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

"Functional screening and in vitro analysis reveals thioesterases with enhanced substrate specificity profiles that improve short-chain fatty acid production in Escherichia coli." The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Abstract 13 Short-chain fatty acid (SCFAs) biosynthesis is pertinent to production of biofuels, industrial 14 compounds, and pharmaceuticals from renewable resources. To expand on Escherichia coli 15 SCFA products, we previously implemented a coenzyme A (CoA)-dependent pathway that 16 condenses acetyl-CoA to a diverse group of short-chain fatty acyl-CoAs. To increase product 17 titers and reduce premature pathway termination products, we describe in vivo and in vitro 18 analyses to understand and improve the specificity of the acyl-CoA thioesterase enzyme, which 19 releases fatty acids from CoA. A total of 62 putative bacterial thioesterases, including 23 from 20 the cow rumen microbiome, were inserted into a pathway that condenses acetyl-CoA to an acyl-21 CoA molecule derived from exogenously provided propionic or isobutyric acid. Functional 22 screening revealed thioesterases that increase production of saturated (valerate), unsaturated 23 (trans-2-pentenoate) and branched (4-methylvalerate) SCFAs compared to overexpression of E. 24 coli thioesterase tesB or native expression of endogenous thioesterases. To determine if altered 25 thioesterase acyl-CoA substrate specificity caused the increase in product titers, six of the most 26 promising enzymes were analyzed in vitro. Biochemical assays revealed that the most productive 27 thioesterases rely on promiscuous activity, but have greater specificity for product-associated 28 acyl-CoAs than for precursor acyl-CoAs. Here we introduce novel thioesterases with improved 29 specificity for saturated, branched and unsaturated short-chain acyl-CoAs, thereby expanding the 30 diversity of potential fatty acid products while increasing titers of current products. The growing 31 uncertainty associated with protein database annotations denotes this study as a model for 32 isolating functional biochemical pathway enzymes in situations where experimental evidence of 33 enzyme function is absent.