Improving Industrially Relevant Phenotypic Traits by Engineering Chromosome Copy Number in Saccharomyces pastorianus

Abstract

The lager-brewing yeast Saccharomyces pastorianus is a hybrid between S. cerevisiae and S. eubayanus with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial Saccharomyces strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of S. pastorianus remains elusive. Here we attempt to delete single copies of chromosomes which are relevant for the production of off-flavor compound diacetyl by centromere silencing. However, the engineered strains display CNV of multiple nontargeted chromosomes. We attribute this unintended CCNV to inherent instability and to a mutagenic effect of electroporation and of centromere-silencing. Regardless, the resulting strains displayed large phenotypic diversity. By growing centromere-silenced cells in repeated sequential batches in medium containing 10% ethanol, mutants with increased ethanol tolerance were obtained. By using CCNV mutagenesis by exposure to the mitotic inhibitor MBC, selection in the same setup yielded even more tolerant mutants that would not classify as genetically modified organisms. These results show that CCNV of alloaneuploid S. pastorianus genomes is highly unstable, and that CCNV mutagenesis can generate broad diversity. Coupled to effective selection or screening, CCNV mutagenesis presents a potent tool for strain improvement.

How to characterize a strain? Clonal heterogeneity in industrial Saccharomyces influences both phenotypes and heterogeneity in phenotypes

“…The latter include ploidy changes, aneuploidies/chromosome copy number variations (CCNVs), loss of heterozygosity (LOH), gross chromosomal rearrangements (GCRs), and mitotic crossing overs (Peter et al, 2018; van den Broek et al, 2015; Zhu et al, 2016). These phenomena can alter their industrial performance and may happen very rapidly (Gorter de Vries et al, 2020; Kadowaki et al, 2017; Large et al, 2020; Morard et al, 2019, 2020; Zhang et al, 2016). Along with point mutations, these GSV events result in clonal populations gradually accumulating differences in various traits, leading to clonal heterogeneity, clonal interference (competition among isogenic asexual lineages), and hence the emergence of so‐called subclonal lineages, reminiscent of the experimental evolution setups conducted with laboratory strains (Blundell et al, 2018; Lang et al, 2011; Large et al, 2020; Payen et al, 2016).…”

Genomic instability in an interspecific hybrid of the genus Saccharomyces: a matter of adaptability

“…In fact, in the strains sequenced to the date, aneuploidy is found in industrial strains that are often polyploid [51–53], which is also the case for allopolyploids of the genus, suggesting a link between genome stability, aneuploidy and hybridization in domestication. Furthermore, large difference of aneuploidy in a population has been coupled with phenotypic diversity, ultimately facilitating large phenotypic leaps [54, 55]. In general, it has been revealed that the genome structure is much less stable than what was classically thought, with increasing evidence that it could be an important aspect of evolution and domestication.…”

Long-insert clone experimental evidence for assembly improvement and chimeric chromosomes detection in an allopentaploid beer yeast

“…For example, genome diversity of lager yeast strains is thought to be limited due to an evolutionary bottleneck that occurred during the industrialization of lager beer production ( Gallone et al 2019 ). However, extra-variation and innovation in the lager beer industry could be achieved by screening for different ploidy levels yeasts in available collections or by artificially increasing the number of chromosome copies by genetic modification, physical or chemical treatments ( Gorter de Vries et al 2017 , 2020 ).…”