Difference between revisions of "Part:BBa K590064"

Latest revision as of 00:44, 2 November 2011

The FabBrick: FabH2, an enzyme for changing up fatty acid biosynthesis

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

Our previous attempts at using FabH2 utilized a construct where FabH2 was coexpressed with Aldehyde Decarbonylase and Acyl-ACP Reductase. This construct(BBa_K590030) did not result in even chain length alkane production, likely due to FabH2 toxicity, leading to low alkane yield (approximately 10 mg/L vs. 170 mg/L for the PetroBrick).

In order to reduce these toxic effects, we cloned FabH2 onto a low copy number pSB3K3 IPTG inducible expression vector to form the FabBrick. 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 time that even chain length alkanes have been recombinantly 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 C14/C16 yield is only approximately 4 mg/L.

GCMS trace confirming C16 alkane produced only upon FabBrick induction. Black trace from the PetroBrick alone. Red trace from the PetroBrick + uninduced FabBrick. Green trace from the PetroBrick + induced FabBrick.

GCMS trace confirming C14 alkane produced only upon FabBrick induction. Black trace from the PetroBrick alone. Red trace from the PetroBrick + uninduced FabBrick. Green trace from the PetroBrick + induced FabBrick.

Reference

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