The β-Oxidation pathway, normally involved in the catabolism of fatty acids, can be functionally made to act as a fermentative, cyclic, elongation pathway via the expression of a trans-enoyl-CoA reductase. Our lab previously constructed a growth coupled strain of Escherichia coli capable of n-alcohol production via the reverse β-oxidation pathway and an acyl-CoA reductase. Because the terminal acyl-CoA reduction to alcohol can occur at any elongation length, these strains generate a wide distribution of different chain lengths of n-alcohols. However, tight control of the average chain length, and product profile is desirable as some chain lengths of alcohols are more economically viable products than others. In this study, we explored the ability to control the product profile of this cyclic pathway by tuning the ratio of the three pathway enzymes through a series of in vitro and in vivo experiments. We reconstituted the pathway in vitro with a purified trifunctional complex (FadBA) responsible for the thiolase, enoyl-CoA hydratase and hydroxyacyl-CoA dehydrogenase activities, a trans-2-enoyl-CoA reductase (TER), and an acyl-CoA reductase (ACR). Using this system we found that by controlling the ratio of these three enzymes, and the ratio of NADH and NADPH reactions we could control the average chain length of the alcohol product profile. The reconstituted pathway can be manipulated to produce a range of profiles, from conditions yielding mostly butanol to conditions producing a majority combination of hexadecanol and octadecanol. Using a combination of data from previous characterization of the individual enzymes, and from our in vitro system, including kinetic parameters, reaction time-courses, product profiles, and LC-MS based measurements of intermediate metabolite concentrations, we developed a kinetic model of the fatty alcohol producing reverse β-oxidation pathway. Finally, to demonstrate that the trends we found in vitro are valid strategies in vivo we constructed a reverse β-oxidation, growth-coupled, strain of E. coli with orthogonal, inducible expression of the three enzyme/enzyme complexes.

Tuning enzyme ratios controls fatty alcohol product profiles from reversed β-oxidation

ACS Spring 2022

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