Part:BBa_K925000

Delta-12 desaturase

Short description

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

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 426
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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.

References

LIVORE V., TRIPODI K., UTARRO A., 2007. Elongation of polyunsaturated fatty acids in trypanosomatids. FEBS Journal, 274: 264–274.

Contribution From NNU-China 2022

Group: [ https://2022.igem.wiki/nnu-china/]

Author: Yaru Jiang

Delta-12 desaturase

        Delta 12 Desaturase (Δ12 desaturase), a trans-membrane enzyme, can introduce a double bond at the Δ-12 site in the hydrocarbon chain of oleic acid (C18:1, Δ9). This converts the substrate to linoleic acid (C18:2 Δ9,12), which is further used as a substrate to produce EPA in the alternating action of desaturases and elongases[1]. Δ12 desaturase from Synechocystis sp PCC 6803 (SyElo9) was registered in 2012. Here, we verified that SyElo9 had functional activity in Y.lipolytica. First, we knocked out the endogenous Δ12 desaturase (encoded by the Fad2 gene) in Y. lipolytica polf to facilitate the examination of the extension activity for SyElo9. The gas chromatography results showed that overexpression of SyElo9 in strain polf ΔylFad2 can convert oleic acid to linoleic acid (Fig 1). Further, the introduction of SyElo9 into the Y. lipolytica polf genome and fermentation revealed a significant increase in the proportion of linoleic acid, up to 59.895% (Fig 1). These results provide references for future iGEM team to select suitable sources of Δ12 desaturase.

Fig 1. The fatty acid distribution in various strains

Reference

[1]Gombos, Z., Wada, H., Varkonyi, Z., Los, D. A., & Murata, N. (1996). Characterization of the Fad12 mutant of Synechocystis that is defective in Δ12 acyl-lipid desaturase activity. Biochimica et Biophysica Acta (BBA)-Lipids and Lipid Metabolism, 1299(1), 117-123.