Genome Shuffling Protocol for the Pentose-Fermenting Yeast Scheffersomyces stipitis

Abstract

This chapter presents the protocol for genome shuffling based on recursive cross-mating in the pentose-fermenting yeast Scheffersomyces (Pichia) stipitis. Genome shuffling involves two stages. In the first stage, a pool of mutants with improved phenotypes is selected. Several rounds of random mutagenesis can be done using different mutagens, and mutant selection can be based on different criteria to generate different mutant cell lines. In the second stage, the genomes of mutants derived from different lines are mated recursively to allow for genetic recombination, followed by screening after each mating cycle to select for improved phenotypes in the recombinants. A number of reports have described genome shuffling based on recursive protoplast fusion in bacteria and yeasts. Recently, we developed mating-based genome shuffling in the pentose-fermenting yeast S. stipitis. We have used this approach to obtain genetically stable mutants of S. stipitis with considerably improved tolerance to hardwood spent sulphite liquor (HW SSL), a pulping waste liquor containing a complex mixture of inhibitory substances. This was achieved in the complete absence of knowledge as to the precise genetic modifications needed to confer HW SSL tolerance. Here we describe the protocols for recursive UV mutagenesis, cross-mating, sporulation and isolation of recombinants with improved phenotypic traits.