(719f) Understanding Kinetic Profile of the Reversed ?-Oxidation Pathway to Improve Fatty Alcohol Bioproduction

The β-Oxidation pathway, normally involved in the catabolism of fatty acids, can be functionally reversed to act as a fermentative, iterative, elongation pathway when driven by the activity of a trans-enoyl-CoA reductase. The carbon-carbon bond formation mediated by thiolases, together with the terminal acyl-CoA reduction that can occur on substrates with varied chain lengths, unlock the potential for the production of a wide range of aliphatic compounds (e.g., fatty acids, alcohols, β-hydroxy-carboxylic acids). The inherent properties and commercial value of these oleochemicals are dependent on their functional groups and, chain length. Therefore, tight control of the average chain length and product profile is desirable. Lacking a termination enzyme with a narrow chain length preference, we sought alternative factors that could influence the product profile and pathway flux in the cyclic pathway. In this study, we reconstituted the reversed β-oxidation (R-βOx) pathway in vitro with a purified tri-functional complex (FadBA) responsible for the thiolase, enoyl-CoA hydratase and hydroxyacyl-CoA dehydrogenase activities, a trans-enoyl-CoA reductase (TER), and an acyl-CoA reductase (ACR). Through a combination of a design-of-experiment (DOE)-based exploration and individual enzyme assays, we were able to determine the rate limiting step of the R-βOx pathway. We also demonstrated that by controlling the enzyme ratios and the ratio of NADH and NADPH, the product profile can be tuned from primarily short-chain (C4 and C6) products, up to long-chain profiles of primarily C14 and longer alcohols. Additionally, we investigated how the chain length and distribution of intermediate metabolites correlate with alcohol product profile via LC-MS quantification. Our results emphasize the necessity of careful consideration of enzyme ratios in future studies, and highlight potential enzyme engineering targets for increasing flux in the alcohol-producing R-βOx pathway.