Phosphoenolpyruvate Carboxykinase as the Sole Anaplerotic Enzyme in Saccharomyces cerevisiae
Open accessAbstract
Pyruvate carboxylase is the sole anaplerotic enzyme in glucose-grown cultures of wild-type Saccharomyces cerevisiae . Pyruvate carboxylase-negative (Pyc − ) S. cerevisiae strains cannot grow on glucose unless media are supplemented with C 4 compounds, such as aspartic acid. In several succinate-producing prokaryotes, phosphoenolpyruvate carboxykinase (PEPCK) fulfills this anaplerotic role. However, the S. cerevisiae PEPCK encoded by PCK1 is repressed by glucose and is considered to have a purely decarboxylating and gluconeogenic function. This study investigates whether and under which conditions PEPCK can replace the anaplerotic function of pyruvate carboxylase in S. cerevisiae . Pyc − S. cerevisiae strains constitutively overexpressing the PEPCK either from S. cerevisiae or from Actinobacillus succinogenes did not grow on glucose as the sole carbon source. However, evolutionary engineering yielded mutants able to grow on glucose as the sole carbon source at a maximum specific growth rate of ca. 0.14 h −1 , one-half that of the (pyruvate carboxylase-positive) reference strain grown under the same conditions. Growth was dependent on high carbon dioxide concentrations, indicating that the reaction catalyzed by PEPCK operates near thermodynamic equilibrium. Analysis and reverse engineering of two independently evolved strains showed that single point mutations in pyruvate kinase, which competes with PEPCK for phosphoenolpyruvate, were sufficient to enable the use of PEPCK as the sole anaplerotic enzyme. The PEPCK reaction produces one ATP per carboxylation event, whereas the original route through pyruvate kinase and pyruvate carboxylase is ATP neutral. This increased ATP yield may prove crucial for engineering of efficient and low-cost anaerobic production of C 4 dicarboxylic acids in S. cerevisiae .
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Early glucose-limited chemostat studies in S. cerevisiae showed that glucose-limited selection led to increased biomass yield and a decreased fermentative capacity.
“Early studies in glucose-limited chemostats revealed that glucose-limited selection led to increased biomass yield and a decreased fermentative capacity [93,98].”
From Adaptive laboratory evolution—Principles and applications for biotechnology (2013) · cites this paper as [93]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
PROTEIN MEASUREMENT WITH THE FOLIN PHENOL REAGENT
Lowry, O. H., Rosebrough, N., Farr, A. L., & Randall, R. (1951) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(19)52451-6 · Cited by 315,771 · see citing works
Sikorski, R. S., & Hieter, P. (1989) · Genetics · doi:10.1093/genetics/122.1.19 · Cited by 8,685 · see citing works
Sherman, F., Fink, G. R., & Hicks, J. B. (1979) · Cold Spring Harbor Laboratory eBooks · Cited by 7,543 · see citing works
Werpy, T. A., & Petersen, G. R. (2004) · doi:10.2172/15008859 · Cited by 3,219 · see citing works
Mumberg, D., Müller, R., & Funk, M. (1995) · Gene · doi:10.1016/0378-1119(95)00037-7 · Cited by 1,972 · see citing works
Verduyn, C., Postma, E., Scheffers, W. A., & van Dijken, J. P. (1992) · Yeast · doi:10.1002/yea.320080703 · Cited by 1,510 · see citing works
Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast.
