Figure 4 : Sensitivity analyses of oleochemical pathways. We used a global sensitivity analysis to examine the influence of enzyme concentrations on three distinct biochemical objectives: total production, unsaturated fraction, and average length of oleochemical products. In our models, total production is most strongly influenced by the concentrations of either (i) the thioesterase and a single product-specific enzyme or (ii) two product-specific enzymes, while unsaturated fraction and average chain length are most sensitive to the concentrations of core FAS enzymes.
profiles. For the methyl ketone pathway, our results indicate that thioesterase concentration has an outsized influence on both total production and chain length, but also suggest that FadD and β-ketoacyl-CoA thioesterase (FadM) can affect both objectives. This finding is consistent with in vivo data showing that the thioesterase, FadD, and FadM need overlapping substrate specificities to maximize the production of specific chain lengths. Importantly, severalin vivo studies have showed that overexpression of acyl-coenzyme A dehydrogenase (FadE) can improve titers of methyl ketones, but our sensitivity analysis does not reproduce this influence; this discrepancy suggests that FadE may be overactive in our model (Yan et al., 2020). We conclude our brief comparison of general effects by noting that in both model and experiment, the concentrations of final enzymes in the FAME and FAEE pathways (i.e., the O-methyltransferase (MT) and the wax synthase (WS), respectively) strongly influence total production, perhaps a result of their low activities, relative to other enzymes (Sherkhanov et al., 2016; Steen et al., 2010).
We used our models to investigate several intriguing effects fromin vivo studies. An experimental study of the alkane pathway examined different expression levels of acyl-ACP reductase (AAR) and (ii) an aldehyde deformylating oxygenase (ADO) by using alternative plasmids, promoters, and pseudo-operon configurations (Song et al., 2016). Shifts in the expression of both enzymes had a prominent influence on total production, but not product profile. We recreated this effect by changing the AAR:ADO ratio in our model; production peaked at an intermediate ratio, while average chain length stayed constant (Figs. 5A-5B). This behavior suggests that AAR and ADO catalyze rate-limiting steps and must be overexpressed at similar levels to improve titer. Their inability to affect product profile, in turn, probably reflects their narrow substrate specificities (i.e., C16 and C18). Intriguingly, the same experimental study also showed that the overexpression of FabH and FabB can reduce alkane production. Our model