A Front-end Desaturase from Chlamydomonas reinhardtii Produces Pinolenic and Coniferonic Acids by ω13 Desaturation in Methylotrophic Yeast and Tobacco
Open Access
- 1 January 2006
- journal article
- research article
- Published by Oxford University Press (OUP) in Plant and Cell Physiology
- Vol. 47 (1), 64-73
- https://doi.org/10.1093/pcp/pci224
Abstract
Pinolenic acid (PA; 18:3Δ5,9,12) and coniferonic acid (CA; 18:4Δ5,9,12,15) are Δ5-unsaturated bis-methylene-interrupted fatty acids (Δ5-UBIFAs) commonly found in pine seed oil. They are assumed to be synthesized from linoleic acid (LA; 18:2Δ9,12) and α-linolenic acid (ALA; 18:3Δ9,12,15), respectively, by Δ5-desaturation. A unicellular green microalga Chlamydomonas reinhardtii also accumulates PA and CA in a betain lipid. The expressed sequence tag (EST) resource of C.reinhardtii led to the isolation of a cDNA clone that encoded a putative fatty acid desaturase named as CrDES containing a cytochrome b5 domain at the N-terminus. When the coding sequence was expressed heterologously in the methylotrophic yeast Pichia pastoris, PA and CA were newly detected and comparable amounts of LA and ALA were reduced, demonstrating that CrDES has Δ5-desaturase activity for both LA and ALA. CrDES expressed in the yeast showed Δ5-desaturase activity on 18:1Δ9 but not 18:1Δ11. Unexpectedly, CrDES also showed Δ7-desaturase activity on 20:2Δ11,14 and 20:3Δ11,14,17 to produce 20:3Δ7,11,14 and 20:4Δ7,11,14,17, respectively. Since both the Δ5 bond in C18 and the Δ7 bond in C20 fatty acids are ‘ω13’ double bonds, these results indicate that CrDES has ω13 desaturase activity for ω9 unsaturated C18/C20 fatty acids, in contrast to the previously reported front-end desaturases. In order to evaluate the activity of CrDES in higher plants, transgenic tobacco plants expressing CrDES were created. PA and CA accumulated in the leaves of transgenic plants. The highest combined yield of PA and CA was 44.7% of total fatty acids, suggesting that PA and CA can be produced in higher plants on a large scale.Keywords
This publication has 50 references indexed in Scilit:
- Chlamydomonas reinhardtii Genome Project. A Guide to the Generation and Use of the cDNA InformationPlant Physiology, 2003
- Functional identification of a fatty acid Δ5 desaturase gene from Caenorhabditis elegansFEBS Letters, 1998
- Isolation of a Δ5-Fatty Acid Desaturase Gene fromMortierella alpinaJournal of Biological Chemistry, 1998
- Gapped BLAST and PSI-BLAST: a new generation of protein database search programsNucleic Acids Research, 1997
- Efficient Promoter Cassettes for Enhanced Expression of Foreign Genes in Dicotyledonous and Monocotyledonous PlantsPlant and Cell Physiology, 1996
- pBINPLUS: An improved plant transformation vector based on pBIN19Transgenic Research, 1995
- CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choiceNucleic Acids Research, 1994
- Isolation and characterization of a cDNA clone corresponding to the Rt locus of Petunia hybridaThe Plant Journal, 1994
- NUTRITIONAL AND PHYSIOLOGICAL SIGNIFICANCE OF LIPIDSJournal of Dispersion Science and Technology, 1989
- The detection and classification of membrane-spanning proteinsBiochimica et Biophysica Acta (BBA) - Biomembranes, 1985