Team:St Andrews/Omega-3-synthesis
From 2012.igem.org
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<img src="https://static.igem.org/mediawiki/2012/5/57/OmegaThreeLogo_100.png" align="left"></img> | <img src="https://static.igem.org/mediawiki/2012/5/57/OmegaThreeLogo_100.png" align="left"></img> | ||
- | <p>ω-3 | + | <p>ω-3 fatty acids are a key component of the human diet. Our team is recreating this synthetic pathway in <i>E. coli</i>, using genes from the cyanobacteria Synechocystis and the trypanosomatid Leishmania major. Combining the DNA code for elongase and desaturase enzymes, we can convert the plain fatty acid of <i>E. coli</i> into highly valuable ω-3 fatty acids.</p> |
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- | <p>Omega-3 fatty acids are an essential part of the human diet (Bender, Bender, 1999). Human beings, | + | <p>Omega-3 fatty acids are an essential part of the human diet (Bender, Bender, 1999). Human beings, like all larger organisms, cannot synthesize ω-3 fatty acids, This is due to a lack of the enzyme Δ15 desaturase, which creates a double bond at the 15th carbon of a long-chain fatty acid. Certain micro-organisms, such as microalgae and cyanobacteria, do contain this desaturase and can thus directly synthesize ω-3 fatty acids (Arts <i>et al.</i>, 2009). ω-3 fatty acids then enter the food chain – algae are eaten by fish, and seafood is subsequently the main source of ω-3 for humans (Tonon et al., 2002).</p> |
- | <p>However, the current economic policies of overfishing are a serious contributor to marine biodestruction. As the human population is estimated to rise to 9.1 billion by 2050 (Cohen, 2003), pressure on fish stock will increase. Additionally, global warming will reduce the availability of ω-3 (Arts et al, 2009): at higher temperatures, microalgae produce less ω-3 desaturated fatty acids. Desaturated carbon chains cause a lower melting temperature in the membrane, which the microorganism wants to avoid by synthesizing more saturated fatty acids in their membranes (Garwin, Cronan, 1980). Thus, the combination of declining fish stock and a decrease in overall ω-3 fatty acids is making the supply for human nutrition a relevant issue. </p> | + | <p>However, the current economic policies of overfishing are a serious contributor to marine biodestruction. As the human population is estimated to rise to 9.1 billion by 2050 (Cohen, 2003), pressure on fish stock will increase. Additionally, global warming will reduce the availability of ω-3 (Arts et al., 2009): at higher temperatures, microalgae produce less ω-3 desaturated fatty acids. Desaturated carbon chains cause a lower melting temperature in the membrane, which the microorganism wants to avoid by synthesizing more saturated fatty acids in their membranes (Garwin, Cronan, 1980). Thus, the combination of declining fish stock and a decrease in overall ω-3 fatty acids is making the supply for human nutrition a relevant issue. </p> |
<p>Harvesting algae directly is costly and ineffective (Borowitzka, 1997). There is much potential in expressing a metabolic pathway for ω-3 fatty acid synthesis in <i>E. coli</i>, which is cheaper and more accessible. </p> | <p>Harvesting algae directly is costly and ineffective (Borowitzka, 1997). There is much potential in expressing a metabolic pathway for ω-3 fatty acid synthesis in <i>E. coli</i>, which is cheaper and more accessible. </p> | ||
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<p><h5>Figure 4: <em>"The metabolic pathway to ω-3 fatty acids"</em></h5></p> | <p><h5>Figure 4: <em>"The metabolic pathway to ω-3 fatty acids"</em></h5></p> | ||
<div class="span5"><p>Figure 4 shows the elongation and desaturation enzymes necessary to convert an 18:1 fatty acid, which <i>E. coli</i> synthesizes, into an poly-unsaturated fatty acid.</p> | <div class="span5"><p>Figure 4 shows the elongation and desaturation enzymes necessary to convert an 18:1 fatty acid, which <i>E. coli</i> synthesizes, into an poly-unsaturated fatty acid.</p> | ||
- | <p><em>modified from Livore et al, 2006</em></p> | + | <p><em>modified from Livore et al., 2006</em></p> |
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<p>The genes for Δ12, Δ15 (ω6) and Δ6 were obtained from <i>Synechocystis sp.</i>, a cyanobacterium. The trypanosomatid <i>Leishmania major</i> provided the DNA for the ELO 6 gene. Additionally, we used <i>Trypanosome cruzi</i> as a secondary source of Δ12.</p> | <p>The genes for Δ12, Δ15 (ω6) and Δ6 were obtained from <i>Synechocystis sp.</i>, a cyanobacterium. The trypanosomatid <i>Leishmania major</i> provided the DNA for the ELO 6 gene. Additionally, we used <i>Trypanosome cruzi</i> as a secondary source of Δ12.</p> | ||
