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Summary  There is significant interest in farming algae for the direct production of biofuels and valuable lipids.  Chlamydomonas reinhardtii  is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants of this organism with a perturbed lipid content are slow and tedious. Here, we present the   C hlamydomonas  high‐lipid sorting ( CH i L i S ) strategy, which enables enrichment of high‐lipid mutants by fluorescence‐activated cell sorting ( FACS ) of pooled mutants stained with the lipid‐sensitive dye  N ile  R ed. This method only takes 5 weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantification of lipid content while preserving cell viability. We improved separation of high‐lipid mutants from the wild type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20‐fold enrichment of the known high‐lipid mutant  sta1  from a mixture of  sta1  and wild‐type cells. We then applied  CH i L i S  to sort thousands of high‐lipid cells from a pool of about 60 000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that about 50% showed a reproducible high‐lipid phenotype. We further characterized nine of the mutants with the highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had a higher triacylglycerol content and perturbed whole‐cell fatty acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of  CH i L i S  opens the door to a systems‐level understanding of green algal lipid biology by enabling genome‐saturating isolation of mutants in key genes.

A fluorescence‐activated cell sorting‐based strategy for rapid isolation of high‐lipid C hlamydomonas mutants