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.
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
By combining a number of genes, we were able to recreate the pathway described above (Fig. 1). The following genes were employed (please click for sequences and KEGG numbers):
Δ6 Synechocystis
ATGCTAACAGCGGAAAGAATTAAATTTACCCAGAAACGGGGGTTTCGTCGGGTACTAAAC
CAACGGGTGGATGCCTACTTTGCCGAGCATGGCCTGACCCAAAGGGATAATCCCTCCATG
TATCTGAAAACCCTGATTATTGTGCTCTGGTTGTTTTCCGCTTGGGCCTTTGTGCTTTTT
GCTCCAGTTATTTTTCCGGTGCGCCTACTGGGTTGTATGGTTTTGGCGATCGCCTTGGCG
GCCTTTTCCTTCAATGTCGGCCACGATGCCAACCACAATGCCTATTCCTCCAATCCCCAC
ATCAACCGGGTTCTGGGCATGACCTACGATTTTGTCGGGTTATCTAGTTTTCTTTGGCGC
TATCGCCACAACTATTTGCACCACACCTACACCAATATTCTTGGCCATGACGTGGAAATC
CATGGAGATGGCGCAGTACGTATGAGTCCTGAACAAGAACATGTTGGTATTTATCGTTTC
CAGCAATTTTATATTTGGGGTTTATATCTTTTCATTCCCTTTTATTGGTTTCTCTACGAT
GTCTACCTAGTGCTTAATAAAGGCAAATATCACGACCATAAAATTCCTCCTTTCCAGCCC
CTAGAATTAGCTAGTTTGCTAGGGATTAAGCTATTATGGCTCGGCTACGTTTTCGGCTTA
CCTCTGGCTCTGGGCTTTTCCATTCCTGAAGTATTAATTGGTGCTTCGGTAACCTATATG
ACCTATGGCATCGTGGTTTGCACCATCTTTATGCTGGCCCATGTGTTGGAATCAACTGAA
TTTCTCACCCCCGATGGTGAATCCGGTGCCATTGATGACGAGTGGGCTATTTGCCAAATT
CGTACCACGGCCAATTTTGCCACCAATAATCCCTTTTGGAACTGGTTTTGTGGCGGTTTA
AATCACCAAGTTACCCACCATCTTTTCCCCAATATTTGTCATATTCACTATCCCCAATTG
GAAAATATTATTAAGGATGTTTGCCAAGAGTTTGGTGTGGAATATAAAGTTTATCCCACC
TTCAAAGCGGCGATCGCCTCTAACTATCGCTGGCTAGAGGCCATGGGCAAAGCATCGTGA
KEGG entry sll0541
Δ12 Synechocystis
ATGACTGCCACGATTCCCCCGTTGACACCAACGGTAACGCCCAGCAATCCCGATCGCCCG
ATTGCGGATCTCAAACTACAAGACATCATTAAAACCCTGCCCAAGGAATGCTTCGAGAAA
AAAGCGAGCAAAGCCTGGGCTTCTGTTTTGATTACCCTAGGGGCGATCGCCGTGGGCTAT
TTGGGCATTATTTATCTGCCCTGGTACTGCTTGCCCATTACCTGGATCTGGACAGGGACA
GCCTTAACGGGGGCCTTCGTTGTCGGCCATGACTGTGGCCATCGCTCCTTTGCTAAAAAA
CGCTGGGTCAATGATTTAGTGGGACATATCGCTTTTGCTCCCCTCATCTACCCTTTCCAT
AGCTGGCGCCTACTCCACGACCACCATCACCTCCACACCAACAAAATTGAGGTTGATAAC
GCCTGGGATCCCTGGAGTGTGGAAGCTTTCCAAGCCAGCCCGGCGATCGTCCGGCTTTTT
TATCGGGCCATCCGGGGTCCCTTCTGGTGGACTGGTTCCATTTTCCATTGGAGCTTAATG
CACTTCAAACTTTCCAACTTTGCCCAAAGGGACCGCAATAAAGTCAAATTATCCATTGCC
GTTGTCTTCCTGTTTGCGGCGATCGCCTTTCCTGCCCTAATTATCACCACAGGGGTGTGG
GGTTTCGTCAAATTTTGGCTAATGCCCTGGTTGGTGTATCACTTTTGGATGAGCACTTTT
ACCATTGTGCACCACACCATTCCCGAAATTCGTTTCCGTCCCGCCGCCGATTGGAGTGCC
GCCGAAGCCCAGTTAAATGGTACTGTTCACTGCGATTATCCCCGTTGGGTGGAAGTGCTC
TGCCATGACATCAACGTCCATATTCCCCACCACCTCTCCGTTGCCATCCCTTCCTATAAC
CTACGACTAGCCCACGGAAGTTTAAAAGAAAACTGGGGACCTTTTCTTTACGAGCGCACC
TTTAACTGGCAATTAATGCAACAAATTAGTGGGCAATGTCATTTATATGACCCCGAACAT
GGCTACCGCACCTTCGGCTCCCTGAAAAAAGTTTAA
KEGG entry 429257.20
Δ15 Synechocystis
