Quantitative analysis and engineering of fatty acid biosynthesis in E. coli

Liu, Tiangang; Vora, Harmit; Khosla, Chaitan

doi:10.1016/j.ymben.2010.02.003

Cited by 190 publications

(169 citation statements)

References 44 publications

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“…Early metabolic engineering studies on fatty acid production have been dedicated to optimizing precursor supply and the specificity of plant fatty acyl-ACP thioesterase 4,6,10,30,31 . Engineering the reversal of b-oxidation pathway 7 and a dynamic sensor-regulator system 32 have also led to the production of fatty-acid-derived long chain chemicals and fuels.…”

Section: Discussionmentioning

confidence: 99%

Modular optimization of multi-gene pathways for fatty acids production in E. coli

et al. 2013

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Section: Discussionmentioning

confidence: 99%

Modular optimization of multi-gene pathways for fatty acids production in E. coli

et al. 2013

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“…A key prerequisite is to maximize the specific activity of the fatty acid synthase (FAS) in the biotransforming microbe. Indeed, recent efforts have shown that genetic manipulation of the E. coli FAS can improve its productivity of free fatty acids and their derivatives (7,16,19,20). Notwithstanding these early successes, considerably greater improvements in atom economy and volumetric productivity are warranted, if fatty acid derivatives are to emerge as prominent fuels in the marketplace.…”

Section: Discussionmentioning

confidence: 99%

“…To estimate the relative protein ratios of the reconstituted FAS, we first sought to measure the mRNA level of each subunit and protein levels of selected representative subunits in E. coli BL21(DE3) and its fatty acid overproducing derivative, TL101/pMSD8/pTL58. [TL101 is a fadE knockout strain, pMSD8 leads to overexpression of the E. coli acetyl-CoA carboxylase, and pTL58 is responsible for overexpression of the E. coli thioesterase as well as a medium chain thioesterase from the camphor plant (7).] To do so, we used quantitative PCR (qPCR) for mRNA measurements and Western blotting for protein concentration analysis.…”

Section: Messenger Rna and Protein Concentrations Of Fatty Acid Synthasementioning

confidence: 99%

“…Two sets of experiments were performed to demonstrate the relevance of the above biochemical analysis to fatty acid production in vivo. First, the intracellular concentration of FabF was increased in E. coli TL101/ pTL58, a fadE knockout of BL21(DE3) that produces relatively high levels of fatty acids as a result of overexpression of both the native TesA thioesterase from E. coli as well as a medium chainspecific thioesterase from the camphor plant (7). Insertion of the fabF gene on pTL58 led to expression of this enzyme under control of the P BAD promoter, which, as predicted, markedly decreased fatty acid productivity (Fig.…”

Section: Effects Of Individual Protein Concentrations On the Steady-smentioning

confidence: 99%

“…Cultures from E. coli BL21(DE3), TL101/pMSD8 (a fadE knockout of BL21(DE3) that overexpresses the acetyl-CoA carboxylase), and TL101/pMSD8/pTL58 (that overexpresses the same carboxylase as well as TesA and a second medium chain-specific thioesterase from the camphor plant ) (7,16) were lysed and centrifuged. The supernatants were subjected to native PAGE, followed by Western blot analysis with an anti-ACP antibody.…”

Section: Effects Of Individual Protein Concentrations On the Steady-smentioning

confidence: 99%

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

Yu¹,

Liu

Zhu

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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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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Recent Progress on Microbial Metabolic Engineering for the Conversion of Lignocellulose Waste for Biofuel Production

Sharma

Reena

Kumar

et al. 2013

Biofuels Production

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Quantitative analysis and engineering of fatty acid biosynthesis in E. coli

Cited by 190 publications

References 44 publications

Modular optimization of multi-gene pathways for fatty acids production in E. coli

Modular optimization of multi-gene pathways for fatty acids production in E. coli

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

Recent Progress on Microbial Metabolic Engineering for the Conversion of Lignocellulose Waste for Biofuel Production

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