Isolation and characterization of carotenoid hyperproducing mutants of yeast by flow cytometry and cell sorting

The carotenoid pigment astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) is an important component in feeds of aquacul-tural animals. It is produced as a secondary metabolite by the yeast Phaffia rhodozyma , and the isolation of rare mutants that produce increased quantities is limited by the lack of genetic selections. As a model system for enriching mutants increased in production of secondary metabolites, we have used quantitative flow cytometry/cell sorting (FCCS) to isolate astaxanthin hyperproducing mutants of the yeast. Experimental conditions were developed that gave a quantitative correlation of fluorescence and carotenoid content. In mutated populations, a 10,000-fold enrichment of carotenoid-overpro-ducing yeasts was obtained. Distinctive differences were detected by FCCS in fluorescence and forward scatter values of mutant and wild-type populations of yeasts. Comparison of wild-type and mutant clones by fluorescence confocal laser microscopy showed that the mutants had more intense fluorescence throughout the cell than the wild-type. Quantitative FCCS is a sensitive method to isolate and characterize carotenoid overproducing mutants and should be useful as a general method for the isolation of mutants increased in other fluorescent metabolites.