A Case Study of Genomic Instability in an Industrial Strain of Saccharomyces cerevisiae

Abstract

The Saccharomyces cerevisiae strain JAY270/PE2 is a highly efficient biocatalyst used in the production of bioethanol from sugarcane feedstock. This strain is heterothallic and diploid, and its genome is characterized by abundant structural and nucleotide polymorphisms between homologous chromosomes. One of the reasons it is favored by many distilleries is that its cells do not normally aggregate, a trait that facilitates cell recycling during batch-fed fermentations. However, long-term propagation makes the yeast population vulnerable to the effects of genomic instability, which may trigger the appearance of undesirable phenotypes such as cellular aggregation. In pure cultures of JAY270, we identified the recurrent appearance of mutants displaying a mother-daughter cell separation defect resulting in rough colonies in agar media and fast sedimentation in liquid culture. We investigated the genetic basis of the colony morphology phenotype and found that JAY270 is heterozygous for a frameshift mutation in the ACE2 gene (ACE2/ace2-A7), which encodes a transcriptional regulator of mother-daughter cell separation. All spontaneous rough colony JAY270-derived isolates analyzed carried copy-neutral loss-of-heterozygosity (LOH) at the region of chromosome XII where ACE2 is located (ace2-A7/ace2-A7). We specifically measured LOH rates at the ACE2 locus, and at three additional chromosomal regions in JAY270 and in a conventional homozygous diploid laboratory strain. This direct comparison showed that LOH rates at all sites were quite similar between the two strain backgrounds. In this case study of genomic instability in an industrial strain, we showed that the JAY270 genome is dynamic and that structural changes to its chromosomes can lead to new phenotypes. However, our analysis also indicated that the inherent level of genomic instability in this industrial strain is normal relative to a laboratory strain. Our work provides an important frame of reference to contextualize the interpretation of instability processes observed in the complex genomes of industrial yeast strains.

Punctuated Aneuploidization of the Budding Yeast Genome

“…We plated cultures to CAN media, screened CAN R colonies to identify rough clones, and determined the Ura +/− phenotype of each. In contrast to the rough colonies recovered from nonselective conditions ( Sampaio et al 2017 ; Rodrigues Prause et al 2018 ), 79% (41/52) of rough CANR colonies had lost sChr12 in addition to jChr5 ( Figure 1B ). Our finding that the selected loss of jChr5 markedly shifted the mutational spectrum of LOH on Chr12R to CCNA was consistent with our above prediction, and indicated that clones harboring one aneuploidy were enriched for the presence of additional unselected aneuploidies.…”

“…When we visually inspected CAN-resistant (CAN R ) colonies, we noted that while the majority had a normal smooth appearance; 1 in ∼450 colonies displayed a distinctive rough morphology ( Figure 1A ). Previously, we reported that this morphological switch is precipitated by interhomolog mitotic recombination (MR), resulting in loss of the wild-type allele of the ACE2 gene encoded on sChr12R and homozygosis of the mutant ace2 -A7 allele on jChr12R ( Rodrigues Prause et al 2018 ). ace2 -A7 cells fail to separate after cytokinesis and consequently form rough colonies ( Nelson et al 2003 ; Rodrigues Prause et al 2018 ).…”

“…Previously, we reported that this morphological switch is precipitated by interhomolog mitotic recombination (MR), resulting in loss of the wild-type allele of the ACE2 gene encoded on sChr12R and homozygosis of the mutant ace2 -A7 allele on jChr12R ( Rodrigues Prause et al 2018 ). ace2 -A7 cells fail to separate after cytokinesis and consequently form rough colonies ( Nelson et al 2003 ; Rodrigues Prause et al 2018 ). In this previous study, rough colonies appeared on nonselective media at a frequency of 1 in ∼10,000 colonies, and were always caused by MR events spanning ACE2 on Chr12R ( Sampaio et al 2017 ; Rodrigues Prause et al 2018 ).…”

“…ace2 -A7 cells fail to separate after cytokinesis and consequently form rough colonies ( Nelson et al 2003 ; Rodrigues Prause et al 2018 ). In this previous study, rough colonies appeared on nonselective media at a frequency of 1 in ∼10,000 colonies, and were always caused by MR events spanning ACE2 on Chr12R ( Sampaio et al 2017 ; Rodrigues Prause et al 2018 ). Rough colonies resulting from complete loss of Chr12 were never observed (0/67 genotyped clones).…”

How to characterize a strain? The neglected influence of clonal heterogeneity on the phenotypes of industrialSaccharomyces

“…comparing Saccharomyces strains rarely address the "founder effect" of using a subclone lineage of a strain (due to methodological constraints) to characterize the strain itself. Only a few studies have focused on heterogeneous subclone lineages as well as cryptic variation of the PE-2 Bioethanol strain or its derivative JAY270 (Reis et al 2014;Rodrigues-Prause et al 2018; Sampaio, Watson and Argueso 2019) and those of the Lager W34/70 strain (Bolat, Walsh and Turtoi 2008;van den Broek et al 2015), while in most other cases, strains are used interchangeably with subclone lineages. In fact, the commonly used and well-known tetraploid Ale strain of our study, has been sequenced and analyzed by three recent studies, all of which found different karyotypes due to apparent genotypic heterogeneity of the given subclone lineages studied by each (Table 2., Figure S1.).…”