title: Isolation and optimisation of the oleaginous yeast Sporobolomyces roseus for biosynthesis of 13C isotopically labelled 18-carbon unsaturated fatty acids and trans 18:1 and 18:2 derivatives through synthesis.
abstract: An oleaginous and psychrotrophic strain (F38-3) of Sporobolomyces roseus Kluyver & van Niel was isolated from a salt marsh environment in Nova Scotia, Canada following a screening program to select for high producers of 18-carbon unsaturated fatty acids. Fatty acid production was characterised as a function of temperature at 20 g glucose L(-1), and optimal yields were obtained at 14°C, achieving 5.7 g dw biomass and 39.2% total fatty acids by dry weight, with 18:1, 18:2 and 18:3 all-cis fatty acids accounting for 49.4%, 14.3% and 6.7% of total fatty acids (TFA), respectively--the highest reported for this species. Production of 18:3 was inversely correlated to growth temperature, rising from 2% of TFA at 30°C to 8.9% at 6°C. Cultivation of isolate F38-3 on universally (13)C (U-(13)C) labelled glucose and subsequent transesterification and isolation of the fatty acid methyl esters (FAMEs) by preparative chromatography yielded pure, highly (13)C-enriched (>90%) 18:1, 18:2 and 18:3 all-cis FAMEs. The U-(13)C 18:1 FAME was catalytically converted to U-(13)C 18:1 trans-9 and purified to >99.5% purity. The U-(13)C 18:2 was converted by alkaline isomerisation into a 50/50 mixture of 18:2 cis-9, trans-11 and 18:2 trans-10, cis-12 isomers and purified to >95.0% purity. Overall, 10%, by weight, of labelled glucose fed to isolate F38-3 was recovered as fatty acid methyl esters and 7.5% as 18-carbon unsaturated fats, and the final isomerisation reactions resulted in yields of 80% or greater. The ultimate goal of the work is to develop methodologies to produce (13)C-labelled metabolic tracers as tools to study the metabolism of trans fats.
authors.affiliation: Institute for National Measurement Standards, National Research Council Canada, Building M-12, 1200 Montreal Rd., Ottawa, ON, K1A 0R6, Canada.
authors.forename: Yi
authors.initials: Y
authors.lastname: Cui
authors.forename: Catharine
authors.initials: C
authors.lastname: Fraser
authors.forename: Graeme
authors.initials: G
authors.lastname: Gardner
authors.forename: Ching-Jang
authors.initials: CJ
authors.lastname: Huang
authors.forename: Michael
authors.initials: M
authors.lastname: Reith
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Windust
mesh_terms: D000818:Animals; D001487:Basidiomycota / Q000302:isolation & purification / Q000378:metabolism*; D002247:Carbon Isotopes; D004951:Esterification; D005227:Fatty Acids / Q000096:biosynthesis / Q000737:chemistry; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis* / Q000737:chemistry; D007536:Isomerism; D009674:Nova Scotia; D044242:Trans Fatty Acids / Q000096:biosynthesis*; D015003:Yeasts / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D002247:Carbon Isotopes; D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D044242:Trans Fatty Acids