Isolation and characterization of carotenoid hyperproducing mutants of yeast by flow cytometry and cell sorting.
The carotenoid pigment astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) is an important component in feeds of aquacultural 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-overproducing 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.
An
G H
GH
Department of Food Microbiology and Toxicology, University of Wisconsin, Madison 53706.
Bielich
J
J
Auerbach
R
R
Johnson
E A
EA
D002338:Carotenoids / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods; D005453:Fluorescence; D008856:Microscopy, Fluorescence; D009154:Mutation; D024341:Xanthophylls; D015003:Yeasts / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019207:beta Carotene / Q000031:analogs & derivatives*
D024341:Xanthophylls; D019207:beta Carotene; D002338:Carotenoids; C005948:astaxanthine