DNA-mediated transformation.

Several methods for DNA-mediated transformation of Pichia pastoris have been developed which vary in type of DNA that is transformable (e.g., linear versus circular) efficiency, cost, and labor and each is described in detail. As in Saccharomyces cerevisiae, gene replacement (also known as gene knock-out) methods provide a unique tool to investigate the function of specific P. pastoris genes. After construction, the function of the deleted gene is investigated from the phenotype of the mutant strain. In S. cerevisiae, an efficient polymerase chain reaction (PCR)-based method for the construction of gene replacement fragments has been developed. Modifications of this PCR method have been developed to adapt this approach to P. pastoris.