Recombination between Homoeologous Chromosomes of Lager Yeasts Leads to Loss of Function of the Hybrid GPH1 Gene

Yeasts used in the production of lagers contain complex allopolyploid genomes, resulting from the fusion of two different yeast species closely related to Saccharomyces cerevisiae and Saccharomyces bayanus. Recombination between the homoeologous chromosomes has generated a number of hybrid chromosomes. These recombination events provide potential for adaptive evolution through the loss or gain of gene function. We have examined the genotypic and phenotypic effects of one of the conserved recombination events that occurred on chromosome XVI in the region of YPR159W and YPR160W. Our analysis shows that the recombination event occurred within the YPR160W gene, which encodes the enzyme glycogen phosphorylase and generates a hybrid gene that does not produce mature mRNA and is nonfunctional due to frameshifts in the coding region. The loss of function of the hybrid gene leads to glycogen levels similar to those found in haploid yeast strains. The implications for the control of glycogen levels in fermentative yeasts are discussed.Yeasts used in the production of lagers, originally referred to as Saccharomyces carlsbergensis, are now classified as Saccharomyces pastorianus (18). Lager yeasts contain complex polyploid genomes with general tetraploid DNA content and are believed to have arisen from a natural fusion of two different haploid species followed by a genome duplication event or alternatively from the fusion of two diploid yeast species (2, 4, 14, 18). Subsequent genome changes, such as chromosome loss and/or duplication and translocations, have resulted in unequal numbers of chromosomes in the present-day strains, a state referred to as aneuploidy.A sequence analysis of individual genes indicated that the parental strains contributing to the hybrid strain closely resemble Saccharomyces cerevisiae and Saccharomyces bayanus (6, 15, 16). A whole-genome sequence analysis of the lager yeast strain Weihenstephan (15) identified the presence of three types of chromosomes, referred to as (i) S. cerevisiae-like chromosomes, (ii) S. bayanus-like chromosomes, and (iii) hybrids resulting from recombination events between the homoeologous parental chromosomes.Competitive genomic hybridization (CGH) analysis of two S. pastorianus strains, named CMBS-33 and 6701, identified as many as 28 specific locations where recombination between homoeologous pairs of chromosomes or chromosomal translocations may have occurred (3). Of the 28 sites identified, 13 occur at unique sites on eight different chromosomes, while the rest are in subtelomeric X elements or within 25 kbp of the telomere. Many of the genes adjacent to the recombination sites encode proteins that play essential roles in fermentation, including ADH2, ADH4, AAD6 and TDH2 (ethanol metabolism), FLO10, and PHD1 (3).We have recently shown that recombination at these “hot spots” can be induced by the exposure of lager yeasts to environmental stresses such as high temperature and high osmotic stress (13). Furthermore, fermentation under stress conditions leads to the amplification and loss of telomeric regions on a selected set of chromosomes and gene amplification radiating from the rRNA locus on chromosome XII and the DUP locus on chromosome I (13). Since a number of genes, including the MAL (maltose utilization) and the FLO (flocculation) genes, encoding proteins required for the fermentation process reside at the telomeres, such genome dynamics can have important consequences for the immediate quality and outcome of fermentation in addition to severe consequences on strain stability and purity.One of the recombination events identified by CGH analysis is located on chromosome XVI in the region of YPR159W and YPR160W. DNA to the left of the region hybridizes to S. cerevisiae microarrays, while genes between YPR160W and YPR190C and encompassing approximately 58 kb of DNA displayed a lack of hybridization to these microarrays, suggestive of a hybrid chromosome (3). Whole-genome sequence analysis of the Weihenstephan strain confirmed the existence of hybrid chromosome XVI and indicated the presence a second type of chromosome XVI containing S. bayanus-like sequences to the left of YPR159W (15, 16).To examine the genotypic and phenotypic outcomes of this recombination event, the right arm of chromosome XVI has been cloned from the yeast strain CMBS-33. Our analysis reveals that the recombination event occurred within the open reading frame (ORF) of YPR160W (GPH1) encoding the enzyme glycogen phosphorylase, which is required for the mobilization of stored glycogen through its conversion into glucose-1-P. The recombination event generates a hybrid gene that does not produce a mature mRNA and is nonfunctional due to frameshifts in the coding region.