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Background  Pyruvate-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 C 2 -compounds (ethanol or acetate) for growth under conditions with low glucose concentrations, which hitherto has limited application in industry.    Results  Genetic 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 C 2 -compound auxotrophy.    Conclusions  In 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.

An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae