title: Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola.
abstract: Production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plant seed oils has been pursued to improve availability of these omega-3 fatty acids that provide important human health benefits. Canola (Brassica napus), through the introduction of 10 enzymes, can convert oleic acid (OLA) into EPA and ultimately DHA through a pathway consisting of two elongation and five desaturation steps. Herein we present an assessment of the substrate specificity of the seven desaturases and three elongases that were introduced into canola by expressing individual proteins in yeast. In vivo feeding experiments were conducted with 14 potential fatty acid intermediates in an OLA to DHA pathway to determine the fatty acid substrate profiles for each enzyme. Membrane fractions were prepared from yeast expression strains and shown to contain active enzymes. The elongases, as expected, extended acyl-CoA substrates in the presence of malonyl-CoA. To distinguish between enzymes that desaturate CoA- and phosphatidylcholine-linked fatty acid substrates, we developed a novel in vitro method. We show that a delta-12 desaturase from Phytophthora sojae, an omega-3 desaturase from Phytophthora infestans and a delta-4 desaturase from Thraustochytrium sp., all prefer phosphatidylcholine-linked acyl substrates with comparatively low use of acyl-CoA substrates. To further validate our method, a delta-9 desaturase from Saccharomyces cerevisiae was confirmed to use acyl-CoA as substrate, but could not use phosphatidylcholine-linked substrates. The results and the assay methods presented herein will be useful in efforts to improve modeling of fatty acid metabolism and production of EPA and DHA in plants.
authors.affiliation: Scandinavian Biotechnology Research (ScanBiRes) AB, 230 53, Alnarp, Sweden. Jenny.LindbergYilmaz@scanbires.com.
authors.forename: Jenny Lindberg
authors.initials: JL
authors.lastname: Yilmaz
authors.affiliation: Bioriginal Food and Science Corporation, Saskatoon, SK, S7N 0W9, Canada.
authors.forename: Ze Long
authors.initials: ZL
authors.lastname: Lim
authors.affiliation: Scandinavian Biotechnology Research (ScanBiRes) AB, 230 53, Alnarp, Sweden.
authors.forename: Mirela
authors.initials: M
authors.lastname: Beganovic
authors.affiliation: BASF Plant Science LP, Research Triangle Park, NC, 27709, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Breazeale
authors.affiliation: BASF Plant Science LP, Research Triangle Park, NC, 27709, USA.
authors.forename: Carl
authors.initials: C
authors.lastname: Andre
authors.affiliation: Department of Plant Breeding, Swedish University of Agricultural Sciences, 230 53, Alnarp, Sweden.
authors.forename: Sten
authors.initials: S
authors.lastname: Stymne
authors.affiliation: Bioriginal Food and Science Corporation, Saskatoon, SK, S7N 0W9, Canada.
authors.forename: Patricia
authors.initials: P
authors.lastname: Vrinten
authors.affiliation: BASF Plant Science LP, Research Triangle Park, NC, 27709, USA.
authors.forename: Toralf
authors.initials: T
authors.lastname: Senger
mesh_terms: D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism*; D029688:Brassica napus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004281:Docosahexaenoic Acids / Q000378:metabolism*; D015118:Eicosapentaenoic Acid / Q000378:metabolism; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D006801:Humans; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D019301:Oleic Acid / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D010940:Plant Proteins; D004281:Docosahexaenoic Acids; D019301:Oleic Acid; D008316:Malonyl Coenzyme A; D015118:Eicosapentaenoic Acid; D044943:Fatty Acid Desaturases; D000123:Acetyltransferases
keywords: Acyl-CoA; Desaturases; Elongases; Genetic engineering; Lipid biochemistry; Nutrition; Phosphatidylcholine; Polyunsaturated fatty acids (PUFA); n-3 Fatty acids