Rothstein, R. (1991) · Methods in Enzymology · doi:10.1016/0076-6879(91)94022-5 · Cited by 1,283 · see citing works
van Dijken, J. P., Bauer, J., Brambilla, L., Duboc, P., Francois, J. M., Gancedo, C., Giuseppin, M. L. F., Heijnen, J. J., Hoare, M., Lange, H. C., et al. (2000) · Enzyme and Microbial Technology · doi:10.1016/s0141-0229(00)00162-9 · Cited by 598 · see citing works
Self-citationZelle, R. M., de Hulster, E. A. F., van Winden, W. A., de Waard, P., Dijkema, C., Winkler, A. A., Geertman, J.-M. A., van Dijken, J. P., Pronk, J. T., & van Maris, A. J. A. (2008) · Applied and Environmental Microbiology · doi:10.1128/aem.02591-07 · Cited by 379 · see citing works
Self-citationJantama, K., Haupt, M. J., Svoronos, S. A., et al. (2007) · Biotechnology and Bioengineering · doi:10.1002/bit.21694 · Cited by 330 · see citing works
WERF, M. J. V. D., Guettler, M. V., Jain, M. K., et al. (1997) · Archives of Microbiology · doi:10.1007/s002030050452 · Cited by 316 · see citing works
Genome-Based Metabolic Engineering of Mannheimia succiniciproducens for Succinic Acid Production
Lee, S. J., Song, H., & Lee, S. Y. (2006) · Applied and Environmental Microbiology · doi:10.1128/aem.72.3.1939-1948.2006 · Cited by 278 · see citing works
Daran-Lapujade, P. A. S., Jansen, M. L. A., Daran, J.-M. G., van Gulik, W. M., de Winde, J. H., & Pronk, J. T. (2004) · Journal of Biological Chemistry · doi:10.1074/jbc.m309578200 · Cited by 274 · see citing works
Self-citationOrganic acids: old metabolites, new themes
Goldberg, I., Rokem, J. S., & Pines, O. (2006) · Journal of Chemical Technology & Biotechnology · doi:10.1002/jctb.1590 · Cited by 257 · see citing works
Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli
Zhang, X., Jantama, K., Moore, J. C., et al. (2009) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0905396106 · Cited by 255 · see citing works
Samuelov, N. S., Lamed, R., Lowe, S. E., & Zeikus, J. G. (1991) · Applied and Environmental Microbiology · doi:10.1128/aem.57.10.3013-3019.1991 · Cited by 216 · see citing works
Lange, H. C., & Heijnen, J. J. (2001) · Biotechnology and Bioengineering · doi:10.1002/bit.10054 · Cited by 198 · see citing works
van Maris, A. J. A., Konings, W. N., van Dijken, J. P., & Pronk, J. T. (2004) · Metabolic Engineering · doi:10.1016/j.ymben.2004.05.001 · Cited by 190 · see citing works
Self-citationSantt, O., Pfirrmann, T., Braun, B., Juretschke, J., Kimmig, P., Scheel, H., Hofmann, K., Thumm, M., & Wolf, D. H. (2008) · Molecular Biology of the Cell · doi:10.1091/mbc.e08-03-0328 · Cited by 182 · see citing works
CO2 sensing in fungi and beyond
Bahn, Y., & Mühlschlegel, F. A. (2006) · Current Opinion in Microbiology · doi:10.1016/j.mib.2006.09.003 · Cited by 160 · see citing works
McKinlay, J. B., Shachar‐Hill, Y., Zeikus, J. G., et al. (2006) · Metabolic Engineering · doi:10.1016/j.ymben.2006.10.006 · Cited by 157 · see citing works
Does elevated atmospheric CO2 concentration inhibit mitochondrial respiration in green plants?
Drake, B. G., Azcón‐Bieto, J., Berry, J. A., et al. (1999) · Plant Cell & Environment · doi:10.1046/j.1365-3040.1999.00438.x · Cited by 156 · see citing works
de Jong-Gubbels, P., Vanrolleghem, P. A., Heijnen, S. J., van Dijken, J. P., & Pronk, J. T. (1995) · Yeast · doi:10.1002/yea.320110503 · Cited by 144 · see citing works
Self-citationThe Carboxylation of Phosphoenolpyruvate and Pyruvate
Cooper, T., Tchen, T. T., Wood, H. G., et al. (1968) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)92022-3 · Cited by 142 · see citing works
Dduntze, W., Neumann, D., Atzpodien, W., et al. (2005) · European Journal of Biochemistry · doi:10.1111/j.1432-1033.1969.tb00658.x · Cited by 138 · see citing works
Canelas, A. B., van Gulik, W. M., & Heijnen, J. J. (2008) · Biotechnology and Bioengineering · doi:10.1002/bit.21813 · Cited by 131 · see citing works
Boles, E., Schulte, F., Miosga, T., Freidel, K., Schlüter, E., Zimmermann, F. K., Hollenberg, C. P., & Heinisch, J. J. (1997) · Journal of Bacteriology · doi:10.1128/jb.179.9.2987-2993.1997 · Cited by 115 · see citing works
