Upgrading the Alkane Precursor Pool through Thioesterase Engineering

In recent years microbially produced alkanes have become popular as direct drop-in fuel compounds. In 2013 Howard and colleagues introduced a highly modular alkane production pathway demonstrating that modification of the fatty acid precursor pool led to altered alkane production. However, they were unable to generate medium-length (C₅ to C₁₁), branched chain compounds. Here I propose to engineer thioesterase specificity to enable production of medium-length branched free fatty acids that can be converted into respective alkanes. In order to identify residues that are involved in specificity determination we expressed chimeras of CpFatB1 (C₈) and CpFatB2 (C₁₄) in Escherichia coli and measured their fatty acid profiles. We found that a region spanning 100 amino acids around the proposed binding pocket highly contributes to substrate specificity. We will use the Rosetta Enzyme Design Application to identify mutants that enable branched-chain activity in four different thioesterases (BsTES, C_4/6; CpFatB1: C₈; UcFatB2: C₁₂ and CpFatB2: C₁₄). Mutated thioesterases will be expressed in a branched-chain fatty acid production strain and free fatty acids will be extracted and measured via GC-MS. This work will amplify the pool of microbial-derived fuel compounds and provide insight into enzyme specificity determination.