A Synthetic Biology Approach to Engineer a Functional Reversal of the β-Oxidation Cycle

While we have recently constructed a functional reversal of cycle as a platform for the production of fuels and chemicals by engineering global regulators, and eliminating native fermentative pathways, the system level approach used makes it. difficult to determine which of the many deregulated enzymes are responsible for product synthesis. This, in turn, limits efforts to fine-tune the synthesis of specific products and. prevents, the transfer of the engineered pathway to other organisms: In the reported here, we overcome the aforementioned limitations by using a synthetic biology approach to 111 construct and functionally characterize a reversal of the beta-Oxidation cycle This was achieved through the in vitro kinetic characterization of each functional unit of the core and termination pathways, followed by their in vivo assembly and functional characterization : With this approach, the four functional units of the core pathway, thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydralase/3-hydroxyacyl-CoA dehydralase and in dehydrogenase/trans-enoyl-CoA reductase, were purified and kinetically characterized in vitro. When these four functional units were assembled in vivo in combination with thioesterases as the termination pathway, the synthesis of a variety of 4-C carboxylic acids from a one-turn functional..reversal of the beta-oxidation cycle was realized; T individual expression and modular construction of these well-defined core components exerted the majority of control over product formation, with only highly selective termination pathways resulting in shifts in product formation. Further control over product synthesis Was demonstrated by overexpressing a long-chain thiolase that enables the operation Of multiple turns of the reversal of the beta-oxidation cycle and hence the synthesis of longer-chain carboxylic acids. The welt-defined and self-contained nature of each functional unit makes the engineered reversal of the beta-oxidation cycle "chassis neutral" and hence transferrable to the host of choice for efficient fuel or chemical production.,