Isolation of Phaffia rhodozyma Mutants with Increased Astaxanthin Content

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Abstract
Plating of the astaxanthin-producing yeast Phaffia rhodozyma onto yeast-malt agar containing 50 μM antimycin A gave rise to colonies of unusual morphology, characterized by a nonpigmented lower smooth surface that developed highly pigmented vertical papillae after 1 to 2 months. Isolation and purification of the pigmented papillae, followed by testing for pigment production in shake flasks, demonstrated that several antimycin isolates were increased two- to fivefold in astaxanthin content compared with the parental natural isolate (UCD-FST 67-385). One of the antimycin strains (ant-1) and a nitrosoguanidine derivative of ant-1 (ant-1-4) produced considerably more astaxanthin than the parent (ant-1 had 800 to 900 μg/g; ant-1-4 had 900 to 1,300 μg/g; and 67-385 had 300 to 450 μg/g). The mutant strains were compared physiologically with the parent. The antimycin mutants grew slower on ammonia, glutamate, or glutamine as nitrogen sources compared with the natural isolate and also had lower cell yields on several carbon sources. Although isolated on antimycin plates, they were found to be more susceptible to antimycin A, apparently owing to the spatial separation of the papillae from the agar. They were also more susceptible than the parent to the respiratory inhibitor thenoyltrifluoroacetone and were slightly more susceptible to cyanide, but did not differ from the natural isolate in susceptibility to azide. The antimycin-derived strains were also killed faster than the parent by hydrogen peroxide. The carotenoid compositions of the parent and the antimycin-derived strains were similar to those previously determined in the type strain (UCD-FST 67-210) except that two carotenoids not previously found in the type strain were present in increased quantities in the antimycin mutants and phoenicoxanthin was a minor component. The chemical properties of the unknown carotenoids suggested that the strains isolated on antimycin agar tended to oxygenate and desaturate carotene precursors to a greater extent than the parent. The physiology of the antimycin isolates and the known specificity of antimycin for cytochrome b in the respiratory chain suggests that alteration of cytochrome b or cytochrome P-450 components involved in oxygenation and desaturation of carotenes in mitochondria are affected, which results in increased astaxanthin production. These astaxanthin-overproducing mutants and more highly pigmented derivative strains could be useful in providing a natural source of astaxanthin for the pen-reared-salmon industry or for other farmed animals that contain astaxanthin as their principal carotenoid. Images