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Biotechnol Biofuels. 2013 Dec 2;6(1):178. doi: 10.1186/1754-6834-6-178.

Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii.

Cagnon C#1,2,3, Mirabella B#1,2,3, Nguyen HM1,2,3,4, Beyly-Adriano A1,2,3, Bouvet S1,2,3, Cuiné S1,2,3, Beisson F1,2,3, Peltier G1,2,3, Li-Beisson Y1,2,3.

Author information

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.
Contributed equally



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.


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.


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.

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