title: Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii.
abstract: BACKGROUND
  Oils produced by microalgae are precursors to biodiesel. To achieve a profitable production of biodiesel from microalgae, identification of factors governing oil synthesis and turnover is desirable. The green microalga Chlamydomonas reinhardtii is amenable to genetic analyses and has recently emerged as a model to study oil metabolism. However, a detailed method to isolate various types of oil mutants that is adapted to Chlamydomonas has not been reported.
  
  
  RESULTS
  We describe here a forward genetic approach to isolate mutants altered in oil synthesis and turnover from C. reinhardtii. It consists of a three-step screening procedure: a primary screen by flow cytometry of Nile red stained transformants grown in 96-deep-well plates under three sequential conditions (presence of nitrogen, then absence of nitrogen, followed by oil remobilization); a confirmation step using Nile red stained biological triplicates; and a validation step consisting of the quantification by thin layer chromatography of oil content of selected strains. Thirty-one mutants were isolated by screening 1,800 transformants generated by random insertional mutagenesis (1.7%). Five showed increased oil accumulation under the nitrogen-replete condition and 13 had altered oil content under nitrogen-depletion. All mutants were affected in oil remobilization.
  
  
  CONCLUSIONS
  This study demonstrates that various types of oil mutants can be isolated in Chlamydomonas based on the method set-up here, including mutants accumulating oil under optimal biomass growth. The strategy conceived and the protocol set-up should be applicable to other microalgal species such as Nannochloropsis and Chlorella, thus serving as a useful tool in Chlamydomonas oil research and algal biotechnology.
authors.affiliation: CEA Cadarache, Institute of Environmental Biology and Biotechnology, Saint-Paul-lez-Durance F-13108, France.|CNRS, UMR7265, Saint-Paul-lez-Durance F-13108, France.|Aix-Marseille Université, Saint-Paul-lez-Durance F-13108, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Cagnon
authors.affiliation: CEA Cadarache, Institute of Environmental Biology and Biotechnology, Saint-Paul-lez-Durance F-13108, France.|CNRS, UMR7265, Saint-Paul-lez-Durance F-13108, France.|Aix-Marseille Université, Saint-Paul-lez-Durance F-13108, France.
authors.forename: Boris
authors.initials: B
authors.lastname: Mirabella
authors.affiliation: CEA Cadarache, Institute of Environmental Biology and Biotechnology, Saint-Paul-lez-Durance F-13108, France.|CNRS, UMR7265, Saint-Paul-lez-Durance F-13108, France.|Aix-Marseille Université, Saint-Paul-lez-Durance F-13108, France.|Present address: Institut des Sciences Moléculaires de Marseille, UMR 7313, Aix-Marseille Université, Marseille, France.
authors.forename: Hoa Mai
authors.initials: HM
authors.lastname: Nguyen
authors.affiliation: CEA Cadarache, Institute of Environmental Biology and Biotechnology, Saint-Paul-lez-Durance F-13108, France.|CNRS, UMR7265, Saint-Paul-lez-Durance F-13108, France.|Aix-Marseille Université, Saint-Paul-lez-Durance F-13108, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Beyly-Adriano
authors.affiliation: CEA Cadarache, Institute of Environmental Biology and Biotechnology, Saint-Paul-lez-Durance F-13108, France.|CNRS, UMR7265, Saint-Paul-lez-Durance F-13108, France.|Aix-Marseille Université, Saint-Paul-lez-Durance F-13108, France.
authors.forename: Séverine
authors.initials: S
authors.lastname: Bouvet
authors.affiliation: CEA Cadarache, Institute of Environmental Biology and Biotechnology, Saint-Paul-lez-Durance F-13108, France.|CNRS, UMR7265, Saint-Paul-lez-Durance F-13108, France.|Aix-Marseille Université, Saint-Paul-lez-Durance F-13108, France.
authors.forename: Stéphan
authors.initials: S
authors.lastname: Cuiné
authors.affiliation: CEA Cadarache, Institute of Environmental Biology and Biotechnology, Saint-Paul-lez-Durance F-13108, France.|CNRS, UMR7265, Saint-Paul-lez-Durance F-13108, France.|Aix-Marseille Université, Saint-Paul-lez-Durance F-13108, France.
authors.forename: Fred
authors.initials: F
authors.lastname: Beisson
authors.affiliation: CEA Cadarache, Institute of Environmental Biology and Biotechnology, Saint-Paul-lez-Durance F-13108, France.|CNRS, UMR7265, Saint-Paul-lez-Durance F-13108, France.|Aix-Marseille Université, Saint-Paul-lez-Durance F-13108, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Peltier
authors.affiliation: CEA Cadarache, Institute of Environmental Biology and Biotechnology, Saint-Paul-lez-Durance F-13108, France.|CNRS, UMR7265, Saint-Paul-lez-Durance F-13108, France.|Aix-Marseille Université, Saint-Paul-lez-Durance F-13108, France.
authors.forename: Yonghua
authors.initials: Y
authors.lastname: Li-Beisson