Biocatalytic systems (e.g., multi-enzyme pathways) 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 <i>Escherichia coli</i>, 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. Multi-parameter 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. Most intriguingly, results indicate that two components primarily influence—and, thus, enable fine control over—total production, while 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 new, experimentally validated strategies for incorporating those considerations into FAS designs.

Kinetically guided, ratiometric tuning of fatty acid biosynthesis

2019 Spring National Meeting

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