Difference between revisions of "Part:BBa K925000"

Revision as of 23:03, 24 September 2012

Delta-12 desaturase

Short description

Delta-12 desaturase involved in an Omega-3 biosynthetic pathway.

Sequence and Features

Description

This part encodes a Δ12 desaturase derived from Synechocystis sp PCC 6803. It is a membrane-bound enzyme able to introduce a double bond at the Δ-12 site in the hydrocarbon chain of oleic acid (18:1, Δ9). This converts the substrate into linoleic acid (18:2 Δ9,12), a polyunsaturated fatty acid (PUFA). Mass spectrometry results show that feeding oleic acid to transformed E. coli, the bacterial membrane composition changes. Linoleic acid is observed.

Characterisation

In order to show desaturase activity of this enzyme, we performed a lipid analysis on fatty acid methyl esters (FAME) by gas chromatography–mass spectrometry GC-MS. Our samples were membrane assays and lipid extracts from E. coli expressing this desaturase.

As a control, the same FAME-GC analysis was performed on unmodified cells, and the lipid profiles were compared. FAME 18:2 (Δ9,12) standard was also run to compare the desaturation pattern to the expected 18:2 of the transformed cells.

Importantly, the cells were grown in the presence of manually added substrate 18:1 (Δ9). These 18:1 fatty acids are not present in unmodified E. coli BL21.

Results

Our results indicate that C18:2 is present in both the lipid extracts and membrane assays from cells transformed with the Δ-12 desaturase, unlike in untransformed cells. Moreover, this 18:2 has the same desaturation pattern as the control, meaning the 18:2 found in the transformed cells is the expected 18:2 (Δ9,12).

Conclusion

Lipid profiles of E. coli transformed with our construct show that this Δ-12 desaturase is able to catalyze the desaturation of oleic acid to give linoleic acid. In this way, this BioBrick^TM can be used to build a biosynthetic pathway for PUFAs and Omega-3 fatty acids, along with other desaturases and elongases.