Identification and functional characterization of the moss Physcomitrella patens Δ⁵-desaturase gene involved in arachidonic and eicosapentaenoic acid biosynthesis

Abstract

The moss Physcomitrella patens contains high levels of arachidonic acid and lesser amounts of eicosapentaenoic acid. Here we report the identification and characterization of a Δ5-desaturase from P. patens that is associated with the synthesis of these fatty acids. A full-length cDNA for this desaturase was identified by data base searches based on homology to sequences of known Δ5-desaturase cDNAs from fungal and algal species. The resulting P. patens cDNA encodes a 480-amino acid polypeptide that contains a predicted N-terminal cytochrome b5-like domain as well as three histidine-rich domains. Expression of the enzyme in Saccharomyces cerevisiae resulted in the production of the Δ5-containing fatty acid arachidonic acid in cells that were provided di-homo-γ-linolenic acid. In addition, the expressed enzyme generated Δ5-desaturation products with the C20 substrates ω-6 eicosadienoic and ω-3 eicosatrienoic acids, but no products were detected with the C18 fatty acid linoleic and α-linolenic acids or with the C22 fatty acid adrenic and docosapentaenoic acids. When the corresponding P. patens genomic sequence was disrupted by replacement through homologous recombination, a dramatic alteration in the fatty acid composition was observed, i.e. an increase in di-homo-γ-linolenic and eicosatetraenoic acids accompanied by a concomitant disappearance of the Δ5-fatty acid arachidonic and eicosapentaenoic acids. In addition, overexpression of the P. patens cDNA in protoplasts isolated from a disrupted line resulted in the restoration of arachidonic acid synthesis.