Ketosynthase mutants enable short-chain fatty acid biosynthesis in E. coli.

Cells build fatty acids in tightly regulated assembly lines, or fatty acid synthases (FASs), in which ß-ketoacyl-acyl carrier protein (ACP) synthases (KSs) catalyze sequential carbon-carbon bond forming reactions that generate acyl-ACPs of varying lengths-precursors for a diverse set of lipids and oleochemicals. To date, most efforts to control fatty acid synthesis in engineered microbes have focused on modifying termination enzymes such as acyl-ACP thioesterases, which release free fatty acids from acyl-ACPs. Changes to the substrate specificity of KSs provide an alternative-and, perhaps, more generalizable-approach that focuses on controlling the acyl-ACPs available for downstream products. This study combines mutants of FabF and FabB, the two elongating KSs of the E. coli FAS, with in vitro and in vivo analyses to explore the use of KS mutants to control fatty acid synthesis. In vitro, single amino acid substitutions in the gating loop and acyl binding pocket of FabF shifted the product profiles of reconstituted FASs toward short chains and showed that KS mutants, alone, can cause large shifts in average length (i.e., 6.5-13.5). FabB, which is essential for unsaturated fatty acid synthesis, blunted this effect in vivo, but exogenously added cis-vaccenic acid (C18:1) enabled sufficient transcriptional repression of FabB to restore it. Strikingly, a single mutant of FabB afforded titers of octanoic acid as high as those generated by an engineered thioesterase. Findings indicate that fatty acid synthesis must be decoupled from microbial growth to resolve the influence of KS mutants on fatty acid profiles but show that these mutants offer a versatile approach for tuning FAS outputs.

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.

Kinetically guided, ratiometric tuning of fatty acid biosynthesis.

Cellular metabolism is a nonlinear reaction network in which dynamic shifts in enzyme concentration help regulate the flux of carbon to different products. Despite the apparent simplicity of these biochemical adjustments, their influence on metabolite biosynthesis tends to be context-dependent, difficult to predict, and challenging to exploit in metabolic engineering. This study combines a detailed kinetic model with a systematic set of in vitro and in vivo analyses to explore the use of enzyme concentration as a control parameter in fatty acid synthesis, an essential metabolic process with important applications in oleochemical production. Compositional analyses of a modeled and experimentally reconstituted fatty acid synthase (FAS) from Escherichia coli indicate that the concentration ratio of two native enzymes-a promiscuous thioesterase and a ketoacyl synthase-can tune the average length of fatty acids, an important design objective of engineered pathways. The influence of this ratio is sensitive to the concentrations of other FAS components, which can narrow or expand the range of accessible chain lengths. Inside the cell, simple changes in enzyme concentration can enhance product-specific titers by as much as 125-fold and elicit shifts in overall product profiles that rival those of thioesterase mutants. This work develops a kinetically guided approach for using ratiometric adjustments in enzyme concentration to control the product profiles of FAS systems and, broadly, provides a detailed framework for understanding how coordinated shifts in enzyme concentration can afford tight control over the outputs of nonlinear metabolic pathways.

High-Throughput, Fluorescence-Based Esterase Activity Assay for Assessing Polysorbate Degradation Risk during Biopharmaceutical Development.

PURPOSE: Hydrolytic degradation of polysorbate during 2-8°C storage of monoclonal antibody drug products has been attributed to residual enzymes (e.g., esterases) from bioprocessing steps. Robust detection of esterase activity using sensitive, non-polysorbate surrogate substrates can provide an alternate method to assess polysorbate degradation risk, if the correlation between the esterase activity and polysorbate degradation is established. METHODS: A general esterase activity assay was developed as a monitoring and characterization tool during bioprocess development of monoclonal antibodies. RESULTS: We report a fluorescence plate-based assay for quantifying esterase activity, utilizing 4-methylumbelliferyl caprylate (MU-C8) as the esterase substrate. The assay was first assessed for substrate, inhibitor and pH specificity using both model enzymes and purified protein samples. The assay was then extensively tested to understand sample matrix effects on activity rates. CONCLUSIONS: The use of this high-throughput method will allow for rapid characterization of protein samples in under three hours. The esterase activity correlated directly with polysorbate degradation and can provide valuable information on polysorbate degradation risk throughout drug development.

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.