Microbial fatty acid derivatives are emerging as promising alternatives to fossil fuel derived transportation fuels. Among bacterial fatty acid synthases (FAS), the Escherichia coli FAS is perhaps the most well studied, but little is known about its steady-state kinetic behavior. Here we describe the reconstitution of E. coli FAS using purified protein components and report detailed kinetic analysis of this reconstituted system. When all ketosynthases are present at 1 μM, the maximum rate of free fatty acid synthesis of the FAS exceeded 100 μM∕ min. The steady-state turnover frequency was not significantly inhibited at high concentrations of any substrate or cofactor. FAS activity was saturated with respect to most individual protein components when their concentrations exceeded 1 μM. The exceptions were FabI and FabZ, which increased FAS activity up to concentrations of 10 μM; FabH and FabF, which decreased FAS activity at concentrations higher than 1 μM; and holo-ACP and TesA, which gave maximum FAS activity at 30 μM concentrations. Analysis of the S36T mutant of the ACP revealed that the unusual dependence of FAS activity on holo-ACP concentration was due, at least in part, to the acyl-phosphopantetheine moiety. MALDI-TOF mass spectrometry analysis of the reaction mixture further revealed medium and long chain fatty acyl-ACP intermediates as predominant ACP species. We speculate that one or more of such intermediates are key allosteric regulators of FAS turnover. Our findings provide a new basis for assessing the scope and limitations of using E. coli as a biocatalyst for the production of diesel-like fuels.D ue to their high energy density and low water solubility, fatty acids are arguably the most appropriate biofuel precursors. Therefore, fatty acid synthases (FASs) have emerged as attractive engineering targets in society's recent quest for transportation fuels from renewable sources. Among different FASs, the Escherichia coli synthase is perhaps most well understood (1). However, notwithstanding extensive analysis of fatty acid biosynthesis and its regulation in E. coli (2-5), little is known about its steady-state kinetic properties. We therefore sought to undertake systematic kinetic analysis of the fully reconstituted E. coli FAS. It was anticipated that such analysis would provide a fundamentally new basis for assessing the scope and limitations of producing fatty acids using E. coli as a biocatalyst.Fatty acid biosynthesis in E. coli is catalyzed by an enzyme system consisting of nine distinct proteins-FabA, FabB, FabD, FabF, FabG, FabH, FabI, FabZ, and ACP (Fig. 1). Together, they convert one equivalent of acetyl-CoA and 6-8 equivalents of malonyl-CoA into C 14 -C 18 acyl-ACP species. One or two reducing equivalents of NAD(P)H are utilized in each round of chain elongation. Whereas most of the resulting fatty acyl chains are directly harnessed for phospholipid biosynthesis, the cytoplasmic mutant of the periplasmic thioesterase, TesA, is capable of releasing free fatty acids via hydrolysis of acyl-...

Polysaccharides are biopolymers made up of a large number of monosaccharides joined together by glycosidic bonds. Polysaccharides are widely distributed in nature: Some, such as peptidoglycan and cellulose, are the components that make up the cell walls of bacteria and plants, and some, such as starch and glycogen, are used as carbohydrate storage in plants and animals. Fungi exist in a variety of natural environments and can exploit a wide range of carbon sources. They play a crucial role in the global carbon cycle because of their ability to break down plant biomass, which is composed primarily of cell wall polysaccharides, including cellulose, hemicellulose, and pectin. Fungi produce a variety of enzymes that in combination degrade cell wall polysaccharides into different monosaccharides. Starch, the main component of grain, is also a polysaccharide that can be broken down into monosaccharides by fungi. These monosaccharides can be used for energy or as precursors for the biosynthesis of biomolecules through a series of enzymatic reactions. Industrial fermentation by microbes has been widely used to produce traditional foods, beverages, and biofuels from starch and to a lesser extent plant biomass. This review focuses on the degradation and utilization of plant homopolysaccharides, cellulose and starch; summarizes the activities of the enzymes involved and the regulation of the induction of the enzymes in well-studied filamentous fungi.

The entire fatty acid biosynthetic pathway from Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from fourteen purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H into the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multi-enzyme system. At steady state, a maximum turnover rate of 0.5 s−1 was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. By altering these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximum turnover rate of the pathway. Our reconstituted system provides a powerful tool to understand and engineer rate-limiting and regulatory steps in this complex and practically significant metabolic pathway.

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