An impaired ubiquitin ligase complex favors initial growth of auxotrophic yeast strains in synthetic grape must
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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.
KeywordsWine yeast Adaptive laboratory evolution (ALE) Experimental evolution Fermentation kinetics Next-generation sequencing (NGS) Rsp5p-Bul1/2p
We are grateful to Cristina Juez and Laura López for technical assistance, and Rafael Torres for advise with NGS data analysis. This work was supported by the Spanish Ministerio de Ciencia e Innovación (grants AGL2009-07327 and AGL2012-32064) and Junta de Andalucía (grant P10-AGR6544). AM was the recipient of a FPI fellowship from the Spanish Ministerio de Economía y Competitividad. MQ and MN were recipients of JAE-Doc fellowships from the Spanish National Research Council (CSIC), co-funded by the European Social Fund of the EU.
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