An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis and expression in Arabidopsis thaliana

Dauenpen Meesapyodsuk and Xiao Qiu ^*

Department of Food & Bioproduct Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan, Canada, S7N5A8; and Bioriginal Food and Science Corporation, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada, S7N0W9

^* Corresponding author; email: xiao.qiu{at}usask.ca.

Claviceps purpurea, a fungal pathogen responsible for ergotdiseases in many agriculturally important cereal crops, produceshigh levels of ricinoleic acid (12-hydroxyoctadec-cis-9-enoicacid) in its sclerotia. It has been believed for many yearsthat the biosynthesis of this fatty acid in C. purpurea involvesa hydration process with linoleic acid as the substrate. Usingdegenerate PCR, we cloned a gene from the sclerotia encodingan enzyme (CpFAH) which has high sequence similarity to theC. purpurea oleate desaturase, but only low similarity to plantoleate hydroxylases. Functional analysis of CpFAH in yeast indicatedit acted predominantly as a hydroxylase, introducing hydroxylgroups at the 12-position of oleic acid and palmitoleic acid.As well, it showed ${Delta}$ ¹² desaturase activities on 16C and 18C monounsaturatedfatty acids and to a much lesser extent, ${omega}$ ³ desaturase activitieson ricinoleic acid. Heterologous expression of CpFAH under theguidance of a seed-specific promoter in Arabidopsis thalianawild type and mutant (fad2/fae1) plants resulted in the accumulationof relatively higher levels of hydroxyl fatty acids in seeds.These data indicate that the biosynthesis of ricinoleic acidin C. purpurea is catalyzed by the fungal desaturase-like hydroxylaseand CpFAH, the first ${Delta}$ ¹² oleate hydroxylase of non-plant origin,is a good candidate for the transgenic production of hydroxylfatty acids in oilseed crops.