Dataset Information

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 ?-3 fatty acids normally found in this organism and showed that this strain contains an inactivating mutation in the CrFAD7 gene, encoding the sole ?-3 desaturase activity in this organism. Second, we showed that some species of Chlamydomonas make ?6-unsaturated polyunsaturated fatty acids (PUFA) rather than the ?5 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.

SUBMITTER: Pflaster EL

PROVIDER: S-EPMC4248691 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A high-throughput fatty acid profiling screen reveals novel variations in fatty acid biosynthesis in Chlamydomonas reinhardtii and related algae.

Pflaster Erin L EL Schwabe Michael J MJ Becker Joyanne J Wilkinson Melissa S MS Parmer Ashley A Clemente Thomas E TE Cahoon Edgar B EB Riekhof Wayne R WR

Eukaryotic cell 20140919 11

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 ...[more]

PMID: 25239975

Similar Datasets

Project description:BackgroundWhen subject to stress conditions such as nutrient limitation microalgae accumulate triacylglycerol (TAG). Fatty acid, a substrate for TAG synthesis is derived from de novo synthesis or by membrane remodeling. The model industrial alga Chlorellasorokiniana accumulates TAG and other storage compounds under nitrogen (N)-limited growth. Molecular mechanisms underlying these processes are still to be elucidated.ResultPreviously we used transcriptomics to explore the regulation of TAG synthesis in C. sorokiniana. Surprisingly, our analysis showed that the expression of several key genes encoding enzymes involved in plastidic fatty acid synthesis are significantly repressed. Metabolic labeling with radiolabeled acetate showed that de novo fatty acid synthesis is indeed downregulated under N-limitation. Likewise, inhibition of the Target of Rapamycin kinase (TOR), a key regulator of metabolism and growth, decreased fatty acid synthesis. We compared the changes in proteins and phosphoprotein abundance using a proteomics and phosphoproteomics approach in C. sorokiniana cells under N-limitation or TOR inhibition and found extensive overlap between the N-limited and TOR-inhibited conditions. We also identified changes in the phosphorylation status of TOR complex proteins, TOR-kinase, and RAPTOR, under N-limitation. This indicates that TOR signaling is altered in a nitrogen-dependent manner. We find that TOR-mediated metabolic remodeling of fatty acid synthesis under N-limitation is conserved in the chlorophyte algae Chlorella sorokiniana and Chlamydomonas reinhardtii.ConclusionOur results indicate that under N-limitation there is significant metabolic remodeling, including fatty acid synthesis, mediated by TOR signaling. This process is conserved across chlorophyte algae. Using proteomic and phosphoproteomic analysis, we show that N-limitation affects TOR signaling and this in-turn affects the metabolic status of the cells. This study presents a link between N-limitation, TOR signaling and fatty acid synthesis in green-lineage.

Project description:BackgroundMicroalgae have the potential to deliver biofuels without the associated competition for land resources. In order to realise the rates and titres necessary for commercial production, however, system-level metabolic engineering will be required. Genome scale metabolic reconstructions have revolutionized microbial metabolic engineering and are used routinely for in silico analysis and design. While genome scale metabolic reconstructions have been developed for many prokaryotes and model eukaryotes, the application to less well characterized eukaryotes such as algae is challenging not at least due to a lack of compartmentalization data.ResultsWe have developed a genome-scale metabolic network model (named AlgaGEM) covering the metabolism for a compartmentalized algae cell based on the Chlamydomonas reinhardtii genome. AlgaGEM is a comprehensive literature-based genome scale metabolic reconstruction that accounts for the functions of 866 unique ORFs, 1862 metabolites, 2249 gene-enzyme-reaction-association entries, and 1725 unique reactions. The reconstruction was compartmentalized into the cytoplasm, mitochondrion, plastid and microbody using available data for algae complemented with compartmentalisation data for Arabidopsis thaliana. AlgaGEM describes a functional primary metabolism of Chlamydomonas and significantly predicts distinct algal behaviours such as the catabolism or secretion rather than recycling of phosphoglycolate in photorespiration. AlgaGEM was validated through the simulation of growth and algae metabolic functions inferred from literature. Using efficient resource utilisation as the optimality criterion, AlgaGEM predicted observed metabolic effects under autotrophic, heterotrophic and mixotrophic conditions. AlgaGEM predicts increased hydrogen production when cyclic electron flow is disrupted as seen in a high producing mutant derived from mutational studies. The model also predicted the physiological pathway for H2 production and identified new targets to further improve H2 yield.ConclusionsAlgaGEM is a viable and comprehensive framework for in silico functional analysis and can be used to derive new, non-trivial hypotheses for exploring this metabolically versatile organism. Flux balance analysis can be used to identify bottlenecks and new targets to metabolically engineer microalgae for production of biofuels.

Dataset Information

A high-throughput fatty acid profiling screen reveals novel variations in fatty acid biosynthesis in Chlamydomonas reinhardtii and related algae.

Publications

A high-throughput fatty acid profiling screen reveals novel variations in fatty acid biosynthesis in Chlamydomonas reinhardtii and related algae.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets