A kinetic framework for modeling oleochemical biosynthesis in Escherichia coli.

Microorganisms build fatty acids with biocatalytic assembly lines, or fatty acid synthases (FASs), that can be repurposed to produce a broad set of fuels and chemicals. Despite their versatility, the product profiles of FAS-based pathways are challenging to adjust without experimental iteration, and off-target products are common. This study uses a detailed kinetic model of the Escherichia coli FAS as a foundation to model nine oleochemical pathways. These models provide good fits to experimental data and help explain unexpected results from in vivo studies. An analysis of pathways for alkanes and fatty acid ethyl esters (FAEEs), for example, suggests that reductions in titer caused by enzyme overexpression-an experimentally consistent phenomenon-can result from shifts in metabolite pools that are incompatible with the substrate specificities of downstream enzymes, and a focused examination of multiple alcohol pathways indicates that coordinated shifts in enzyme concentrations provide a general means of tuning the product profiles of pathways with promiscuous components. The study concludes by integrating all models into a graphical user interface. The models supplied by this work provide a versatile kinetic framework for studying oleochemical pathways in different biochemical contexts.

A kinetic rationale for functional redundancy in fatty acid biosynthesis.

Cells build fatty acids with biocatalytic assembly lines in which a subset of enzymes often exhibit overlapping activities (e.g., two enzymes catalyze one or more identical reactions). Although the discrete enzymes that make up fatty acid pathways are well characterized, the importance of catalytic overlap between them is poorly understood. We developed a detailed kinetic model of the fatty acid synthase (FAS) of Escherichia coli and paired that model with a fully reconstituted in vitro system to examine the capabilities afforded by functional redundancy in fatty acid synthesis. The model captures-and helps explain-the effects of experimental perturbations to FAS systems and provides a powerful tool for guiding experimental investigations of fatty acid assembly. Compositional analyses carried out in silico and in vitro indicate that FASs with multiple partially redundant enzymes enable tighter (i.e., more independent and/or broader range) control of distinct biochemical objectives-the total production, unsaturated fraction, and average length of fatty acids-than FASs with only a single multifunctional version of each enzyme (i.e., one enzyme with the catalytic capabilities of two partially redundant enzymes). Maximal production of unsaturated fatty acids, for example, requires a second dehydratase that is not essential for their synthesis. This work provides a kinetic, control-theoretic rationale for the inclusion of partially redundant enzymes in fatty acid pathways and supplies a valuable framework for carrying out detailed studies of FAS kinetics.

Abietane-Type Diterpenoids Inhibit Protein Tyrosine Phosphatases by Stabilizing an Inactive Enzyme Conformation.

Protein tyrosine phosphatases (PTPs) contribute to a striking variety of human diseases, yet they remain vexingly difficult to inhibit with uncharged, cell-permeable molecules; no inhibitors of PTPs have been approved for clinical use. This study uses a broad set of biophysical analyses to evaluate the use of abietane-type diterpenoids, a biologically active class of phytometabolites with largely nonpolar structures, for the development of pharmaceutically relevant PTP inhibitors. Results of nuclear magnetic resonance analyses, mutational studies, and molecular dynamics simulations indicate that abietic acid can inhibit protein tyrosine phosphatase 1B, a negative regulator of insulin signaling and an elusive drug target, by binding to its active site in a non-substrate-like manner that stabilizes the catalytically essential WPD loop in an inactive conformation; detailed kinetic studies, in turn, show that minor changes in the structures of abietane-type diterpenoids (e.g., the addition of hydrogens) can improve potency (i.e., lower IC50) by 7-fold. These findings elucidate a previously uncharacterized mechanism of diterpenoid-mediated inhibition and suggest, more broadly, that abietane-type diterpenoids are a promising source of structurally diverse-and, intriguingly, microbially synthesizable-molecules on which to base the design of new PTP-inhibiting therapeutics.