There is significant interest in farming algae for the direct production of biofuels and valuable lipids. @entity2060 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 @entity2060 high-lipid sorting (CHiLiS) strategy, which enables enrichment of high-lipid mutants by fluorescence-activated cell sorting (FACS) of pooled mutants stained with the lipid-sensitive dye Nile Red. 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 @entity68182 from a mixture of @entity68182 and wild-type cells. We then applied CHiLiS 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 @entity1674 content and perturbed whole-cell @entity844 composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of CHiLiS 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 XXXX mutants.

@entity68182 :: ('5719082(Tax:3055)', 'Gene') :: ['sta1'] @entity1674 :: ('MESH:D014280', 'Chemical') :: ['triacylglycerol'] @entity844 :: ('CHEBI:35366', 'Chemical') :: ['fatty acid'] @entity2060 :: ('3055', 'Species') :: ['Chlamydomonas', 'Chlamydomonas reinhardtii']
@entity2060 :: (3055,Species) :: ['Chlamydomonas']