- | <p>However, our first successful ligations of Δ12 did not yield us with the expected 18:2 fatty acid. We hypothesized that E. | + | <p>However, our first successful ligations of Δ12 did not yield us with the expected 18:2 fatty acid. We hypothesized that <i>E. coli</i>’s inherent 18-carbon chain fatty acid might not be suited as a substrate for Δ12 – the double bond is in a different position, the 11th. Therefore, we "fed" our cells with suitable 18:1, to then observe 18:2 fatty acid, and ultimately ω-3 desaturation, in the mass spec results!</p> |
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<p> We were able to ligate these genes into pET-15b vector first. After a number of expression attempts, some initial conclusions were reached. All experiments done on ELO6 failed. Also, Δ12 from T. cruzi gave overall weaker results than the same gene from Synechocystis. As such, latter work was only carried out on genes from Synechocystis.<p> | <p> We were able to ligate these genes into pET-15b vector first. After a number of expression attempts, some initial conclusions were reached. All experiments done on ELO6 failed. Also, Δ12 from T. cruzi gave overall weaker results than the same gene from Synechocystis. As such, latter work was only carried out on genes from Synechocystis.<p> | ||
<p> Δ6, Δ12, and Δ15 desaturases were successfully ligated into two distinct pET-Duet vectors (vectors with two multicloning sites), one of them containing Δ12 and Δ15 desaturases, and the other Duet vector ligated only to Δ6 desaturase.<p> | <p> Δ6, Δ12, and Δ15 desaturases were successfully ligated into two distinct pET-Duet vectors (vectors with two multicloning sites), one of them containing Δ12 and Δ15 desaturases, and the other Duet vector ligated only to Δ6 desaturase.<p> | ||
- | <p> After transformation into cell strain BL21(DE3), even though protein expression was clear, functionality could not be established. It was hypothesized that the naturally-occuring 18:1 fatty acid in E. coli is the wrong substrate for the desaturases. This fatty acid has its desaturation at the 11th carbon. Thus, the <i>E. coli</i> were fed 18:1 (Δ9).<p> | + | <p> After transformation into cell strain BL21(DE3), even though protein expression was clear, functionality could not be established. It was hypothesized that the naturally-occuring 18:1 fatty acid in <i>E. coli</i> is the wrong substrate for the desaturases. This fatty acid has its desaturation at the 11th carbon. Thus, the <i>E. coli</i> were fed 18:1 (Δ9).<p> |
<p> Characterisation followed by analysing lipid composition of transformed <i>E. coli</i>. Lipid profiles of membrane assays and lipid extracts from cells were obtained using Fatty Acid Methyl Ester (FAME) Analysis by GC-MS.<p> | <p> Characterisation followed by analysing lipid composition of transformed <i>E. coli</i>. Lipid profiles of membrane assays and lipid extracts from cells were obtained using Fatty Acid Methyl Ester (FAME) Analysis by GC-MS.<p> | ||
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</div> | </div> | ||
- | <p> | + | <p>Arts, <i>et al.</i>, 2009. Lipids in Aquatic Ecosystems. New York. Springer.</p> |
- | <p> | + | <p>Bender D. A. and Bender, A. E, 1999. Benders' dictionary of nutrition and food technology. Cambridge: CRC Press.</p> |
- | <p> | + | <p>Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 7;72:248-54.</p> |
- | <p> | + | <p>Cohen, J., 2003. Human Population: The Next Half Century. Science, New Series, Vol. 302, No. 5648. Pp. 1172-1175.</p> |
- | <p> | + | <p>Garwin J. L. and Cronan J. E. Jr, 1980. Thermal modulation of fatty acid synthesis in <i>Escherichia coli</i> does not involve de novo enzyme synthesis. J Bacteriol, 141(3): 1457–1459.</p> |
- | <p> | + | <p>Livore V., Tripodi K., Utarro A., 2007. Elongation of polyunsaturated fatty acids in trypanosomatids. FEBS Journal, 274: 264–274.</p> |
- | <p> | + | <p>Marr A., Ingraham J., 1969. Effect of temperature on the composition of fatty acids in <i>Escherichia coli</i>. J. Bactiol. 84(6). Pp. 1260–1267.</p> |
- | <p> | + | <p>Tonon T., <i>et al.</i>, 2002. Long chain polyunsaturated fatty acid production and partitioning to triacylglycerols in four microalgae. Phytochemistry, Vol 61 Iss 1. Pgs 15-24.</p> |
</section> | </section> | ||
Revision as of 19:28, 25 September 2012
Omega-3 fatty acid synthesis
Introduction
![](https://static.igem.org/mediawiki/2012/5/57/OmegaThreeLogo_100.png)
ω-3 fatty acids are a key component of the human diet. Our team is recreating this synthetic pathway in E. coli, using genes from the cyanobacteria Synechocystis and the trypanosomatid Leishmania major. Combining the DNA code for elongase and desaturase enzymes, we can convert the plain fatty acid of E. coli into highly valuable ω-3 fatty acids.
Project Description
-
Figure 1: "Synechocystis sp."
