Figure 5 . Concentration effects in alkane and FAEE pathways. The FAS supplemented with (A-C) an acyl-ACP reductase (AAR) and an aldehyde deformylating oxygenase (ADO) or (D-F) an acyl-ACP thioesterase specific for medium chains (TesA), a fatty-acid-CoA ligase (FadD), and a wax synthase (WS). (A) Alkane production peaks at a high AAR:ADO ratio, which does not alter (B) average chain length. (C) Overexpression of β-ketoacyl-ACP synthase III (FabH) or β-ketoacyl-ACP synthase I (FabB) decrease alkane production in vivo (black) and in our model (i.e., 1 μM to 100 μM; white with black hatching). (D) Increasing concentrations of WS increase FAEE production, while TesA decreases it; FadD has no effect. (E) High concentrations of TesA increase the production of short-chain acyl-CoAs at the expense of long-chain acyl-CoAs, the preferred substrates of WS. (F) FAEE production decreases as a result. In A-F, the concentrations of pathway enzymes not mentioned are unchanged from Figs. 2B and 3B.
recreates this effect and suggests that it results from a reduction in appropriately sized acyl-ACPs (Fig. 5C). In brief, FabH overexpression increases short-chain acyl-ACPs, at the expense of long chains, while FabB overexpression sequesters long-chain acyl-ACPs in FabBᐧACP complexes (Fig. S10). Both effects reduce the pool of substrates for AAR.
Next, we turned to the FAEE pathway. In an early study of this pathway, TesA overexpression reduced FAEE titers (Steen et al., 2010). We speculated that this effect, which is recreated in our model (Fig. 5D), might result from a mismatch between (i) the pool of available acyl-ACPs and (ii) the substrate specificity of WS. Our model allowed us to explore this mismatch. As expected, high concentrations of TesA increased the production of short-chain acyl-ACPs at the expense of long chains, which are the preferred substrates of WS (Figs. 5E-5F). Our focused analyses of alkane and FAEE production illustrates the importance of kinetic models that capture changes in concentrations of length-specific metabolic intermediates.