(288d) Complementary Enzyme and Metabolic Engineering Strategies for Highly Selective Oleochemical Bioprocesses

Fatty acids and fatty acid derivatives, known as oleochemicals, comprise a $13 billion global market and service a broad array of applications, ranging from fuels to personal care products. The underrepresentation of the medium-chain (eight-to-twelve-carbon) compounds in conventional oleochemical sources has led to resource-intensive processes and unsustainable land-use practices to keep pace with demand for the medium-chain oleochemical products. Industrial biotechnology, however, provides a scalable, sustainable, and selective platform to these products; both the fatty acid biosynthesis cycle and the reverse β-oxidation cycle have been manipulated in various microbial hosts to produce fatty acids and fatty acid derivatives at high titers and productivities. Furthermore, enzyme engineering has been demonstrated as a tool to enhance the selectivity of these biosynthetic routes toward medium-chain products. The goal of this project is to elucidate complementary enzyme and metabolic engineering strategies to achieve highly selective platforms for production of medium-chain oleochemicals.

In the synthetic biology workhorse, Escherichia coli, specificity for medium-chain fatty acids has been primarily demonstrated via mutagenesis of the acyl-ACP thioesterase. Here, we combine random mutagenesis with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a high-throughput screening method to identify an acyl-ACP thioesterase from Umbellularia californica (BTE) with enhanced specificity for 12-carbon substrates. However, to obtain a highly selective platform E. coli strain, other components in fatty acid biosynthesis must be addressed. We employ various metabolic engineering strategies to this end, including the elimination of free fatty acids produced by native thioesterases as well as reassigning desaturation in phospholipid metabolism. Thus, to attain narrower product distributions in vivo, we express our engineered BTE in an E. coli strain deficient in the native thioesterases which may lead to accumulation of long-chain free fatty acids. In addition, the expression of BTE in E. coli results in a mixed product distribution of saturated and unsaturated free fatty acids because the native branchpoint for lipid desaturation occurs relatively early in fatty acid biosynthesis at 10-carbon acyl-ACP substrates. To resolve this, we reassign the essential desaturation to occur later in fatty acid biosynthesis, enabling the 12-carbon thioesterase to only catalyze the cleavage of saturated acyl-ACPs. Finally, we evaluate the effects of acyl-ACP substrate availability in E. coli on the free fatty acid distribution by reconstituting representative and synthetic acyl-ACP concentrations in vitro. By combining these enzyme and metabolic engineering approaches, we aim to identify effective an strategy for developing high-yielding bioprocesses for production of medium-chain free fatty acids.