Coding
FabH2

Part:BBa_K590034

Designed by: Matthew Harger   Group: iGEM11_Washington   (2011-09-15)
Revision as of 22:06, 26 October 2011 by Hargem (Talk | contribs) (→‎Usage and Biology)

FabH2

This part encodes FabH2. [http://2011.igem.org/Team:Washington 2011 University of Washington iGEM Team] has produced even chain length alkanes using this part and the Petrobrick. In addition, expression of this part and the Petrobrick should theoretically produce branched chain alkanes, but we have not been able to demonstrate this effect, possibly due to the absence of the appropriate substrates in E. coli

Usage and Biology

FabH2 is from Bacillus subtilis. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in linear fatty acids. However, FabH2 can also use Isobutyryl-CoA, Isovaleryl-CoA, and 2-Methylbutyryl-CoA (products from Valine, Leucine, and Isoleucine degradation), resulting in 2-methyl branched fatty acid production[1]. In addition, FabH2 has been hypothesized to start fatty acid elongation with a straight 3-carbon unit(propionyl-CoA), yielding odd chain length fatty acids, which could be converted into even chain length alkanes by the Petrobrick. Expression of FabH2 on the same high copy number constitutive plasmid as the PetroBrick( as in Part BBa_K590030) results in slow cell growth, and low alkane yield( under 10 mg/L vs. approximately170 mg/L in the Petrobrick).

Growth curve showing that cells overexpressing FabH2 are growth deficient. ADC AAR DrR FabH2

By moving FabH2 into a low copy number inducible vector(As in The FabBrick) ,we were able to get C14 and C16 alkane production, completing the alkane spectrum from C13 to C17. This is the first time that even chain length alkanes have been recombinately produced.This construct was co-transformed with the Petrobrick in XL1-Blue E. coli. When FabH2 was induced by adding 5uM IPTG, a peak corresponding to the C16 were observed. This was confirmed from the MS spectrum, which had an overall fingerprint consistent with alkane, and a parent ion at a mass of 226, confirming the identity as C16 alkane. In addition, a peak corresponding to the C14 alkane was observed, completing the alkane spectrum from C13 to C17. This is the first tim e that even chain length alkanes have been recombinately produced. This part requires further optimization in order to further increase total alkane yield( currently at approximately 40 mg/L vs 170 mg/L for the Petrobrick), and to increase the amount of C14/C16 alkane yield, as current yield is only approximately 4 mg/L.



GCMS trace confirming C16 alkane produced only upon FabBrick induction.


MS spectrum verifies peak contains C16 alkane.


Zoomed in spectrum. Note the parent ion at a mass of 226.

Reference

1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.








Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 310
    Illegal AgeI site found at 949
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 355
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Parameters
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