Revision as of 16:12, 19 September 2012

Omega-3 fatty acid synthesis

ω-3 Fatty acids are an essential component of our diet and are paramount to maintaining human health. 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

Synechocystis sp.
Trypanosome cruzi
Leishmania major

Omega-3 fatty acids are an essential part of the human diet (Bender, Bender, 1999). Human beings, as 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 microrganisms, 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 sbsequently 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

Diagram showing 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 at the 9th carbon (18:1) (Marr, Ingraham, 1969). Valuable ω-3 fatty acids require 3 double bonds, and need to have a chain length between 18 and 22 carbons. Desaturases, elongases Different kind in e coli

The genes for Δ12, Δ15 (ω6) and Δ6 were obtained from Synechocystis sp., a cyanobacteria. The trypanosomatid Leishmania major provided the DNA for the Elo 6 gene. Additionally, we used Trypanosome cruzi as a secondary source of Δ12.

Please see the Lab Book.

Methods

Sequences Primers Sequencing results Lipid analysis Mass Spectrometry Sequences

Lipid analysis

Lipids were extracted from transformed E. coli using the Blight/Dyer method. (Please refer to the Lipid extraction lab book entry for further detail.)

We then analyzed the the fatty acid content of the transformed cells to determine whether it differed from the previously analyzed background lipid composition. This was done using mass spectrometry.

Mass Spectrometry

Biobricks

References

ARTS, et al, 2009. Lipids in Aquatic Ecosystems. New York. Springer.

BENDER D. A. and BENDER, A. E, 1999. Benders' dictionary of nutrition and food technology. Cambridge: CRC Press.

COHEN, J., 2003. Human Population: The Next Half Century. Science, New Series, Vol. 302, No. 5648. Pp. 1172-1175.

GARWIN J. L. and CRONAN J. E. Jr, 1980. Thermal modulation of fatty acid synthesis in Escherichia coli does not involve de novo enzyme synthesis. J Bacteriol, 141(3): 1457–1459.

LIVORE V., TRIPODI K., UTARRO A., 2007. Elongation of polyunsaturated fatty acids in trypanosomatids. FEBS Journal, 274: 264–274.

MARR A., INGRAHAM J., 1969. Effect of temperature on the composition of fatty acids in Escherichia coli. J. Bactiol. 84(6). Pp. 1260–1267.

TONON T., et al, 2002. Long chain polyunsaturated fatty acid production and partitioning to triacylglycerols in four microalgae. Phytochemistry, Vol 61 Iss 1. Pgs 15-24.

Team:St Andrews/Omega-3-synthesis

From 2012.igem.org