Cloning of Δ12‐ and Δ6‐desaturases from Mortierella alpina and recombinant production of γ‐linolenic acid in Saccharomyces cerevisiae

Abstract

Two cDNA clones with homology to known desaturase genes were isolated from the fungus Mortierella alpina. The open reading frame in one clone encoded 399 amino acids and exhibited delta12-desaturase activity when expressed in Saccharomyces cerevisiae in the presence of endogenous fatty acid substrate oleic acid. The insert in another clone contained an open reading frame encoding 457 amino acids and exhibited delta6-desaturase activity in S. cerevisiae in the presence of exogenous fatty acid substrate linoleic acid. Expression of the delta12-desaturase gene under appropriate media and temperature conditions led to the production of linoleic acid at levels up to 25% of the total fatty acids in yeast. When linoleic acid was provided as an exogenous substrate to the yeast cultures expressing the delta6-desaturase activity, the level of gamma-linolenic acid reached 10% of the total yeast fatty acids. Co-expression of both the delta6- and delta12-desaturase cDNA resulted in the endogenous production of gamma-linolenic acid. The yields of gamma-linolenic acid reached as high as 8% of total fatty acids in yeast.

Characterization and comparison of fatty acyl Δ6 desaturase cDNAs from freshwater and marine teleost fish species

“…Full-length cDNAs for Δ6 desaturases have been isolated from the filamentous fungus Mortierella alpina (Huang et al, 1999), the nematode Caenorhabditis elegans (Napier et al, 1998), rat (Aki et al, 1999), mouse and human (Cho et al, 1999a). Fatty acid Δ5 desaturase genes have been isolated from M. alpina (Michaelson et al, 1998a), C. elegans (Michaelson et al, 1998b;Watts and Browse, 1999) and human (Cho et al, 1999b;Leonard et al, 2000).…”

Substrate specificity and preference of Δ6‐desaturase of Mucor rouxii

“…The yeast transformed with the empty vector, YEp356 plasmid, was used as a control. Rate of substrate conversion (% conversion rate = [product formed/(substrate + product formed)] · 100) was calculated as described previously [10].…”

“…-desaturases of Mortierella alpina [10] and Ceratodon purpureus [15], which are evolutionary related to the Mucor D 6 -desaturase (Fig. 1).…”

“…Subsequently, these two synthesized products can be further modified to form arachidonic (C20:4D 5,8,11,14 ) and eicosapentanoic acids (C20:5D 5,8,11,14,17 ), respectively [3,4]. Genes encoding membrane-bound D 6 -desaturases have been cloned from several organisms, including higher plants [5,6], worms [7], mammals [8], and fungi [9,10]. Structural analyses showed that they share three conserved histidine-rich motifs, two-long hydrophobic regions and a N-terminal cytochrome b 5 -like domain.…”

“…-position [5][6][7][8][9][10]. Despite conserved characteristics found in other D 6 -desaturases, variation in terms of substrate specificity and preference have been also reported [5,[11][12][13][14][15].…”