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. Conclusion 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.

Microalgal lipid droplets: composition, diversity, biogenesis and functions

“…The occurrence and formation of LDs can be traced easily by microscopic examination or through flow cytometry, taking advantage of the lipophilic property of dyes such as Nile red or Bodipy (Cagnon et al 2013;Greenspan et al 1985;Mou et al 2012;Xie et al 2014;Yan et al 2013;Zhang et al 2014). Structure of LDs has been studied under Confocal fluorescent or Transmission Electron Microscopes (TEM) (Fig.…”

Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii

“…Cells were usually maintained in Tris-acetatephosphate medium (Harris, 2001) in Erlenmeyer flasks at 25°C with shaking at 120 rpm under constant illumination (150 mmol photons m 22 s 21 ). For photoautotrophic growth, cultures grown in minimal medium were kept under identical conditions with additional supply of 1.8% CO 2 in air (Cagnon et al, 2013). Cell numbers were counted using a Beckman-Coulter multisizer.…”

Lipid droplet mobilization: The different ways to loosen the purse strings

“…The relationship between storage lipid hydrolysis and cell proliferation is also documented in microalgae [12,58,134]. Nutrient deprivation, such as nitrogen starvation, triggers TAG accumulation and induces growth arrest and cellular quiescence.…”