3.1 | A Refined Model for Fatty Acid Synthesis
In prior work, we developed a detailed kinetic model of the FAS ofE. coli by modeling the nine enzymes necessary to convert malonyl-CoA and acetyl-CoA to free fatty acids (Fig. 1) (Mains et al., 2022; Ruppe et al., 2020; Ruppe and Fox, 2018). The model uses rate equations based on detailed reaction mechanisms reported in the literature and includes separate association and dissociation steps for all heteromeric complexes. In this study, we refined our model by incorporating commonly ignored secondary activities of FabZ (β-hydroxyacyl-ACP dehydratase), FabB (β-ketoacyl-ACP synthase I), and FabF (β-ketoacyl-ACP synthase II).
We began by focusing on unsaturated fatty acid synthesis. Previous studies indicate that FabF and FabZ can produce unsaturated fatty acids, although less efficiently than FabA (β-hydroxy-decanoyl-ACP dehydratase) and FabB (Cronan et al., 1969; Feng and Cronan, 2009; Silbert and Vagelos, 1967). We incorporated this set of secondary activities by adding rate equations describing (i) the allylic rearrangement oftrans -dec-2-enoyl-ACP to cis -dec-3-enoyl ACP by FabZ and (ii) the condensation of cis -dec-3-enoyl-ACP with malonyl-ACP by FabF. The updated model captured the relative differences in unsaturated fractions between reconstituted FASs lacking FabA, FabB, FabZ, or FabF but underpredicted concentrations of unsaturated products (Tables S3 and S4). This finding is consistent with our observation that in vitro systems tend to produce larger unsaturated fractions thanin vivo systems (Mains et al., 2022; Ruppe et al., 2020), which we used to optimize the original model (and which might reasonably bias the final model toward smaller unsaturated fractions)
Next, we incorporated the contributions of FabF and FabB to acyl chain initiation. Despite their primary role in acyl-ACP elongation, FabF and FabB can also generate β-ketobutyryl-ACP, an intermediate that initiates fatty acid synthesis (i.e., it is the primary product of β-ketoacyl-ACP synthase III, or FabH). To add this activity, we introduced reactions for (i) FabB- and FabF-catalyzed decarboxylation of malonyl-ACP, (ii) FabB-catalyzed condensation of malonyl-ACP with acetyl-ACP or acetyl-CoA to form β-ketobutyryl-ACP, and (iii) FabF- catalyzed condensation of malonyl-ACP with acetyl-ACP to form β-ketobutyryl-ACP (Table S1; (Mains et al., 2022)). We fit this newly expanded model to initial rates and product profiles of experimentally reconstituted FASs (Fig. S1). The final optimized model recreated an unusual C18-rich product profile to which it was not fit (Fig. S3)—an indication that it adequately captures the contributions of different core FAS enzymes to fatty acid synthesis.