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

Abstract

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.

Expression of Lactococcus lactis NADH oxidase increases 2,3-butanediol production in Pdc-deficient Saccharomyces cerevisiae

“…Mth1p regulates glucose transporters by inactivating the transcriptional regulator Rgt1 (Moriya and Johnston, 2004). Reduced glucose influx by the MTH1 mutation in Pdc-deficient S. cerevisiae strains partially alleviated the demand for C 2 -compounds and retardation of cell growth on glucose (Oud et al, 2012). In this study, however, the BD5_Con strain with the wild type MTH1 could grow on a synthetic glucose medium despite of the low growth rate (Fig.…”

“…As a result, the Pdc-deficient S. cerevisiae strains are unable to grow in batch cultures on defined media with glucose as a sole carbon source (Flikweert et al, 1996). As recently reported, the mutation of MTH1 allows the Pdc-deficient S. cerevisiae strains to overcome the growth defects on glucose (Oud et al, 2012). Mth1p regulates glucose transporters by inactivating the transcriptional regulator Rgt1 (Moriya and Johnston, 2004).…”

Enhanced production of 2,3-butanediol by engineered Saccharomyces cerevisiae through fine-tuning of pyruvate decarboxylase and NADH oxidase activities

“…In particular, a mutation (G241C) was employed for improving 2,3-BD production by the S. cerevisiae strain because the MTH1 mutation could overcome the C 2 -auxotrophy of Pdc-deficient S. cerevisiae [12, 21]. However, the MTH1 mutation resulted in reduced expression levels of hexose transporters in S. cerevisiae [11, 20], causing slow glucose uptake rates.…”

“…In the presence of extracellular glucose, signal transduction via the glucose sensors (Rgt2/Snf3) and casein kinases (Yck1/2) induces phosphorylation of Mth1 to be degraded [19]. The degradation of Mth1 led to the down-regulation of hexose transporter genes ( HXT s) [20], resulting in decreased glucose influx rate in spite of extracellular glucose [21]. A slow glucose uptake rate caused by the MTH1 mutation might be responsible for restoration of growth defect by the Pdc-deficient strains on glucose [12].…”

“…A slow glucose uptake rate caused by the MTH1 mutation might be responsible for restoration of growth defect by the Pdc-deficient strains on glucose [12]. Additionally, although the exact mechanism remains unknown, the mutant MTH1 could partially relieve the C 2 -auxotrophy of Pdc-deficient S. cerevisiae [12, 21]. The mutation in MTH1 might be regarded as an indispensable strategy for Pdc-deficient S. cerevisiae to grow on glucose, but the slow glucose consumption rates caused by the MTH1 mutation resulted in much lower 2,3-BD productivity [12] than that by bacterial 2,3-BD production systems [22, 23].…”