Skip to main content

Cloning of a Novel Omega-6 Desaturase from Flax (Linum usitatissimum L.) and Its Functional Analysis in Saccharomyces cerevisiae

Molecular Biotechnology volume 42, pages 168–174 (2009)Cite this article

Abstract

The Δ12 desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18:1) to linoleic acid (18:2). Several members of this family are known from plants like Arabidopsis and Soybean. Using primers from conserved C- and N-terminal regions, we have cloned a novel Δ12 desaturase gene amplified from flax genomic DNA, denoted as LuFAD2-2. This intron-less gene is 1,149-base pair long encoding 382 amino acids—putative membrane-bound Δ12 desaturase protein. Sequence comparisons show that the novel sequence has 85% similarity with previously reported flax Δ12 desaturase at amino acid level and shows typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that are universally present among plant desaturases. The signature amino acid sequence ‘YNNKL’ was also found to be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining tree generated from the sequence alignment grouped LuFAD2-2 among the other FAD2 sequences from Ricinus, Hevea, Jatropha, and Vernicia. When LuFAD2-2 and LuFAD2 were expressed in Saccharomyces cerevisiae, they could convert the oleic acid to linoleic acid, with an average conversion rate of 5.25 and 8.85%, respectively. However, exogenously supplied linoleic acid was feebly converted to linolenic acid suggesting that LuFAD2-2 encodes a functional FAD2 enzyme and has substrate specificity similar to LuFAD2.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Abbreviations

ALA:

Alpha linolenic acid; 18:3

ER:

Endoplasmic reticulum

FAD:

Fatty acid desaturase

FAD2:

Omega-6 desaturase

LA:

Linoleic acid; 18:2

PUFA:

Polyunsaturated fatty acid 16:0; Palmitic acid, 18:0; Stearic acid, 18:1; Oleic acid

References

  1. Ohlrogge, J., & Browse, J. (1995). Lipid biosynthesis. The Plant Cell, 7, 957–970.

    Article  CAS  Google Scholar 

  2. Tang, G. Q., Novitzky, W. P., Griffing, H. C., Huber, S. C., & Dewey, R. E. (2005). Oleate desaturase enzymes of soybean: Evidence of regulation through differential stability and phosphorylation. The Plant Journal, 44, 433–446. doi:10.1111/j.1365-313X.2005.02535.x.

    Article  CAS  Google Scholar 

  3. Schlueter, J. A., Vasylenko-Sanders, I. F., Deshpande, S., Yi, J., Siegfried, M., Roe, B. A., Schlueter, S. D., Scheffler, B. E., & Shoemaker, R. C. (2007). The FAD2 gene family of soybean: Insights into the structural and functional divergence of a paleopolyploid genome. The Plant Genome [A Supplement to Crop Science], 47. http://www.crops.org/genome/. doi:10.2135/cropsci2005.07.0206tpg.

  4. Hernández, M. L., Mancha, M., & Martínez-Rivas, J. M. (2005). Molecular cloning and characterization of genes encoding two microsomal oleate desaturase (FAD2) from olive. Phytochemistry, 66, 1417–1426. doi:10.1016/j.phytochem.2005.04.004.

    Article  Google Scholar 

  5. Martinez-Rivas, J. M., Sperling, P., Luhs, W., & Heinz, E. (2001). Spatial and temporal regulation of three different microsomal oleate desaturase genes (FAD2) from normal-type and high-oleic varieties of sunflower (Helianthus annuus L.). Molecular Breeding, 8(2), 159–168. doi:10.1023/A:1013324329322.

    Article  CAS  Google Scholar 

  6. Fofana, B., Duguid, S., & Cloutier, S. (2004). Cloning of fatty acid biosynthetic genes β-ketoacyl CoA synthase, fatty acid elongase, stearoyl-ACP desaturase and fatty acid desaturase and analysis of expression in the early developmental stages of flax (Linum usitatissimum L) seeds. Plant Science, 166, 1487–1496. doi:10.1016/j.plantsci.2004.01.025.

    Article  CAS  Google Scholar 

  7. Krasowska, A., Dziadkowiec, D., Polinceusz, A., Plonka, A., & Łukaszewicz, M. (2007). Cloning of flax oleic fatty acid desaturase and its expression in yeast. Journal of the American Oil Chemists’ Society, 84, 809–816. doi:10.1007/s11746-007-1106-9.

    Article  CAS  Google Scholar 

  8. Murray, M. G., & Thompson, W. F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8(19), 4321–4325. doi:10.1093/nar/8.19.4321.

    Article  CAS  Google Scholar 

  9. Manku, M. S., Horrobin, D. F., Huang, Y. S., & Morse, N. (1983). Fatty acids in plasma and red cell membranes in normal humans. Lipids, 18, 906–908. doi:10.1007/BF02534572.

    Article  CAS  Google Scholar 

  10. Shanklin, J., Whittle, E., & Fox, B. G. (1994). Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase. Biochemistry, 33, 12787–12794. doi:10.1021/bi00209a009.

    Article  CAS  Google Scholar 

  11. Emanuelsson, O., Nielsen, H., Brunak, S., & Heijne, G. (2000). Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. Journal of Molecular Biology, 300, 1005–1016. doi:10.1006/jmbi.2000.3903.

    Article  CAS  Google Scholar 

  12. Emanuelsson, O., Brunak, S., Heijne, G., & Nielsen, H. (2007). Locating proteins in the cell using TargetP, SignalP, and related tools. Nature Protocols, 2, 953–971. doi:10.1038/nprot.2007.131.

