Biodiesel can be made using free fatty acids that are microbially produced and consume photosynthetic biomass as a feedstock. The aliphatic chain length in the fatty acid is dictated by a fatty acyl-acyl carrier protein (ACP) thioesterase. Within the Escherichia coli fatty acid biosynthesis pathway, fatty acyl-ACP thioesterase I (TesA) is relatively promiscuous and exhibits mild specificity for myristoyl-ACP (14:0). Here, we employed computational tools to suggest TesA redesigns that would reduce binding to myristoyl-ACP while improving binding for shorter-chain acyl-ACPs, specifically lauroyl-ACP (12:0). By limiting TesA promiscuity, we can adjust the fatty acid profile and furthermore control the chemical properties of the end product (biodiesel or high-value chemicals). Our approach incorporated a predict-design-test mechanism, where libraries of computationally- designed mutants were tested experimentally, and these results were subsequently used to fine-tune our computational methods. We found that seven of the ten tested designs in the third round had improved specificity for lauroyl-ACP over myristoyl-ACP. Relative to wild-type, the top two mutants exhibited 200% and 171% increases in lauroyl-ACP:myristoyl-ACP specificity while maintaining total fatty acid production (4% decrease and 15% increase, respectively). These successful results benchmark the utility of computations in protein engineering.

Computational redesign of acyl-ACP thioesterase for medium-chain fatty acid production

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.