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Directed Evolution of Pyruvate Decarboxylase-Negative Saccharomyces cerevisiae , Yielding a C 2 -Independent, Glucose-Tolerant, and Pyruvate-Hyperproducing Yeast
Open accessAbstract
The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc − ) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc − S. cerevisiae strains have two growth defects: (i) growth on synthetic medium in glucose-limited chemostat cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C 2 compound, these strains cannot grow in batch cultures on synthetic medium with glucose. We used two subsequent phenotypic selection strategies to obtain a Pdc − strain without these growth defects. An acetate-independent Pdc − mutant was obtained via (otherwise) glucose-limited chemostat cultivation by progressively lowering the acetate content in the feed. Transcriptome analysis did not reveal the mechanisms behind the C 2 independence. Further selection for glucose tolerance in shake flasks resulted in a Pdc − S. cerevisiae mutant (TAM) that could grow in batch cultures (μ max = 0.20 h −1 ) on synthetic medium, with glucose as the sole carbon source. Although the exact molecular mechanisms underlying the glucose-tolerant phenotype were not resolved, transcriptome analysis of the TAM strain revealed increased transcript levels of many glucose-repressible genes relative to the isogenic wild type in nitrogen-limited chemostat cultures with excess glucose. In pH-controlled aerobic batch cultures, the TAM strain produced large amounts of pyruvate. By repeated glucose feeding, a pyruvate concentration of 135 g liter −1 was obtained, with a specific pyruvate production rate of 6 to 7 mmol g of biomass −1 h −1 during the exponential-growth phase and an overall yield of 0.54 g of pyruvate g of glucose −1 .
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“Adaptive changes towards ethanol tolerance were linked to the up-regulation of oxyR and nrdR, indicating major changes in the cellular respiratory system but also changes in amino acid metabolism [85].”
From Adaptive laboratory evolution—Principles and applications for biotechnology (2013) · cites this paper as [85]
In the citing paper
“Furthermore, changes in enterobactin biosynthetic processes (ent genes) were also observed in independent studies [84,85,91].”
From Adaptive laboratory evolution—Principles and applications for biotechnology (2013) · cites this paper as [85]
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Metadata
- Authors
- van Maris, A. J. A., Geertman, J.-M. A., Vermeulen, A., Groothuizen, M. K., Winkler, A. A., Piper, M. D. W., van Dijken, J. P., & Pronk, J. T.
- Affiliations
- Department of Biotechnology, Delft University of Technology, NL-2628 BC Delft
- Delft University of Technology (NL)
- BIRD Engineering B.V., NL-3044 CK Rotterdam, The Netherlands
- Corresponding author
- Pronk, Jack T.
- Venue
- Applied and Environmental Microbiology
- Publisher
- American Society for Microbiology
- Published
- 2004-01
- Volume
- 70
- Issue
- 1
- Pages
- 159-166
- ISSN
- 0099-2240 (ISSN-L)
- 1098-5336
- PMC
- PMC321313
- OpenAlex
- W2119059442
- Cited by
- 227
- References
- 51
- Citations by year
- 2026: 3 · 2025: 5 · 2024: 10 · 2023: 5 · 2022: 15 · 2021: 25 · 2020: 19 · 2019: 5 · 2018: 16 · 2017: 16 · 2016: 15 · 2015: 16 · 2014: 14 · 2013: 10 · 2012: 16
- FWCI
- 1.42
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Applied and Environmental Microbiology (publishedVersion, public-domain)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- UCL Discovery (University College London) (submittedVersion)
- Research Repository (Delft University of Technology) (submittedVersion)
- Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Physical Sciences › Engineering › Biomedical Engineering › Biofuel production and bioconversion
- Keywords
- Saccharomyces cerevisiae, glucose tolerance, phenotypic selection, glucose repression, glucose-limited chemostat, batch culture, yeast metabolic engineering
- MeSH terms
- Culture Media, Glucose, Mutation, Pyruvate Decarboxylase, Saccharomyces cerevisiae, Transcription, Genetic, Gene Expression Regulation, Fungal, Directed Molecular Evolution, Pyruvic Acid, Proteome, Saccharomyces cerevisiae Proteins
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