Genetic transformation of xylose-fermenting yeastPichia stipitis

Abstract

A plasmid-mediated transformation system has been developed for the xylose-fermenting yeastPichia stipitis. We found that plasmid vectors containing theSaccharomyces cerevisiae 2 μ replicon and the kanamycin resistance gene (Km^R) could be introduced into thePichia cells and maintained as extrachromosomal elements.Pichia transformants containing such vectors will be resistant to the antibiotic geneticin that can be inactivated by the protein product of Km^R. Plasmids identical to those used for transformation can be recovered from thePichia transformants. Protocols for transformation ofP. stipitis by the CaCl₂-polyethylene glycol-protoplast process or by direct electroporation of intactPichia cells have both been developed.