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Cited by 104Fungal and yeast genetics researchMicrobial Metabolic Engineering and BioproductionBiofuel production and bioconversion

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

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Oud, Bart, Flores, Carmen-Lisset, Gancedo, Carlos, Zhang, Xiuying, Trueheart, Joshua, Daran, Jean-Marc, Pronk, Jack T, van Maris, Antonius JA·2012·Microbial Cell Factories
DOI 10.1186/1475-2859-11-131PMC PMC3503853
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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 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.

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Crossref · 10.1186/1475-2859-11-131

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Figures

Figure 1 Alignment of the Mth1 amino acid sequences from TAM and RWB837. TAM is a Pdc- strain evolved for growth on glucose and RWB837 is the ancestral Pdc- strain from which the TAM strain was derived. The casein kinase I consensus phosphorylation site is underlined and bold. The deleted region is rich in amino acid stretches in serine and other amino acids which may form PEST regions that are associated with proteins that have short half-lives [50,53].
Figure 1 Alignment of the Mth1 amino acid sequences from TAM and RWB837. TAM is a Pdc- strain evolved for growth on glucose and RWB837 is the ancestral Pdc- strain from which the TAM strain was derived. The casein kinase I consensus phosphorylation site is underlined and bold. The deleted region is rich in amino acid stretches in serine and other amino acids which may form PEST regions that are associated with proteins that have short half-lives [50,53].CC BY · PubMed Central
Figure 2 Growth ofS. cerevisiaestrains with differentMTH1alleles on synthetic medium agarose plates with 20 g l-1glucose as the sole carbon source (left plate), 20 g l-1glucose supplemented with 0.3% (v/v) ethanol (middle plate) or 2% (v/v) glycerol and 0.3% (v/v) ethanol (right plate). The strains used were: IMI078 (Pdc-MTH1-ΔT), IMI082 (Pdc-), IMI083 (evolved Pdc-), IMZ104 (Pdc- overexpression MTH1), IMZ103 (Pdc- empty plasmid), CEN.PK113-7D (Pdc+ reference). Plates were incubated at least 3 days at 30°C.
Figure 2 Growth ofS. cerevisiaestrains with differentMTH1alleles on synthetic medium agarose plates with 20 g l-1glucose as the sole carbon source (left plate), 20 g l-1glucose supplemented with 0.3% (v/v) ethanol (middle plate) or 2% (v/v) glycerol and 0.3% (v/v) ethanol (right plate). The strains used were: IMI078 (Pdc-MTH1-ΔT), IMI082 (Pdc-), IMI083 (evolved Pdc-), IMZ104 (Pdc- overexpression MTH1), IMZ103 (Pdc- empty plasmid), CEN.PK113-7D (Pdc+ reference). Plates were incubated at least 3 days at 30°C.CC BY · PubMed Central
Figure 3 Growth profile of IMI078 (A; Pdc-MTH1-ΔT) and IMI083 (B; evolved Pdc-) on high concentrations of glucose. Cultivation in an aerated pH-controlled (pH 5) bioreactor with 7.5 g l-1 glucose and 0.3% (v/v) ethanol. The results are from one representative experiment. Duplicate experiments deviated <5% in titers, CO2 production and OD660.
Figure 3 Growth profile of IMI078 (A; Pdc-MTH1-ΔT) and IMI083 (B; evolved Pdc-) on high concentrations of glucose. Cultivation in an aerated pH-controlled (pH 5) bioreactor with 7.5 g l-1 glucose and 0.3% (v/v) ethanol. The results are from one representative experiment. Duplicate experiments deviated <5% in titers, CO2 production and OD660.CC BY · PubMed Central
Figure 4 Growth profile of IMI078 (Pdc-MTH1-ΔT) on glucose as the sole carbon source. Cultivation in an aerated pH-controlled (pH 5) bioreactor growing on 7.5 g l-1 glucose without external C2-sources. The depicted results are from one experiment of a set of four replicates, which all had identical specific growth rates (0.097 ± 0.007 h-1). The lag phase (rCO2 > 1 mmol l-1 h-1) of the replicates varied between 20 and 80 h after inoculation.
Figure 4 Growth profile of IMI078 (Pdc-MTH1-ΔT) on glucose as the sole carbon source. Cultivation in an aerated pH-controlled (pH 5) bioreactor growing on 7.5 g l-1 glucose without external C2-sources. The depicted results are from one experiment of a set of four replicates, which all had identical specific growth rates (0.097 ± 0.007 h-1). The lag phase (rCO2 > 1 mmol l-1 h-1) of the replicates varied between 20 and 80 h after inoculation.CC BY · PubMed Central

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Research works printed but not linked: 1

  1. Schulte F, Ciriacy M: HTR1/MTH1 encodes a repressor for HXT genes. Yeast. 1995, 11: S239-

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Metadata

Authors
Oud, B., Flores, C.-L., Gancedo, C., Zhang, X., Trueheart, J., Daran, J.-M., Pronk, J. T., & van Maris, A. J.
Affiliations
  • Delft University of Technology (NL)
  • Instituto de Investigaciones Biomédicas Sols-Morreale (ES)
ORCID
Venue
Microbial Cell Factories
Publisher
Springer Science and Business Media LLC
Published
2012-09-15
Volume
11
Issue
1
Pages
131-131
ISSN
1475-2859 (ISSN-L)
DOI
10.1186/1475-2859-11-131
PMC
PMC3503853
OpenAlex
W2127470474
Cited by
104
References
61
Citations by year
2026: 2 · 2025: 3 · 2024: 5 · 2023: 2 · 2022: 5 · 2021: 24 · 2020: 9 · 2019: 4 · 2018: 7 · 2017: 8 · 2016: 11 · 2015: 7 · 2014: 10 · 2013: 6
FWCI
2.96
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Microbial Cell Factories (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Research Repository (Delft University of Technology) (submittedVersion, other-oa)
  • Data Archiving and Networked Services (DANS) (submittedVersion, cc-by)
Funders
Ministerio de Ciencia e Innovación
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Physical Sciences › Engineering › Biomedical Engineering › Biofuel production and bioconversion
Keywords
MTH1, Saccharomyces cerevisiae, glucose tolerance, glucose detection, transcription factors, protein stability, phosphorylation sites, reverse engineering, glucose repression, ethanol tolerance, glucose uptake, hexose transport, metabolic engineering
MeSH terms
Alleles, Amino Acid Sequence, Fermentation, Glucose, Molecular Sequence Data, Mutation, Pyruvate Decarboxylase, Saccharomyces cerevisiae, Sequence Alignment, Saccharomyces cerevisiae Proteins, Adaptor Proteins, Signal Transducing, Metabolic Engineering

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