Analysis of Interdependent Kinetic Controls of Fatty Acid Synthases

Biocatalytic systems (e.g., multienzyme pathways or complexes)
enable the conversion of simple sugars into complex products under
ambient conditions and, thus, represent promising platforms for the
synthesis of renewable fuels and chemicals. Unfortunately, to date,
many of these systems have proven difficult to engineer without a
detailed understanding of the kinetic relationships that regulate
the concerted action of their constituent enzymes. This study develops
a mechanistic kinetic model of the fatty acid synthase (FAS) of Escherichia coli and uses that model to determine
how different FAS components work together to control the production
of free fatty acidsprecursors to a wide range of oleochemicals.
Perturbational analyses indicate that the modification or overexpression
of a single FAS component can depress fatty acid production (a commonly
observed phenomenon) by sequestering the proteins with which it interacts
and/or by depleting common substrate pools. Compositional studies,
in turn, suggest that simple changes in the ratios of FAS components
can alter the average length of fatty acids but show that specialized
enzymes (i.e., highly specific ketoacyl synthases or thioesterases)
are required for narrow product profiles. Intriguingly, a sensitivity
analysis indicates that two components primarily influenceand,
thus, enable fine control overtotal production, but suggests
that the enzymes that regulate product profile are more broadly influential.
Findings thus reveal the general importance of kinetic considerations
in efforts to engineer fatty acid biosynthesis and provide strategiesand
a kinetic modelfor incorporating those considerations into
FAS designs.