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

Bart Oud12, Carmen-Lisset Flores3, Carlos Gancedo3, Xiuying Zhang45, Joshua Trueheart4, Jean-Marc Daran12, Jack T Pronk12 and Antonius JA van Maris12*

Author Affiliations

1 Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands

2 Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA, Delft, The Netherlands

3 Department of Metabolism and Cell Signaling, Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-UAM, 28029, Madrid, Spain

4 Microbia Inc., 60 Westview Street, Lexington, MA, 02421, USA

5 Present address: Merrimack Pharmaceutical Inc., One Kendall Square, Suite B7201, Cambridge, MA, 02139, USA

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Microbial Cell Factories 2012, 11:131  doi:10.1186/1475-2859-11-131

Published: 15 September 2012

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.

Keywords:
Inverse metabolic engineering; Reverse metabolic engineering; Whole genome sequencing; Glucose tolerance; by-product reduction; MTH1 allele