3.4 | A comparison of competing objectives in alternative alcohol pathways.
Our prior work on fatty acid biosynthesis indicates that coordinated shifts in enzyme concentration can tune average chain length—a critical design objective of engineered systems—without altering total production (Mains et al., 2022). We sought to evaluate this engineering strategy in four pathways for alcohol biosynthesis, where control over length remains difficult. We began by supplementing our first alcohol pathway, which relies on FadD, a CAR, and an aldehyde reductases (AHR), with three additional pathways that extend the core FAS with 1-4 enzymes: (i) an acyl-ACP reductase (ATR), (ii) a TesA, FadD, and an acyl-CoA reductase (ACR2), or (iii) TesA, FadD, a different acyl-CoA reductase (ACR1), and an aldehyde reductase (AHR). As before, we added new enzymes to our base model and optimized poorly characterized kinetic parameters with fits to experimental data (Fig. S5). With our four alternative models in hand, we examined the range of average chain lengths accessible with simple shifts in enzyme concentration. Intriguingly, for all pathways, the breadth of chain lengths decreases as total production increases, and the highest production levels culminate in a single average chain length (Fig. 6). This tradeoff is sharpest for the ATR and ACR1 pathways (Figs. 6B and 6D), where critical enzymes—namely ATR and ACR1—have very narrow substrate specificities. The CAR and ACR2 pathways, by contrast, enable broad control until very high production levels, with the CAR pathway providing the greatest flexibility (Fig. 6A and 6C). The control afforded by this pathway reflects the broad substrate specificities of all enzymes downstream of the FAS.
An analysis of enzyme compositions at high and low average chain lengths reveals the mechanisms of control (Dataset S1). For the CAR, ACR1, and ACR2 pathways, high concentrations of TesA and low concentrations of FabF and FabB facilitate the production of short-chain alcohols, while the opposite shifts encourage long-chain products. This trend is consistent with influence of TesA, FabF, and FabB on fatty acid biosynthesis (Mains et al., 2022). Results also suggest that a reduction in FabA concentration and increase in FabZ can promote the production of short-chain alcohols—perhaps, the result of the preference of FabZ for short substrates—but this effect is less pronounced (i.e., changes in enzyme concentrations are smaller than those of TesA, FabF, and FabB). For the ATR pathway, which has no thioesterase, chain length appeared most sensitive to the concentrations of FabB and FabA (with trends consistent with those exhibited by the other pathways).
For each pathway, we used a total enzyme concentration informed by the overexpression strategy described in an experimental study. The ATR and ACR1 enzymes, however, are not particularly active, and high enzyme concentrations could blunt the tradeoff between chain