High‐throughput fluorescence‐activated cell sorting for lipid hyperaccumulating Chlamydomonas reinhardtii mutants

Abstract

SummaryThe genetically tractable microalga Chlamydomonas reinhardtii has many advantages as a model for renewable bioproducts and/or biofuels production. However, one limitation of C. reinhardtii is its relatively low-lipid content compared with some other algal species. To overcome this limitation, we combined ethane methyl sulfonate mutagenesis with fluorescence-activated cell sorting (FACS) of cells stained with the lipophilic stain Nile Red to isolate lipid hyperaccumulating mutants of C. reinhardtii. By manipulating the FACS gates, we sorted mutagenized cells with extremely high Nile Red fluorescence signals that were rarely detected in nonmutagenized populations. This strategy successfully isolated several putative lipid hyperaccumulating mutants exhibiting 23% to 58% (dry weight basis) higher fatty acid contents than their progenitor strains. Significantly, for most mutants, nitrogen starvation was not required to attain high-lipid content nor was there a requirement for a deficiency in starch accumulation. Microscopy of Nile Red stained cells revealed that some mutants exhibit an increase in the number of lipid bodies, which correlated with TLC analysis of triacyglycerol content. Increased lipid content could also arise through increased biomass production. Collectively, our findings highlight the ability to enhance intracellular lipid accumulation in algae using random mutagenesis in conjunction with a robust FACS and lipid yield verification regime. Our lipid hyperaccumulating mutants could serve as a genetic resource for stacking additional desirable traits to further increase lipid production and for identifying genes contributing to lipid hyperaccumulation, without lengthy lipid-induction periods.

Long-duration effect of multi-factor stresses on the cellular biochemistry, oil-yielding performance and morphology of Nannochloropsis oculata

“…Previous reports often qualitatively describe lipids by the NR staining method [45–47]. By combining the conventional methods and the fluorometric staining using NR, this study identified linear regression equations with high goodness of fit between FI and the oil-yielding performance indexes (i.e., TL content, TAG content, and biodiesel production) (Table 3).…”

Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas

“…Previous studies have assessed the responses of metabolic genes and proteins under non-steady state conditions when TAG accumulates (Miller et al, 2010;Boyle et al, 2012;Msanne et al, 2012;Blaby et al, 2013;Wase et al, 2014) or identified mutants that are defective for TAG accumulation or catabolism (Li et al, 2012;Boyle et al, 2012;Tsai et al, 2014;Xie et al, 2014;Ngan et al, 2015;Kajikawa et al, 2015). The relationship between starch synthesis and TAGs has also been investigated, and it appears that fixed carbon can be shunted toward TAGs when starch synthesis is blocked (Li et al, 2010a(Li et al, , 2010bWork et al, 2010;Siaut et al, 2011;Goodenough et al, 2014).…”

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.…”