thioesterases. To determine if altered thioesterase acyl-CoA substrate specificity caused the increase in product titers, six of the most promising enzymes were analyzed in vitro. Biochemical assays revealed that the most productive thioesterases rely on promiscuous activity, but have greater specificity for product-associated acyl-CoAs than for precursor acyl-CoAs. Here we introduce novel thioesterases with improved specificity for saturated, branched and unsaturated short-chain acyl-CoAs, thereby expanding the diversity of potential fatty acid products while increasing titers of current products. The growing uncertainty associated with protein database annotations denotes this study as a model for isolating functional biochemical pathway enzymes in situations where experimental evidence of enzyme function is absent.