Abstract: Transcription promoter and terminator sequences from the Pichia methanolica glyceraldehyde-3-phosphate dehydrogenase 1 gene (GAP1 gene) are disclosed. The sequences are useful within DNA constructs for the production of proteins of interest in cultured P. methanolica cells. Within the expression vectors, a GAP1 promoter and/or a GAP1 terminator is operably linked to a DNA segment encoding the protein of interest.

Abstract: Protease-deficient strains of the methylotrophic yeast Pichia methanolica and materials and methods for generating such strains are disclosed. The strains have a functional deficiency in a vacuolar protease, such as proteinase A or proteinase B. The strains are useful as hosts for the expression of heterologous genes encoding proteins of commercial or other interest.

Abstract: Pichia methanolica cells in which an alcohol oxidase gene has been disrupted are disclosed. The cells may also be deficient in vacuolar protease activity. The cells are useful as hosts in recombinant protein production methods.

Abstract: Methods for transforming Pichia methanolica, and DNA molecules useful in transformation of P. methanolica, are disclosed. P. methanolica cells are exposed, in the presence of DNA molecules, to a pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm and a pulse duration of from 1 to 40 milliseconds, whereby the DNA molecules are introduced into the cells. The DNA molecules may comprise an expression unit that includes a transcription promoter of a P. methanolica gene operably linked to a segment encoding a polypeptide or protein of interest. The DNA molecules may also encode a selectable marker, such as a P. methanolica ADE2 gene. Cells transformed according to the invention may be used in production systems for the preparation of proteins of commercial importance.