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Selection for carotenogenesis in Phaffia rhodozyma was achieved by exposure of yeast strains to dark chemical reactions that generate singlet oxygen. Incubation of a mixture of P. rhodozyma strains containing varying levels of carotenoids in hypochlorous acid or hydrogen peroxide resulted in weak selection for pigmented strains. However, the combination of hydrogen peroxide and hypochlorous acid was strongly selective for carotenogenesis and gave a monoculture of a carotenoid-hyperproducer. Exposure of the yeast to ozone for 10 to 20 min also selected for a hyperproducing strain. These selections were relieved by 1,4-diazabicyclo(2.2.2)-octane, a specific quencher of singlet oxygen or by L-ascorbic acid. Continuous growth of P. rhodozyma on agar plates in an ozone/air atmosphere for 5 d decreased astaxanthin and total carotenoid levels and increased the levels of carotenoid biosynthetic intermediates. Repeated rounds of random mutagenesis followed by ozone exposure yielded mutant strains with higher pigmentation than control cultures. Our results support the hypothesis that a primary function of carotenoids in P. rhodozyma is to protect against singlet oxygen generated in the natural environment of the yeast and that a practical method for preventing strain degeneration during industrial fermentations may be achieved by generation of singlet oxygen using simple chemical supplements or by bubbling ozone through P. rhodozyma cultures during fermentation.

Selection for carotenogenesis in the yeast Phaffia rhodozyma by dark-generated singlet oxygen