A fluorescence-activated cell sorting-based strategy for rapid isolation of high-lipid Chlamydomonas mutants.

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 Chlamydomonas 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 sta1 from a mixture of sta1 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 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 CHiLiS opens the door to a systems-level understanding of green algal lipid biology by enabling genome-saturating isolation of mutants in key genes.

Re-engineering multicloning sites for function and convenience.

Multicloning sites (MCSs) in standard expression vectors are widely used and thought to be benign, non-interacting elements that exist for mere convenience. However, MCSs impose a necessary distance between promoter elements and genes of interest. As a result, the choice of cloning site defines the genetic context and may introduce significant mRNA secondary structure in the 5'-untranslated region leading to strong translation inhibition. Here, we demonstrate the first performance-based assessment of MCSs in yeast, showing that commonly used MCSs can induce dramatic reductions in protein expression, and that this inhibition is highly promoter and gene dependent. In response, we develop and apply a novel predictive model of structure-based translation inhibition to design improved MCSs for significantly higher and more consistent protein expression. In doing so, we were able to minimize the inhibitory effects of MCSs with the yeast TEF, CYC and GPD promoters. These results highlight the non-interchangeable nature of biological parts and represent the first complete, global redesign of a genetic circuit of such widespread importance as a multicloning site. The improved translational control offered by these designed MCSs is paramount to obtaining high titers of heterologous proteins in eukaryotes and to enabling precise control of genetic circuits.