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.