Team:St Andrews/Omega-3-synthesis

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<p>Chart 1. Chart 1. C18:3 refers to an 18 carbon backbone with 3 double bonds. C18:2 and C18:1 would have 2 double bonds and 1 respectively. The upper portion of this figure shows the peaks that would be expected for the various fatty acid methyl esters.The lower 4 panels show mass spec fingerprints that are indicative of our fatty acids of interest.  
<p>Chart 1. Chart 1. C18:3 refers to an 18 carbon backbone with 3 double bonds. C18:2 and C18:1 would have 2 double bonds and 1 respectively. The upper portion of this figure shows the peaks that would be expected for the various fatty acid methyl esters.The lower 4 panels show mass spec fingerprints that are indicative of our fatty acids of interest.  
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<p>Chart 2. All of the cellular lipids were extracted and their fatty acid methyl esters produced. These were analysed by mass spec. In the control cells no C18:2 is present. When Delta 6 is expressed, a peak indicative of C18:2 is present. However, the primary fatty acid is still C18:1. With the expression of Delta 12 and the addition of C18:1 with the carbon carbon double bond at carbon 9 we get great abundance of C18:2. The lowest graph shows that Delta 12 expressed with Delta 15 acts in a complementary fashion and C18:3 is produced.</p>
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<p>Chart 2. All of the cellular lipids were extracted and their fatty acid methyl esters produced. These were analysed by mass spec. In the control cells no C18:2 is present. When Delta 6 is expressed, a peak indicative of C18:2 is present. However, the primary fatty acid is still C18:1. With the expression of Delta 12 and the addition of C18:1 with the carbon carbon double bond at carbon 9 we get great abundance of C18:2. The lowest graph shows that Delta 12 expressed with Delta 15 acts in a complementary fashion and C18:3 - Omega 3 - is produced. </p>
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Revision as of 21:16, 24 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.

  • 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.

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. The double bonds needs to start at the thrid carbon, counting from the end of the carbon chain.

In order to have synthesize ω-3 fatty acids, we needed to introduce enzymes that could elongate and desaturate fatty acid substrates. As seen in the figure above, this pathway can be recreated by a number of desaturases and elongases.

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. (cf. Fig. 2)

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 – perhaps the double bond is in a different position. Therefore, we "fed" our cells with suitable 18:1, to then observe 18:2 fatty acid in the mass spec results!

The below figure shows the results of a PCR extraction of our genes of choice, done with GoTaq HotStart PCR kit at 2 different annealing temperatues: Δ12 (48°C) - Δ12 (56°C) - Δ15 (48°C) - Δ15 (56°C) - Δ6 (48°C) - Δ6 (56°C).

Sequences

Δ12 T. Cruzi Δ12 Synechocystis Δ15 Synechocystis Δ6 Synechocystis ELO 6 L. major

Primers

All primers are notated 5' to 3'. Initially, we worked with NdeI and XhoI as the restriction sites.

Δ12 T. Cruzi forward
Δ12 T. Cruzi reverse
Δ12 Synechocystis forward
Δ12 Synechocystis reverse
Δ15 Synechocystis forward
Δ15 Synechocystis reverse
Δ6 Synechocystis forward
Δ6 Synechocystis reverse
ELO6 L. major forward
ELO6 L. major reverse

When using the pET-duet vector, we needed additionally primers for the alternative restriction sites and His-tags. For Δ6 and Δ12, we cut with HindIII and EcoRI; NdeI and XhoI were used for Δ15 and ELO6.

ELO6 L. major forward
Δ15 T. Cruzi forward
Δ6 T. Cruzi forward
Δ6 T. Cruzi reverse
Δ12 T. Cruzi forward
Δ12 T. Cruzi reverse

For the submission vector, we once again required new primers, using restriction sites EcoRI and PstI. For Δ6 and Δ12 forward, we used the primers from the duet vector (see above).

Δ6 Synechocystis reverse
Δ15 Synechocystis forward
Δ15 and Δ12 Synechocystis reverse

Sequencing results

16/7/12 Δ15

    Δ15 and Δ6 desaturases from Synechocystis in pET-15b were sent off for sequencing. Once again, the results were discouraging even though protein and lipid analysis showed the opposite. BLAST showed that the gene found in both samples corresponded to a T. brucei HMG-CoA reductase which we thought we had cut out from the plasmid. We thus performed a PCR, using the vectors sent off for sequencing as a template.

    Sequence

16/7/12 Δ6

    Sequence

29/6/12 ELO6

    After showing successful insertion of L. major Δ6 elongase in pET-15b, the sample was sent off for sequencing. Unfortunately, the gene sequenced was not the Δ6 elongase we were hoping to find, but a T. brucei mevalonate kinase, which was previously inserted in the pET-15b vectors we had been using. Thus, we digested some fresh pET-15b to be used in the future.

    Sequence

23/7/12 Δ12

    15b forward gave 0 nucleotides

    15b reverse gave 0 nucleotides

    20b forward gave a 41 nucleotide result.

    Sequence

    A BLAST search showed this to be compatible with Crocosphaera watsonii (genome shotgun sequence, 21%), a diazotrophic cyanobacteria.

    20b reverse gave 0 nucleotides

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

Chart 1. Chart 1. C18:3 refers to an 18 carbon backbone with 3 double bonds. C18:2 and C18:1 would have 2 double bonds and 1 respectively. The upper portion of this figure shows the peaks that would be expected for the various fatty acid methyl esters.The lower 4 panels show mass spec fingerprints that are indicative of our fatty acids of interest.

Chart 2. All of the cellular lipids were extracted and their fatty acid methyl esters produced. These were analysed by mass spec. In the control cells no C18:2 is present. When Delta 6 is expressed, a peak indicative of C18:2 is present. However, the primary fatty acid is still C18:1. With the expression of Delta 12 and the addition of C18:1 with the carbon carbon double bond at carbon 9 we get great abundance of C18:2. The lowest graph shows that Delta 12 expressed with Delta 15 acts in a complementary fashion and C18:3 - Omega 3 - is produced.

TODO

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.

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University of St Andrews, 2012.

Contact us: igem2012@st-andrews.ac.uk, Twitter, Facebook

This iGEM team has been funded by the MSD Scottish Life Sciences Fund. The opinions expressed by this iGEM team are those of the team members and do not necessarily represent those of Merck Sharp & Dohme Limited, nor its Affiliates.