    Article  CAS  Google Scholar 

  13. McCartney, A. W., Dyer, J. M., Dhanoa, P. K., Kim, P. K., Andrews, D. W., McNew, J. A., et al. (2004). Membrane-bound fatty acid desaturases are inserted co-translationally into the ER and contain different ER retrieval motifs at their carboxy termini. The Plant Journal, 37, 156–173. doi:10.1111/j.1365-313X.2004.01949.x.

    Article  CAS  Google Scholar 

  14. Fofana, B., Cloutier, S., Duguid, S., Ching, J., & Rampitsch, C. (2006). Gene expression of stearoyl-ACP desaturase and Δ12 fatty acid desaturase 2 is modulated during seed development of flax (Linum usitatissimum). Lipids, 41, 705–712. doi:10.1007/s11745-006-5021-x.

    Article  CAS  Google Scholar 

  15. Covello, P. S., & Reed, D. W. (1996). Functional expression of the extraplastidial Arabidopsis thaliana oleate desaturase gene (FAD2) in Saccharomyces cerevisiae. Plant Physiology, 111, 223–226. doi:10.1104/pp.111.1.223.

    Article  CAS  Google Scholar 

  16. Dyer, J. M., Chapital, D. C., Kuan, J. W., Mullen, R. T., Turner, C., McKeon, T. A., et al. (2002). Molecular analysis of a bifunctional fatty acid conjugase/desaturase from tung. Implications for the evolution of plant fatty acid diversity. Plant Physiology, 130, 2027–2038. doi:10.1104/pp.102.010835.

    Article  CAS  Google Scholar 

  17. Vrinten, P., Hu, Z., Munchinsky, M. A., Rowland, G., & Qiu, X. (2005). Two FAD3 desaturase genes control the level of linolenic acid in flax seed. Plant Physiology, 139, 79–87. doi:10.1104/pp.105.064451.

    Article  CAS  Google Scholar 

  18. Zhang, H. T., Yang, J. S., Shan, L., & Bla, Y. P. (2006). Functional expression of a ώ-3 fatty acid desaturase gene from glycine max in Saccharomyces cerevisiae. Chinese Journal of Biotechnology, 22, 33–38. doi:10.1016/S1872-2075(06)60004-6.

    Article  CAS  Google Scholar 

  19. Dyer, J. M., Chapital, D. C., Kuan, J. W., Shepherd, H. S., Tang, F., & Pepperman, A. B. (2004). Production of linolenic acid in yeast cells expressing an omega-3 desaturase from tung (Aleurites fordii). Journal of the American Oil Chemists’ Society, 81, 647–651. doi:10.1007/s11746-004-956-x.

    Article  CAS  Google Scholar 

  20. Reed, D. W., Schäfer, U. A., & Covello, P. S. (2000). Characterization of the Brassica napus extraplastidial linoleate desaturase by expression in Saccharomyces cerevisiae. Plant Physiology, 122(3), 715–720. doi:10.1104/pp.122.3.715.

    Article  CAS  Google Scholar 

  21. Damude, H. G., Zhang, H., Farrall, L., Ripp, K. G., Tomb, J. F., Hollerbach, D., et al. (2006). Identification of bifunctional Δ12/ώ3 fatty acid desaturase for improving the ratio of ώ3 to ώ6 fatty acids in microbes and plants. Proceedings of the National Academy of Sciences of the United States of America, 103(25), 9446–9451. doi:10.1073/pnas.0511079103.

    Article  CAS  Google Scholar 

  22. Sayanova, O., Haslam, R., Guschina, I., Lioyd, D., Christie, W. W., Harwood, J. L., et al. (2006). A bifunctional Δ12/Δ15 desaturase from Acanthamoeba castellanii directs the synthesis of highly unusual n-1 series unsaturated fatty acids. The Journal of Biological Chemistry, 281(48), 36533–36541. doi:10.1074/jbc.M605158200.

    Article  CAS  Google Scholar 

  23. Lopez Alonso, D., Garcia-Maroto, F., Rodriguez-Ruiz, J., Garrido, J. A., & Vilches, M. A. (2003). Evolution of the membrane-bound fatty acid desaturase. Biochemical Systematics and Ecology, 31, 1111–1124. doi:10.1016/S0305-1978(03)00041-3.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The work in this manuscript is an outcome of the R&D project on flax supported financially by Department of Biotechnology, Government of India. The inputs provided by BVU also were crucial for completing the project. We thank Dr. P. B. Ghorpade, Flax Breeder, Nagpur for supplying seeds of the flax variety NL 97. Technical support by Ms. Ankita Khare and Mrs. Ashwini Rajwade is acknowledged.

Author information

Affiliations

  1. Interactive Research School for Health Affairs, Medical College Campus, Bharati Vidyapeeth University, Pune-Satara Road, Pune, 411043, India

    Rupali M. Khadake, Prabhakar K. Ranjekar & Abhay M. Harsulkar

Authors
  1. Rupali M. Khadake
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Prabhakar K. Ranjekar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Abhay M. Harsulkar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Abhay M. Harsulkar.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Khadake, R.M., Ranjekar, P.K. & Harsulkar, A.M. Cloning of a Novel Omega-6 Desaturase from Flax (Linum usitatissimum L.) and Its Functional Analysis in Saccharomyces cerevisiae . Mol Biotechnol 42, 168–174 (2009). https://doi.org/10.1007/s12033-009-9150-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-009-9150-3

Keywords

  • FAD2
  • Omega-6 desaturase
  • Flax (Linum usitatissimum L.)
  • Linoleic acid
  • Oleic acid
  • S. cerevisiae