Anaplerotic Role for Cytosolic Malic Enzyme in Engineered Saccharomyces cerevisiae Strains
Open accessAbstract
Malic enzyme catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO 2 . The Saccharomyces cerevisiae MAE1 gene encodes a mitochondrial malic enzyme whose proposed physiological roles are related to the oxidative, malate-decarboxylating reaction. Hitherto, the inability of pyruvate carboxylase-negative (Pyc − ) S. cerevisiae strains to grow on glucose suggested that Mae1p cannot act as a pyruvate-carboxylating, anaplerotic enzyme. In this study, relocation of malic enzyme to the cytosol and creation of thermodynamically favorable conditions for pyruvate carboxylation by metabolic engineering, process design, and adaptive evolution, enabled malic enzyme to act as the sole anaplerotic enzyme in S. cerevisiae . The Escherichia coli NADH-dependent sfcA malic enzyme was expressed in a Pyc − S. cerevisiae background. When PDC2 , a transcriptional regulator of pyruvate decarboxylase genes, was deleted to increase intracellular pyruvate levels and cells were grown under a CO 2 atmosphere to favor carboxylation, adaptive evolution yielded a strain that grew on glucose (specific growth rate, 0.06 ± 0.01 h −1 ). Growth of the evolved strain was enabled by a single point mutation (Asp336Gly) that switched the cofactor preference of E. coli malic enzyme from NADH to NADPH. Consistently, cytosolic relocalization of the native Mae1p, which can use both NADH and NADPH, in a pyc1,2Δ pdc2 Δ strain grown under a CO 2 atmosphere, also enabled slow-growth on glucose. Although growth rates of these strains are still low, the higher ATP efficiency of carboxylation via malic enzyme, compared to the pyruvate carboxylase pathway, may contribute to metabolic engineering of S. cerevisiae for anaerobic, high-yield C 4 -dicarboxylic acid production.
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Other studies used ethylmethanesulfonate (EMS) treated Bacillus boroniphilus to select for increased boron resistance or EMS-treated S. cerevisiae in order to achieve increased nutrient utilization and stress resistance.
“Other studies used ethylmethanesulfonate (EMS) treated Bacillus boroniphilus to select for increased boron resistance [88] or EMS-treated S. cerevisiae in order to achieve increased nutrient utilization and stress resistance [95,113,114].”
From Adaptive laboratory evolution—Principles and applications for biotechnology (2013) · cites this paper as [95]
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- Authors
- Zelle, R. M., Harrison, J. C., Pronk, J. T., & van Maris, A. J. A.
- Affiliations
- Delft University of Technology (NL)
- Microbia, Inc., 60 Westview Street, Lexington, Massachusetts 02421
- Department of Biotechnology, Delft University of Technology and Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, Netherlands
- Venue
- Applied and Environmental Microbiology
- Publisher
- American Society for Microbiology
- Published
- 2011-02
- Volume
- 77
- Issue
- 3
- Pages
- 732-738
- ISSN
- 0099-2240 (ISSN-L)
- 1098-5336
- PMC
- PMC3028732
- OpenAlex
- W2131708560
- Cited by
- 58
- References
- 40
- Citations by year
- 2026: 3 · 2024: 4 · 2023: 5 · 2022: 2 · 2021: 6 · 2020: 2 · 2019: 3 · 2018: 1 · 2017: 2 · 2016: 4 · 2015: 4 · 2014: 6 · 2013: 8 · 2012: 4
- FWCI
- 2.08
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Applied and Environmental Microbiology (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Research Repository (Delft University of Technology) (submittedVersion)
- Zenodo (CERN European Organization for Nuclear Research) (submittedVersion, other-oa)
- Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
- Funders
- Technische Universiteit Delft
- 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
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial metabolism and enzyme function
- Keywords
- malic enzyme, anaplerosis, pyruvate carboxylation, Saccharomyces cerevisiae, SFCA, adaptive evolution, metabolic engineering
- MeSH terms
- Anaerobiosis, Biotechnology, Carbon Dioxide, Cytosol, Escherichia coli, Genetic Engineering, Glucose, Malate Dehydrogenase, Malates, Saccharomyces cerevisiae, Point Mutation, Pyruvic Acid, Saccharomyces cerevisiae Proteins
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