Relative Activities of the β‑Ketoacyl-CoA and Acyl-CoA Reductases Influence the Product Profile and Flux in a Reversed β‑Oxidation Pathway

The β-oxidation pathway, normally involved in the
catabolism
of fatty acids, can be functionally made to act as a fermentative,
iterative, elongation pathway when driven by the activity of a trans-enoyl-CoA reductase (TER). The terminal acyl-CoA reduction
to alcohol can occur on substrates with varied chain lengths, leading
to a broad distribution of fermentation products in vivo. Tight control of the average chain length and product profile is
desirable, as the chain length greatly influences the molecular properties
and the commercial value. 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 iterative 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 TER, and an acyl-CoA
reductase. Using this system, we determined the rate-limiting step
of the elongation cycle and demonstrated that by controlling the ratio
of these three enzymes and the ratio of NADH and NADPH, we can influence
the average chain length of the alcohol product profile.