Difference between revisions of "Part:BBa K771305"

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TesA with Membrane Anchor 2 (without MS2)

TesA is a cytoplasmic mutant of the periplasmic thioesterase. It is capable of releasing free fatty acids, preventing the fatty acids from being directly harnessed for phospholipid biosynthesis.

Membrane anchor 2 without MS2 is described at BBa_K771007.

TesA is linked to C terminal of Membrane anchor 2 without MS2 by Flexible Linker 3(Fl3).

Fatty acid Biosynthetic Pathway

TesA involves in the biosynthesis of fatty acids in E.coli. The biosynthesis pathway is shown as below:

Fatty acid biosynthesis pathways in E.coli, showing the role of FabG, FabZ, FabI and TesA.

The E. coli fatty acid synthesis is initiated when holo-ACP, NADPH and NADH, acetyl-CoA and malonyl-CoA undergo condensation and subsequent reduction to form butyryl-ACP. These reactions are catalyzed by the malonyl-CoA:ACP transacylase FabD, the ketosynthase FabH, the NADPH-dependent ketoreductase FabG, either the dual-function dehydratase/isomerase FabA or the monofunctional dehydratase FabZ, and the NADH-dependent enoyl reductase FabI. Then the butyryl-ACP is extended via 5-7 rounds of analogous reactions to produce a C14 to C18-ACP either fully saturated or monounsaturated. These extension cycles are catalyzed by either the ketosynthase FabB or FabF in collaboration with FabD, FabG, FabA or FabZ, and FabI. Finally, the full-length fatty acid is released from the corresponding fatty acyl-ACP via hydrolysis by C16-specific thioesterase,TesA.

Result

The solo introduce of TesA with Membrane Anchor 2 (without MS2) alone

Fig.2 shows fatty acid content in supernatant of three groups to evaluate the advantages of membrane anchored TesA. A indicates C16/C18 fatty acids content among three groups. B stands for the total amount of fatty acids among three groups.

As Fig.2 A indicates, 20 hours after induction, E.coli with membrane anchored TesA experienced a 50% increase in C-16, C-18 fatty acids content and total fatty acids (Fig.4 B) in supernatant, compared with E.coli with free TesA.Fatty acids yielded from supernatant went up to 0.71mg/(L·OD). The result support that membrane anchored TesA could efficiently transfer fatty acyl-ACP into desirable fatty acids right beneath inner membrane, thus making it much easier for the final products to diffuse into periplasmic space.

Fig.3 shows fatty acid content in the sediment of three groups to evaluate the advantages of membrane anchored TesA. A indicates C16/C18 fatty acids content among three groups. B stands for the total amount of fatty acids among three groups.

Fig.3, on the other hand, shows fatty acids content in sedimentation also went up by 40%, up to 5.02mg/(L·OD). The result is still within our expectation since fatty acyl-ACP has been removed from the reaction system to form fatty acids. As a result, the chemical equilibrium shifts and more fatty acids would be accumulated. The cooperation of TesA with Membrane Anchor 2 (without MS2), FabG with Membrane Anchor 3, FabZ with Membrane Anchor 4, FabI with Membrane Anchor 4(without VVD) could significantly enhance the production of fatty acid by 9 fold compared with enzymes without membrane anchor.

Fatty acid biosynthesis pathways in E.coli, showing the role of FabG, FabZ, FabI and TesA.

Fatty acid biosynthesis pathways in E.coli, showing the role of FabG, FabZ, FabI and TesA.

Sequence and Features