Classical Genetics of Pichia pastoris.

A significant advantage of P. pastoris as an experimental system and an eukaryotic expression platform is the ability to readily bring to bear both classical and molecular genetic approaches to a research problem and to develop technologies for manufacturing important proteins. Although the advent of yeast molecular biology has introduced new and exciting capabilities, classical genetics remains the approach of choice in many instances. These include the generation of random mutations by induced mutagenesis, alteration of any gene in the genome by targeted gene disruption or tagging, the removal of unwanted genetic background by backcrossing, the analysis of gene and protein interactions using mating and complementation, as well as the construction of strains with new combinations of mutant alleles and expressed proteins or polypeptides. This chapter describes the basis of P. pastoris classical genetics. Various methods of introducing of mutations into a strain genome are described. How classical genetic approaches can be applied to modern molecular studies and how they can be instrumental in the development of industrial strains to produce complex proteins will be discussed. Some basic information about available wild-type and genetically modified strains, as well as a method for their long-term storage, is provided.An understanding of the P. pastoris life cycle and its manipulation is the key to genetic analysis and strain construction. In this chapter, a procedure for knocking out an essential gene is described, together with how to determine if the genomic locus involved is essential. The colony screening method to detect the secretion of a specific recombinant protein via the "Yeastern blot" is outlined. An application of P. pastoris classical genetics to the creation and production of therapeutic antibodies and other complex proteins is also discussed.