-
Figure 2: "Trypanosome cruzi"
-
Figure 3: "Leishmania major"
Omega-3 fatty acids are an essential part of the human diet (Bender, Bender, 1999). Human beings, like all larger organisms, cannot synthesize ω-3 fatty acids, This is due to a lack of the enzyme Δ15 desaturase, which creates a double bond at the 15th carbon of a long-chain fatty acid. Certain micro-organisms, such as microalgae and cyanobacteria, do contain this desaturase and can thus directly synthesize ω-3 fatty acids (Arts et al., 2009). ω-3 fatty acids then enter the food chain – algae are eaten by fish, and seafood is subsequently the main source of ω-3 for humans (Tonon et al., 2002).
However, the current economic policies of overfishing are a serious contributor to marine biodestruction. As the human population is estimated to rise to 9.1 billion by 2050 (Cohen, 2003), pressure on fish stock will increase. Additionally, global warming will reduce the availability of ω-3 (Arts et al., 2009): at higher temperatures, microalgae produce less ω-3 desaturated fatty acids. Desaturated carbon chains cause a lower melting temperature in the membrane, which the microorganism wants to avoid by synthesizing more saturated fatty acids in their membranes (Garwin, Cronan, 1980). Thus, the combination of declining fish stock and a decrease in overall ω-3 fatty acids is making the supply for human nutrition a relevant issue.
Harvesting algae directly is costly and ineffective (Borowitzka, 1997). There is much potential in expressing a metabolic pathway for ω-3 fatty acid synthesis in E. coli, which is cheaper and more accessible.
Synthesizing the pathway
![](https://static.igem.org/mediawiki/2012/8/88/Pathway.gif)
Figure 4: "The metabolic pathway to ω-3 fatty acids"
Figure 4 shows the elongation and desaturation enzymes necessary to convert an 18:1 fatty acid, which E. coli synthesizes, into an poly-unsaturated fatty acid.
modified from Livore et al., 2006
E. coli naturally synthesize poly-unsaturated fatty acids up to a carbon chain length of 18, with a single desaturation (18:1) (Marr, Ingraham, 1969). Valuable ω-3 fatty acids require a double bonds at the third carbon from the end of its carbon chain and can have >20 carbons.
In order to have E. coli synthesize ω-3 fatty acids, we needed to introduce enzymes that could elongate and desaturate fatty acid substrates (cf. Fig. 4).
The genes for Δ12, Δ15 (ω6) and Δ6 were obtained from Synechocystis sp., a cyanobacterium. The trypanosomatid Leishmania major provided the DNA for the ELO 6 gene. Additionally, we used Trypanosome cruzi as a secondary source of Δ12.
However, our first successful ligations of Δ12 did not yield us with the expected 18:2 fatty acid. We hypothesized that E. coli’s inherent 18-carbon chain fatty acid might not be suited as a substrate for Δ12 – the double bond is in a different position, the 11th. Therefore, we "fed" our cells with suitable 18:1, to then observe 18:2 fatty acid, and ultimately ω-3 desaturation, in the mass spec results!
Methods
The following genes were employed (please click for sequences and KEGG numbers):
These genes were amplified through PCR (Promega, GoTaq HotStart) at temperatures 48°C and 56°C.
We were able to ligate these genes into pET-15b vector first. After a number of expression attempts, some initial conclusions were reached. All experiments done on ELO6 failed. Also, Δ12 from T. cruzi gave overall weaker results than the same gene from Synechocystis. As such, latter work was only carried out on genes from Synechocystis.
Δ6, Δ12, and Δ15 desaturases were successfully ligated into two distinct pET-Duet vectors (vectors with two multicloning sites), one of them containing Δ12 and Δ15 desaturases, and the other Duet vector ligated only to Δ6 desaturase.
After transformation into cell strain BL21(DE3), even though protein expression was clear, functionality could not be established. It was hypothesized that the naturally-occuring 18:1 fatty acid in E. coli is the wrong substrate for the desaturases. This fatty acid has its desaturation at the 11th carbon. Thus, the E. coli were fed 18:1 (Δ9).
Characterisation followed by analysing lipid composition of transformed E. coli. Lipid profiles of membrane assays and lipid extracts from cells were obtained using Fatty Acid Methyl Ester (FAME) Analysis by GC-MS.
After characterization, we finally ligated separately each of our desaturases into the submission vector pSB1C3.
![](https://static.igem.org/mediawiki/2012/6/61/PCR_of_genes.png)
Fig. 5: "UV photograph of PCR results"
The figure shows the results of a PCR extraction of our genes of choice, done with GoTaq HotStart PCR kit at 2 different annealing temperatures: Δ12 (48°C) - Δ12 (56°C) - Δ15 (48°C) - Δ15 (56°C) - Δ6 (48°C) - Δ6 (56°C).
Primers
All primers are notated 5' to 3'. Initially, we worked with NdeI and XhoI as the restriction sites.