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The growth of Pichiapastoris in a mixture of either glycerol or glucose and methanol follows a diauxic growth, with C1, utilizing enzymes being repressed. Therefore, these carbon sources can not be used as a mixture with methanol to simultaneously grow P. pastoris and induce C1, utilizing enzymes, especially in shake flask cultures of AOX-deficient P. pastoris. Among the alternative carbon sources tested, alanine, sorbitol, mannitol and trehalose, did not repress β-gal production when methanol was used as an inducer in mut- strain of P. pastoris. Our results show that either one of alanine, sorbitol, mannitol or trehalose can be used as a sole carbon and energy source for P. pastoris, although the doubling time on trehalose was very long. Mut- strains growing in media containing trehalose, alanine, sorbitol and mannitol with methanol (0.5%) as an inducing agent expressed as much or higher amount of β-gal as compared to the mut+ growing in methanol containing media.