Team:NTU Taida/Circuit1

From 2014.igem.org

NTU-Taida

Fad System

  • Inspiration :

    A locus (fadL) that is required for the utilization of long-chain fatty acids(LCFA) has been mapped and partially characterized in an Escherichia coli mutant . We have shown that the product of the fadL gene is localized in the outer membrane, forms a specific channel for the uptake of long-chain fatty acids. Derivatives of this mutant that can grow on decanoate(termed fadR) are capable to transport long-chain fatty acids into the cell by fadL and fadR sequence.

  • How it works :

    1 LCFA passes the outer membrane through FadL.

    2 LCFA adsorbs to the periplasmic side of the inner membrane.

    3 LCFA moves to the cytosolic side of the innermembrane by flip-flopmechanism.

    4 FadD abstracts LCFA to synthesize LCFA-CoA which can derepress the fad regulon, including fadL, fadD, and fadE. It is also used in lipid biosynthesis.

    5 Dehydrogenation by FadE produces fatty enoyl-CoA, the first compound in the β oxidation pathway

    Through several assay studying, we conclude that the key component for E-coli to absorb LCFA were FadL and FadR. After absorption, E-coliwill breaks down the fatty acids into acyl-coAs. Acyl-coAs bind to the FadR repressor and free the pfadBA promoter, resulting in the transcription of gene sequences below.

  • Background Knowledge :

    Exogenous fatty acids and its acid derivatives influence a wide variety of cellular processes. The processes governing the transport of fatty acids from the extracellular milieu across the membrane are distinct from those underpinning the transport of hydrophilic substrates such as sugars and amino acids. Investigations into fatty acid transport must address three central issues, which are unique to this process:

    (i) the low solubility of fatty acids under aqueous conditions;

    (ii) the physical and chemical parameters of fatty acids, which allows them to readily partition into a lipid bilayer; and

    (iii) the identification of membrane-bound and membrane-associated proteins, which are likely to play pivotal roles in this process.

    In addition, diversity of lipid and protein species in various biological membranes must be a central consideration for investigations directed at defining the biochemical mechanisms governing fatty acid transport .

    Fatty Acid Transport Defined: Given that fatty acids bind to and flip between the two membrane leaflets, it is imperative to define the fatty acid transport process. For the purposes of discussion, fatty acid transport is defined as the net movement of the fatty acid from the outside of the cell to the inside of the cell — or, more simply stated, the movement of the fatty acid from the extracellular space into the intracellular cytosolic compartment.

    The focus of this review is the net movement of exogenous fatty acids across the membrane, with a specific focus on the role of fatty acid transport proteins and fatty acyl coenzyme A (CoA) synthetases. There is considerable evidence showing that the fatty acid transport proteins FadL (from gram-negative bacteria) and Fat1p (the yeast orthologue of mammalian fatty acid transport proteins [FATP]) function in concert with FACS as components of a fatty acid transport apparatus, which results in concomitant transport and activation to CoA thioesters by a process described as vectorial esterification.

  • Reference :

    Nunn, William D., and Robert W. Simons. "Transport of long-chain fatty acids by Escherichia coli: mapping and characterization of mutants in the fadL gene." Proceedings of the National Academy of Sciences 75.7 (1978): 3377-3381. Black, Paul N. "Primary sequence of the Escherichia coli fadL gene encoding an outer membrane protein required for long-chain fatty acid transport." Journal of bacteriology 173.2 (1991): 435-442.

    Maloy, S. R., et al. "Transport of long and medium chain fatty acids by Escherichia coli K12." Journal of Biological Chemistry 256.8 (1981): 3735-3742. APA Black, Paul N., and Concetta C. DiRusso. "Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification." Microbiology and Molecular Biology Reviews 67.3 (2003): 454-472.

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