Significance The shift from unicellular to multicellular life forms represents a key innovation step in the evolution of life on Earth. However, knowledge on the evolutionary pressures resulting in the selection of multicellular life forms and the underlying molecular mechanisms is far from complete. Our study provides a complete identification of the specific genetic changes by which the unicellular eukaryote S. cerevisiae can acquire a multicellular, fast-sedimenting phenotype. We demonstrated that a minimal evolutionary mechanism encompassed a deregulation of the late step of the cell cycle through mutation in ACE2 followed by whole genome duplication.

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Successful reverse engineering of mutants that have been obtained by nontargeted strain improvement has long presented a major challenge in yeast biotechnology. This paper reviews the use of genome-wide approaches for analysis of Saccharomyces cerevisiae strains originating from evolutionary engineering or random mutagenesis. On the basis of an evaluation of the strengths and weaknesses of different methods, we conclude that for the initial identification of relevant genetic changes, whole genome sequencing is superior to other analytical techniques, such as transcriptome, metabolome, proteome, or array-based genome analysis. Key advantages of this technique over gene expression analysis include the independency of genome sequences on experimental context and the possibility to directly and precisely reproduce the identified changes in naive strains. The predictive value of genome-wide analysis of strains with industrially relevant characteristics can be further improved by classical genetics or simultaneous analysis of strains derived from parallel, independent strain improvement lineages.

BackgroundPyruvate-decarboxylase negative (Pdc-) strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc-S. cerevisiae an interesting platform for efficient conversion of glucose towards pyruvate-derived products without formation of ethanol as a by-product. However, Pdc- strains cannot grow on high glucose concentrations and require C2-compounds (ethanol or acetate) for growth under conditions with low glucose concentrations, which hitherto has limited application in industry.ResultsGenetic analysis of a Pdc- strain previously evolved to overcome these deficiencies revealed a 225bp in-frame internal deletion in MTH1, encoding a transcriptional regulator involved in glucose sensing. This internal deletion contains a phosphorylation site required for degradation, thereby hypothetically resulting in increased stability of the protein. Reverse engineering of this alternative MTH1 allele into a non-evolved Pdc- strain enabled growth on 20 g l-1 glucose and 0.3% (v/v) ethanol at a maximum specific growth rate (0.24 h-1) similar to that of the evolved Pdc- strain (0.23 h-1). Furthermore, the reverse engineered Pdc- strain grew on glucose as sole carbon source, albeit at a lower specific growth rate (0.10 h-1) than the evolved strain (0.20 h-1). The observation that overexpression of the wild-type MTH1 allele also restored growth of Pdc-S. cerevisiae on glucose is consistent with the hypothesis that the internal deletion results in decreased degradation of Mth1. Reduced degradation of Mth1 has been shown to result in deregulation of hexose transport. In Pdc- strains, reduced glucose uptake may prevent intracellular accumulation of pyruvate and/or redox problems, while release of glucose repression due to the MTH1 internal deletion may contribute to alleviation of the C2-compound auxotrophy.ConclusionsIn this study we have discovered and characterised a mutation in MTH1 enabling Pdc- strains to grow on glucose as the sole carbon source. This successful example of reverse engineering not only increases the understanding of the glucose tolerance of evolved Pdc-S. cerevisiae, but also allows introduction of this portable genetic element into various industrial yeast strains, thereby simplifying metabolic engineering strategies.