Difference between revisions of "Part:BBa K925000"
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===Description=== | ===Description=== | ||
| − | This part encodes a Δ12 desaturase derived from Synechocystis sp PCC 6803. | + | |
| + | 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 <i>E. coli</i>, the bacterial membrane composition changes. Linoleic acid is observed. | ||
===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 <i>E. coli</i> expressing this desaturase. | 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 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. | 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). 18:1 fatty acids are not present in unmodified <i>E. coli</i> BL21. | + | 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. |
[[Image:D12.png]] | [[Image:D12.png]] | ||
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===Results=== | ===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). | 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=== | ===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<sup>TM</sup> can be used to build a biosynthetic pathway for PUFAs and Omega-3 fatty acids, along with other desaturases and elongases. | 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<sup>TM</sup> 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/ iGEM Team NNU-China 2022] | ||
| + | |||
| + | '''Author''': Yaru Jiang | ||
| + | |||
| + | ===Characterization from iGEM22-NNU-China=== | ||
| + | |||
| + | ===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. | ||
| + | <html> | ||
| + | <div align="center"> | ||
| + | <figure> | ||
| + | <img src="https://static.igem.wiki/teams/4343/wiki/contribution-3.png" width="60%" style="float:center"> | ||
| + | <figcaption> | ||
| + | <p style="font-size:1rem"> | ||
| + | </p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | </html> | ||
| + | <div align="center"> | ||
| + | :'''Fig 1. The fatty acid distribution in various strains''' | ||
| + | </div> | ||
| + | <p><b><h2>Reference</h2></b></p> | ||
| + | <p>[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.</p> | ||
| + | |||
| + | <!-- --> | ||
Latest revision as of 02:04, 14 October 2022
Delta-12 desaturase
Contents
Short description
Delta-12 desaturase involved in an Omega-3 biosynthetic pathway.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 426
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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 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.
Contribution From NNU-China 2022
Group:iGEM Team NNU-China 2022
Author: Yaru Jiang
Characterization from iGEM22-NNU-China
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.