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.

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.