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Plant and Cell Physiology Advance Access published online on November 2, 2005
Plant and Cell Physiology, doi:10.1093/pcp/pci224
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Plant and Cell Physiology 2005 © The Japanese Society of Plant Physiologists (JSPP); all rights reserved.
Received August 12, 2005
Accepted October 27, 2005

Regular Paper

A Front-End Desaturase from Chlamydomonas reinhardtii Produces Pinolenic and Coniferonic Acids by {omega}13 Desaturation in Methylotrophic Yeast and Tobacco

Masataka Kajikawa 1, Katsuyuki T. Yamato 1, Yoshito Kohzu 1, Shin-ichiro Shoji 1, Keisuke Matsui 2, Yoshikazu Tanaka 2, Yasuyoshi Sakai 3, and Hideya Fukuzawa 1*

1 Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
2 Institute for Fundamental Research, Suntory Limited, Osaka 618-8503, Japan
3 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

* To whom correspondence should be addressed.
Hideya Fukuzawa, E-mail: fukuzawa{at}lif.kyoto-u.ac.jp


  Abstract

Pinolenic acid (PA, 18:3{Delta}5,9,12) and coniferonic acid (CA, 18:4{Delta}5,9,12,15) are {Delta}5-unsaturated bis-methylene-interrupted fatty acids ({Delta}5-UBIFAs) commonly found in pine seed oil. They are assumed to be synthesized from linoleic acid (LA, 18:2{Delta}9,12) and {alpha}-linolenic acid (ALA, 18:3{Delta}9,12,15) by {Delta}5-desaturation, respectively. A unicellular green microalga Chlamydomonas reinhardtii also accumulates PA and CA in a betain lipid. The 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 the comparable amounts of LA and ALA were reduced, demonstrating that CrDES has {Delta}5-desaturase activity for both LA and ALA. CrDES expressed in the yeast showed {Delta}5-desaturase activity on 18:1{Delta}9 but not 18:1{Delta}11. Unexpectedly, CrDES also showed {Delta}7-desaturase activity on 20:2{Delta}11,14 and 20:3{Delta}11,14,17 to produce 20:3{Delta}7,11,14 and 20:4{Delta}7,11,14,17, respectively. Since both the {Delta}5 bond in C18 and the {Delta}7 bond in C20 fatty acids are "{omega}13" double bonds, these results indicate that CrDES has {omega}13 desaturase activity for {omega}9 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: Chlamydomonas reinhardtii; {Delta}5-unsaturated bis-methylene-interrupted fatty acids; fatty acid desaturase; Pichia pastoris; tobacco plant.
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