Evolution of a Yeast With Industrial Background Under Winemaking Conditions Leads to Diploidization and Chromosomal Copy Number Variation.

Industrial wine yeast strains show genome particularities, with strains showing polyploid genomes or chromosome copy number variations, being easier to identify. Although these genomic structures have classically been considered transitory steps in the genomic adaptation to new environmental conditions, they may be more stable than thought. These yeasts are highly specialized strains able to cope with the different stresses associated with the fermentation process, from the high osmolarity to the final ethanol content. In this work, we use adaptive laboratory evolution, focusing on the initial steps of the fermentation process, where growth rate is maximum, to provide new insights into the role of the different genomic and chromosomic rearrangements that occur during adaptation to wine conditions, and providing an understanding of the chronology of the different evolutionary steps.

An impaired ubiquitin ligase complex favors initial growth of auxotrophic yeast strains in synthetic grape must.

We used experimental evolution in order to identify genes involved in the adaptation of Saccharomyces cerevisiae to the early stages of alcoholic fermentation. Evolution experiments were run for about 200 generations, in continuous culture conditions emulating the initial stages of wine fermentation. We performed whole-genome sequencing of four adapted strains from three independent evolution experiments. Mutations identified in these strains pointed to the Rsp5p-Bul1/2p ubiquitin ligase complex as the preferred evolutionary target under these experimental conditions. Rsp5p is a multifunctional enzyme able to ubiquitinate target proteins participating in different cellular processes, while Bul1p is an Rsp5p substrate adaptor specifically involved in the ubiquitin-dependent internalization of Gap1p and other plasma membrane permeases. While a loss-of-function mutation in BUL1 seems to be enough to confer a selective advantage under these assay conditions, this did not seem to be the case for RSP5 mutated strains, which required additional mutations, probably compensating for the detrimental effect of altered Rsp5p activity on essential cellular functions. The power of this experimental approach is illustrated by the identification of four independent mutants, each with a limited number of SNPs, affected within the same pathway. However, in order to obtain information relevant for a specific biotechnological process, caution must be taken in the choice of the background yeast genotype (as shown in this case for auxotrophies). In addition, the use of very stable continuous fermentation conditions might lead to the selection of a rather limited number of adaptive responses that would mask other possible targets for genetic improvement.

Genome-wide study of the adaptation of Saccharomyces cerevisiae to the early stages of wine fermentation.

This work was designed to identify yeast cellular functions specifically affected by the stress factors predominating during the early stages of wine fermentation, and genes required for optimal growth under these conditions. The main experimental method was quantitative fitness analysis by means of competition experiments in continuous culture of whole genome barcoded yeast knockout collections. This methodology allowed the identification of haploinsufficient genes, and homozygous deletions resulting in growth impairment in synthetic must. However, genes identified as haploproficient, or homozygous deletions resulting in fitness advantage, were of little predictive power concerning optimal growth in this medium. The relevance of these functions for enological performance of yeast was assessed in batch cultures with single strains. Previous studies addressing yeast adaptation to winemaking conditions by quantitative fitness analysis were not specifically focused on the proliferative stages. In some instances our results highlight the importance of genes not previously linked to winemaking. In other cases they are complementary to those reported in previous studies concerning, for example, the relevance of some genes involved in vacuolar, peroxisomal, or ribosomal functions. Our results indicate that adaptation to the quickly changing growth conditions during grape must fermentation require the function of different gene sets in different moments of the process. Transport processes and glucose signaling seem to be negatively affected by the stress factors encountered by yeast in synthetic must. Vacuolar activity is important for continued growth during the transition to stationary phase. Finally, reduced biogenesis of peroxisomes also seems to be advantageous. However, in contrast to what was described for later stages, reduced protein synthesis is not advantageous for the early (proliferative) stages of the fermentation process. Finally, we found adenine and lysine to be in short supply for yeast growth in some natural grape musts.