GTGCGTCTAGAAATTTCATCGCCTCAAACAAAGCTTCCTTACCCCAAAACTGAAGAATTA
CCATTTACCCTCCAAGAGCTCAGAAACGCTATTCCAGCGGATTGTTTTGAGCCATCGGTA
GTCCGGTCCTTGGGCTACTTTTTTTTGGATGTTGGTTTAATTGCCGGGTTTTATGCTCTA
GCGGCCTACCTTGATTCCTGGTTCTTCTATCCGATTTTTTGGTTAATTCAGGGAACCCTA
TTCTGGTCCCTGTTTGTGGTGGGCCATGATTGTGGCCATGGCTCCTTTTCCAAATCCAAA
ACCCTTAATAATTGGATTGGTCATCTCAGCCACACGCCAATTTTGGTGCCTTACCATGGC
TGGCGTATTAGTCATCGTACTCACCATGCCAACACGGGCAATATCGACACCGACGAAAGT
TGGTATCCAGTGTCGGAGCAAAAATATAACCAAATGGCCTGGTATGAAAAACTTCTACGT
TTTTACTTGCCTCTGATCGCCTACCCCATTTATCTATTTCGGCGATCGCCAAACCGGCAA
GGCTCCCATTTCATGCCCGGCAGTCCCCTATTCCGTCCCGGAGAAAAAGCAGCTGTTCTC
ACCAGCACCTTTGCCCTTGCAGCCTTTGTCGGCTTCCTTGGCTTTTTAACTTGGCAATTT
GGCTGGCTATTTTTGCTGAAATTTTATGTTGCCCCCTACCTCGTGTTTGTGGTGTGGTTA
GATTTGGTCACATTTTTACATCACACTGAAGACAATATCCCTTGGTATCGTGGTGATGAC
TGGTATTTTCTCAAAGGTGCCCTCTCCACCATTGATCGGGATTACGGCTTCATTAACCCC
ATTCACCATGACATTGGCACCCACGTCGCCCACCATATTTTCTCGAATATGCCCCACTAC
AAGTTACGCCGGGCGACTGAAGCCATCAAGCCCATTTTAGGGGAATATTATCGATATTCT
GACGAGCCAATTTGGCAAGCTTTTTTTAAGTCCTACTGGGCTTGCCATTTTGTTCCTAAT
CAAGGTTCAGGGGTCTATTACCAATCCCCATCCAATGGTGGATATCAAAAGAAACCTTAA
KEGG entry s111441
ELO 6 L. major
ATGAAGGTCATCGTCGCTTCTGGCCCGGACGGTGCCCGCAAGCACGAGGTGGAGCTGGCA
GCCAACGCCACGCTCGCAGATCTGAAGAAGGCCTACCAACGGGGTGTGGACGTGCACCGC
AAGTCGTTCAAGGTTCCCAGCGCGGAGTCGCCGCTGCCAGGTGCGGATAGTGGCAAGCTG
CGCCCGAACCTCATTACTCTGTCAGATAAGGTGCCCCTGTCGCAGCAGGGGGTGAAGGAT
GGCTCGGTGATCACTTACAAGGACCTCGGCCCGCAGATCGGCTACCGCACGGTGTTCTAC
GTCGAGTATGCCGGCCCCATCGCCTTCATGCTGCTGTACGCCATGCGCCCTTCGCTCATC
TACGGCTCTGCCCCGATGCCGGCTTACGGCTACACGCAGAAGCTATACATTGGCCTCTTC
CTCGCCCACTTCTTAAAGCGCGAGCTCGAGTCCATGTTTGTGCACAAGTTCTCGCACCCA
ACGATGCCGATGCGCAACATCTTCAAGAACTGCATCTACTACTGGTCCTTCGCCGCCTTC
ATCGGCTACGTGCTGTGCAGTCCTTCATTCACGCCGACCAGCACCATGCAGTCAAACTTC
GGCGCCGTGGTCATGGTCATCAACGAGCTGCTGAACTTCGCGGTGCACTACCAGCTTAGC
ACGATGCGCAAGTCCGATGGTGACACCACCCGCAACGTGCCGCAAGGCCCTCTGTTCGCC
TTCGTCTCGTGCCCGAACTACTTCTTTGAGATTATGTCGTGGGTGTCCTTTTCCATCGGC
ACAAATATGTTATCCTCCTGGTTCTTCACACTCGCCGGTTTCGTGCAGATGGCGGACTGG
GCGAAGAAGAAGCACCGGGGCTACGTCAAGGCGGACCCGGCCAATAAAAAGAAGGCCGCC
ATTCTGCCCTTCATCATGTAG
KEGG entry LmjF32.1160
These genes were extracted using PCR (Promega, GoTaq HotStart) at temperatures 48°C and 56°C. Both vectors pET-15b and pET-20b were tested for expression. The recombinant plasmids were ligated into two E. coli strains: BL21(DE3) and BL21(DE3)pLysS.
Primers
All primers are notated 5' to 3'. Initially, we worked with NdeI and XhoI as the restriction sites.
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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