Difference between revisions of "Part:BBa K925003"

 
 
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<partinfo>BBa_K925003 short</partinfo>
 
<partinfo>BBa_K925003 short</partinfo>
  
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This part encodes a &#916;6 desaturase derived from Synechocystis sp PCC 6803. The enzyme is a membrane-bound protein able to introduce a double bond at the &#916;-12 site in the hydrocarbon chain of oleic acid (18:1, &#916;9). This converts the substrate into linoleic acid (18:2 &#916;9,12), 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.
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===Short description===
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Delta-6 desaturase involved in an Omega-3 biosynthetic pathway.
  
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===Usage and Biology===
 
  
 
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===Description===
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===Functional Parameters===
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<partinfo>BBa_K925003 parameters</partinfo>
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This part encodes a Δ6 desaturase derived from Synechocystis sp PCC 6803. The enzyme is a membrane-bound protein 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 <i>E. coli</i>, the bacterial membrane composition changes. Linoleic acid is observed.
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===Characterisation===
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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.
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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.
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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 <i>E. coli</i> BL21.
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[[Image:D6.png]]
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[[Image:FAME-_GC_MS_STANDARDSc.png]]
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===Results===
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Our results indicate that, both in lipid extracts and in our membrane assays derived from cells transformed with Δ-6 desaturase, C18:2 is present, unlike in unmodified cells. Moreover, this 18:2 has the same unsaturation pattern as our standard, meaning the 18:2 found in the transformed cells is the expected 18:2 (Δ9,12).
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===Conclusion===
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Lipid profiles of <i>E. coli</i> transformed with our construct show that this Δ-6 desaturase is able to catalyze the desaturation of oleic acid to give linoleic acid. We also suspect this enzyme is able to desaturate alpha-linoleic acid (18:3; Δ9,12,15) further to an 18:4; Δ6,9,12,15 if co-expressed with Δ12 and Δ15 desaturases (BBa_K925000 and BBa_K925001, respectively) in the presence of 18:1 (Δ9) (Livore, 2006).
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In this way, this BioBrick<sup>TM</sup> can be used to build a biosynthetic pathway for PUFAs and Omega-3 fatty acids, along with other desaturases and elongases.
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===References===
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LIVORE V., TRIPODI K., UTARRO A., 2007. Elongation of polyunsaturated fatty acids in trypanosomatids. FEBS Journal, 274: 264–274.

Latest revision as of 23:17, 24 September 2012

Delta-6 desaturase

Short description

Delta-6 desaturase involved in an Omega-3 biosynthetic pathway.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Description

This part encodes a Δ6 desaturase derived from Synechocystis sp PCC 6803. The enzyme is a membrane-bound protein 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.

D6.png

FAME- GC MS STANDARDSc.png

Results

Our results indicate that, both in lipid extracts and in our membrane assays derived from cells transformed with Δ-6 desaturase, C18:2 is present, unlike in unmodified cells. Moreover, this 18:2 has the same unsaturation pattern as our standard, 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-6 desaturase is able to catalyze the desaturation of oleic acid to give linoleic acid. We also suspect this enzyme is able to desaturate alpha-linoleic acid (18:3; Δ9,12,15) further to an 18:4; Δ6,9,12,15 if co-expressed with Δ12 and Δ15 desaturases (BBa_K925000 and BBa_K925001, respectively) in the presence of 18:1 (Δ9) (Livore, 2006). In this way, this BioBrickTM 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.