An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis, and expression in Arabidopsis.
Claviceps purpurea, a fungal pathogen responsible for ergot diseases in many agriculturally important cereal crops, produces high levels of ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. It has been believed for many years that the biosynthesis of this fatty acid in C. purpurea involves a hydration process with linoleic acid as the substrate. Using degenerate polymerase chain reaction, we cloned a gene from the sclerotia encoding an enzyme (CpFAH) that has high sequence similarity to the C. purpurea oleate desaturase, but only low similarity to plant oleate hydroxylases. Functional analysis of CpFAH in yeast (Saccharomyces cerevisiae) indicated it acted predominantly as a hydroxylase, introducing hydroxyl groups at the 12-position of oleic acid and palmitoleic acid. As well, it showed Delta(12) desaturase activities on 16C and 18C monounsaturated fatty acids and, to a much lesser extent, omega(3) desaturase activities on ricinoleic acid. Heterologous expression of CpFAH under the guidance of a seed-specific promoter in Arabidopsis (Arabidopsis thaliana) wild-type and mutant (fad2/fae1) plants resulted in the accumulation of relatively higher levels of hydroxyl fatty acids in seeds. These data indicate that the biosynthesis of ricinoleic acid in C. purpurea is catalyzed by the fungal desaturase-like hydroxylase, and CpFAH, the first Delta(12) oleate hydroxylase of nonplant origin, is a good candidate for the transgenic production of hydroxyl fatty acids in oilseed crops.
Meesapyodsuk
Dauenpen
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Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8.
Qiu
Xiao
X
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D002967:Claviceps / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D019301:Oleic Acid / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012639:Seeds / Q000378:metabolism
D005656:Fungal Proteins; D019301:Oleic Acid; D044943:Fatty Acid Desaturases