Team:St Andrews/Omega-3-synthesis
From 2012.igem.org
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
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Figure 1: "Synechocystis sp."
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Figure 2: "Trypanosome cruzi"
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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): in response to high temperatures, microalgae produce less ω-3 desaturated fatty acids and more saturated fatty acids, as desaturated carbon chains cause a lower melting temperature in 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 provide 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.
The genes were initially ligated into pET-15b vector. 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.
Then, Δ6, Δ12, and Δ15 desaturases were successfully ligated into two distinct pET-Duet vectors (vectors with two multicloning sites). Once vector contained Δ12 and Δ15 desaturases, and the other was ligated only with Δ6 desaturase.
Protein expression was clear after transformation into cell strain BL21(DE3) and induction by IPTG. However, 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, not at the 9th position required for a substrate. Thus, the E. coli were fed 18:1 (Δ9).
Characteritsation was done 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 ligated 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.