3.3 | A sensitivity analysis of different models.
Enzyme concentration is an important design consideration for engineered pathways, where it can be controlled with carefully selected promoters, ribosome binding sites, and dynamic transcriptional systems (Meyer et al., 2019; Salis et al., 2009; Zhao et al., 2018). We used a global sensitivity analysis to determine how changes in enzyme concentrations influence three important engineering objectives—the total production, unsaturated fraction, and average length of oleochemical products (Fig. 4). Our results indicate that total production is most sensitive to the concentrations of acyl-ACP thioesterase and downstream oleochemical-specific enzymes, while product profile (i.e., unsaturated fraction and average chain length) is most sensitive to concentrations of acyl-ACP thioesterase and core FAS enzymes. These sensitivities are consistent with the experimentally observed improvements in titer (and constant product profiles) that result from the overexpression of oleochemical-specific enzymes (Song et al., 2016; Yan et al., 2020).
The specific effects revealed by our analysis are consistent within vivo studies. We will briefly comment on a few examples: Starting with the alcohol pathway, we found that concentrations of TesA, CAR, and AHR have a pronounced influence total production, while TesA and FabF affect average chain length. The original study of this pathway did not examine each enzyme in isolation—a difficult feat for an experimental analysis—but it found that the incorporation of a highly active AHR could increase alcohol production (Kalim Akhtara et al., 2013); additionally, shifts in fatty acid compositions produced corresponding shifts in alcohol