In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli

Abstract

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-...

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