Print Email Facebook Twitter An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae Title An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae Author Oud, B. Flores, C.L. Gancedo, C. Zhang, X. Trueheart, J. Daran, J.M. Pronk, J.T. Van Maris, A.J.A. Faculty Applied Sciences Department BT/Biotechnology Date 2012-09-15 Abstract 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 C2-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 C2-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. Subject inverse metabolic engineeringreverse metabolic engineeringwhole genome sequencingglucose toleranceby-product reductionMTH1 allele To reference this document use: http://resolver.tudelft.nl/uuid:b4fe13a8-7cce-416f-802a-6fd618726d56 DOI https://doi.org/10.1186/1475-2859-11-131 Publisher BioMed Central ISSN 1475-2859 Source http://www.microbialcellfactories.com/content/11/1/131 Source Microbial Cell Factories, 11, 2012 Part of collection Institutional Repository Document type journal article Rights © 2012 The Author(s)Licensee BioMed Central Ltd. - This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Files PDF vanMaris_2012.pdf 944.34 KB Close viewer /islandora/object/uuid:b4fe13a8-7cce-416f-802a-6fd618726d56/datastream/OBJ/view