title: A high-throughput fatty acid profiling screen reveals novel variations in fatty acid biosynthesis in Chlamydomonas reinhardtii and related algae.
abstract: Analysis of fatty acid methyl esters (FAMEs) by gas chromatography (GC) is a common technique for the quantitative and qualitative analysis of acyl lipids. Methods for FAME preparation are typically time-consuming and labor-intensive and require multiple transfers of reagents and products between reaction tubes and autosampler vials. In order to increase throughput and lower the time and materials costs required for FAME preparation prior to GC analysis, we have developed a method in which 10-to-20-mg samples of microbial biomass are transferred to standard GC autosampler vials, transesterified using an emulsion of methanolic trimethylsulfonium hydroxide and hexane, and analyzed directly by GC without further sample handling. This method gives results that are essentially identical to those obtained by the more labor- and material-intensive FAME preparation methods, such as transmethylation with methanolic HCl. We applied this method to the screening of laboratory and environmental isolates of the green alga Chlamydomonas for variations in fatty acid composition. This screening method facilitated two novel discoveries. First, we identified a common laboratory strain of C. reinhardtii, CC-620, completely lacking all ω-3 fatty acids normally found in this organism and showed that this strain contains an inactivating mutation in the CrFAD7 gene, encoding the sole ω-3 desaturase activity in this organism. Second, we showed that some species of Chlamydomonas make Δ6-unsaturated polyunsaturated fatty acids (PUFA) rather than the Δ5 species normally made by the previously characterized laboratory strains of Chlamydomonas, suggesting that there is species-specific variation in the regiospecificity and substrate selectivity of front-end desaturases in this algal genus.
authors.affiliation: School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
authors.forename: Erin L
authors.initials: EL
authors.lastname: Pflaster
authors.affiliation: School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Schwabe
authors.affiliation: School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, USA Department of Biological Sciences, Grinnell College, Grinnell, Iowa, USA.
authors.forename: Joyanne
authors.initials: J
authors.lastname: Becker
authors.affiliation: School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Wilkinson
authors.affiliation: School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, USA Department of Biological Sciences, Fort Valley State University, Fort Valley, Georgia, USA.
authors.forename: Ashley
authors.initials: A
authors.lastname: Parmer
authors.affiliation: Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Clemente
authors.affiliation: Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
authors.forename: Edgar B
authors.initials: EB
authors.lastname: Cahoon
authors.affiliation: School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, USA wriekhof2@unl.edu.
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Riekhof
mesh_terms: D000595:Amino Acid Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D002849:Chromatography, Gas / Q000379:methods*; D044943:Fatty Acid Desaturases / Q000235:genetics*; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis*; D006586:Hexanes / Q000737:chemistry; D016415:Sequence Alignment; D013379:Substrate Specificity; D013452:Sulfonium Compounds / Q000737:chemistry
chemical_list: D005231:Fatty Acids, Unsaturated; D006586:Hexanes; D013452:Sulfonium Compounds; C009581:trimethylsulfonium; D044943:Fatty Acid Desaturases; C078565:omega-3 fatty acid desaturase