Difference between revisions of "Part:BBa K925001"
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===Description=== | ===Description=== | ||
| − | This part encodes a delta-15 desaturase derived from Synechocystis sp PCC 6803. The enzyme is able to introduce a double bond at the Δ-15 site in the hydrocarbon chain of linoleic acid (18:2 ; Δ9,12). This converts the substrate into alpha-linoleic acid (18:3 ; Δ9,12,15), an ω -3 polyunsaturated fatty acid (PUFA). Mass spectrometry results show that when feeding oleic acid to E. coli transformed with this part in addition to Δ -12 desaturase (BBa_K925000), alpha-linoleic acid is observed in membrane lipids. | + | This part encodes a delta-15 desaturase derived from Synechocystis sp PCC 6803. The membrane-bound enzyme is able to introduce a double bond at the Δ-15 site in the hydrocarbon chain of linoleic acid (18:2 ; Δ9,12). This converts the substrate into alpha-linoleic acid (18:3 ; Δ9,12,15), an ω -3 polyunsaturated fatty acid (PUFA). Mass spectrometry results show that when feeding oleic acid to <i>E. coli</i> transformed with this part in addition to Δ -12 desaturase (BBa_K925000), alpha-linoleic acid is observed in membrane lipids. |
===Characterisation=== | ===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 well as to Δ -12 desaturase. This was so that the product of Δ-12 desaturase (18:2 ; Δ9,12) could be used by Δ-15 for the formation of 18:3 ; Δ9,12,15. | + | 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 <i>E. coli</i> expressing this desaturase, as well as to Δ -12 desaturase. This was so that the product of Δ-12 desaturase (18:2 ; Δ9,12) could be used by Δ-15 for the formation of 18:3 ; Δ9,12,15. |
| − | Importantly, the cells were grown in the presence of Δ12 desaturase’s substrate 18:1 (Δ9) so that this could be incorporated to the membranes, as this fatty acid is not present in unmodified E. coli BL21. | + | Importantly, the cells were grown in the presence of Δ12 desaturase’s substrate 18:1 (Δ9) so that this could be incorporated to the membranes, as this fatty acid is not present in unmodified <i>E. coli</i> BL21. |
As a control the same FAME-GC analysis was performed in unmodified cells, and the lipid profiles were compared. Additionally, FAME 18:3 (Δ9,12,15) standard was run to compare to the desaturation pattern to that of the 18:3 expected to be observed in the transformed cells. | As a control the same FAME-GC analysis was performed in unmodified cells, and the lipid profiles were compared. Additionally, FAME 18:3 (Δ9,12,15) standard was run to compare to the desaturation pattern to that of the 18:3 expected to be observed in the transformed cells. | ||
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===Conclusion=== | ===Conclusion=== | ||
| − | Lipid profiles of E. coli transformed with our construct show that this Δ-15 desaturase is able to catalyze the desaturation of linoleic acid to give alpha-linoleic acid, an ω-3 fatty acid. In this way, this BioBrick can be used with BBa_K925000 to build a biosynthetic pathway for PUFAs and ω -3 fatty acids, along with other desaturases and elongases. | + | Lipid profiles of <i>E. coli</i> transformed with our construct show that this Δ-15 desaturase is able to catalyze the desaturation of linoleic acid to give alpha-linoleic acid, an ω-3 fatty acid. In this way, this BioBrick<sup>TM</sup> can be used with BBa_K925000 to build a biosynthetic pathway for PUFAs and ω -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. | ||
Latest revision as of 23:18, 24 September 2012
Delta 15 desaturase
Short description
Delta-15 desaturase involved in an ω-3 biosynthetic pathway.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Description
This part encodes a delta-15 desaturase derived from Synechocystis sp PCC 6803. The membrane-bound enzyme is able to introduce a double bond at the Δ-15 site in the hydrocarbon chain of linoleic acid (18:2 ; Δ9,12). This converts the substrate into alpha-linoleic acid (18:3 ; Δ9,12,15), an ω -3 polyunsaturated fatty acid (PUFA). Mass spectrometry results show that when feeding oleic acid to E. coli transformed with this part in addition to Δ -12 desaturase (BBa_K925000), alpha-linoleic acid is observed in membrane lipids.
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 well as to Δ -12 desaturase. This was so that the product of Δ-12 desaturase (18:2 ; Δ9,12) could be used by Δ-15 for the formation of 18:3 ; Δ9,12,15.
Importantly, the cells were grown in the presence of Δ12 desaturase’s substrate 18:1 (Δ9) so that this could be incorporated to the membranes, as this fatty acid is not present in unmodified E. coli BL21.
As a control the same FAME-GC analysis was performed in unmodified cells, and the lipid profiles were compared. Additionally, FAME 18:3 (Δ9,12,15) standard was run to compare to the desaturation pattern to that of the 18:3 expected to be observed in the transformed cells.
Results
Our results indicate that, both in lipid extracts and in our membrane assays derived from cells transformed with Δ-12 and Δ-15 desaturase, C18:3 is present, unlike in untransformed cells. Moreover, this 18:3 has the same unsaturation pattern as our standard, meaning the 18:3 found in the transformed cells is the expected 18:3 (Δ9,12,15).
Conclusion
Lipid profiles of E. coli transformed with our construct show that this Δ-15 desaturase is able to catalyze the desaturation of linoleic acid to give alpha-linoleic acid, an ω-3 fatty acid. In this way, this BioBrickTM can be used with BBa_K925000 to build a biosynthetic pathway for PUFAs and ω -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.