Monday, July 27, 2009
P20

Genome shuffling of Pichia stipitis for improved tolerance to hardwood spent sulphite liquor

Paramjit K. Bajwa1, Dominic Pinel2, Vincent J.J. Martin2, Jack T. Trevors1, and H. Lee1. (1) Environmental Biology, University of Guelph, Bovey Building, Guelph, ON N1G2W1, Canada, (2) Biology, Concordia University, 7141 Sherbrooke St W., Montreal, QC H4B 1R6, Canada

Abstract: Genome shuffling is a powerful microbial strain improvement technique that uses iterative cycles of genome recombination and selection to combine the useful alleles of many parental strains into single cells showing the desired phenotype. In genome shuffling, an initial pool of mutants is screened to select for strains showing improved phenotypes. The genomes of these selected strains are then recombined iteratively to increase the chances of generating cells with multiple beneficial mutations. In this study, we used genome shuffling based on the natural mating and sporulation ability of the pentose-fermenting yeast Pichia stipitis to isolate stable mutants with improved tolerance to inhibitors in hardwood spent sulfite liquor (HW SSL).  Six UV-induced mutants of P. stipitis were used as the starting strains for 4 rounds of genome shuffling.  After every round, improved strains were selected based on their growth on HW SSL gradient plates. Improved strains selected after each round served as the pool for next round of shuffling. Tolerance to HW SSL increased with each round of shuffling. Fermentation performance of the 2 best HW SSL tolerant strains was tested in defined media and in diluted HW SSL. Both mutant strains utilized 4% (w/v) of xylose or glucose more efficiently and produced more ethanol than the WT. The genome shuffled strains produced ethanol in 85% and 90% (v/v) HW SSL. In contrast, the WT was unable to produce ethanol at 60% (v/v) or higher concentrations of HW SSL.  

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