The Carboxylation of Phosphoenolpyruvate and Pyruvate
Cooper, T., & Wood, H. G. (1971) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)61931-3 · Cited by 100 · see citing works
Stucka, R., Dequin, S., Salmon, J.-M., et al. (1991) · Molecular and General Genetics MGG · doi:10.1007/bf00272171 · Cited by 95 · see citing works
The ICL1 gene from Saccharomyces cerevisiae
Fernández, E., MORENO, F., & Rodicio, R. (1992) · European Journal of Biochemistry · doi:10.1111/j.1432-1033.1992.tb16720.x · Cited by 94 · see citing works
Hartig, A., Simon, M. M., Schuster, T., Daugherty, J. R., Yoo, H. S., & Cooper, T. (1992) · Nucleic Acids Research · doi:10.1093/nar/20.21.5677 · Cited by 92 · see citing works
Aguilera, J., van Dijken, J. P., de Winde, J. H., & Pronk, J. T. (2005) · Biochemical Journal · doi:10.1042/bj20050556 · Cited by 88 · see citing works
Self-citationBrewster, N. K., Val, D. L., Walker, M. E., et al. (1994) · Archives of Biochemistry and Biophysics · doi:10.1006/abbi.1994.1209 · Cited by 85 · see citing works
van Maris, A. J. A., Bakker, B. M., Brandt, M., Boorsma, A., de Mattos, M. J. T., Grivell, L. A., Pronk, J. T., & Blom, J. (2001) · FEMS Yeast Research · doi:10.1111/j.1567-1364.2001.tb00025.x · Cited by 75 · see citing works
Self-citationYin, Z., Smith, R. J., & Brown, A. J. P. (1996) · Molecular Microbiology · doi:10.1111/j.1365-2958.1996.tb02514.x · Cited by 74 · see citing works
Zelle, R. M., de Hulster, E. A. F., Kloezen, W., Pronk, J. T., & van Maris, A. J. A. (2009) · Applied and Environmental Microbiology · doi:10.1128/aem.02396-09 · Cited by 69 · see citing works
Self-citationBendall, D. S., Ranson, S. L., & Walker, D. A. (1960) · Biochemical Journal · doi:10.1042/bj0760221 · Cited by 58 · see citing works
Structure and regulation of the isocitrate lyase gene ICL1 from the yeast Saccharomyces cerevisiae
Schöler, A., & Schüller, H. J. (1993) · Current Genetics · doi:10.1007/bf00312621 · Cited by 55 · see citing works
The Levels of Yeast Gluconeogenic mRNAs Respond to Environmental Factors
Mercado, J. J., Smith, R. J., Sagliocco, F. A., et al. (1994) · European Journal of Biochemistry · doi:10.1111/j.1432-1033.1994.00473.x · Cited by 51 · see citing works
Bicarbonate and the Pathway of Glutamate Oxidation in Isolated Rat‐Liver Mitochondria
Wanders, R. J. A., Meijer, A. J., Groen, A. K., & Tager, J. M. (1983) · European Journal of Biochemistry · doi:10.1111/j.1432-1033.1983.tb07455.x · Cited by 49 · see citing works
Phosphoenolpyruvate carboxykinase in gluconeogenesis and its repression by hexoses in yeasts
de Torrontegui, G., Palacián, E., & Losada, M. (1966) · Biochemical and Biophysical Research Communications · doi:10.1016/0006-291x(66)90437-2 · Cited by 36 · see citing works
Blázquez, M. A., Gamo, F., & Gancedo, C. (1995) · FEBS Letters · doi:10.1016/0014-5793(95)01337-7 · Cited by 30 · see citing works
de Jong-Gubbels, P., Bauer, J., Niederberger, P., Stückrath, I., Kötter, P., van Dijken, J. P., & Pronk, J. T. (1998) · Antonie van Leeuwenhoek · doi:10.1023/a:1001772613615 · Cited by 24 · see citing works
Self-citationFlores, C., & Gancedo, C. (1997) · FEBS Letters · doi:10.1016/s0014-5793(97)00854-5 · Cited by 14 · see citing works
Bauer, J., Luttik, M. A. H., Flores, C., et al. (1999) · FEMS Microbiology Letters · doi:10.1111/j.1574-6968.1999.tb08715.x · Cited by 12 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
Adaptive laboratory evolution—Principles and applications for biotechnology
Dragosits, M., & Mattanovich, D. (2013) · Microbial Cell Factories · doi:10.1186/1475-2859-12-64 · Cited by 827 · see citing works
Oud, B., Maris, A. J. A., Daran, J.-M., & Pronk, J. T. (2012) · FEMS Yeast Research · doi:10.1111/j.1567-1364.2011.00776.x · Cited by 82 · see citing works
Self-citationAnaplerotic Role for Cytosolic Malic Enzyme in Engineered <i>Saccharomyces cerevisiae</i> Strains
Zelle, R. M., Harrison, J. C., Pronk, J. T., & van Maris, A. J. A. (2011) · Applied and Environmental Microbiology · doi:10.1128/aem.02132-10 · Cited by 58 · see citing works
Self-citation
- Authors
- Zelle, R. M., Trueheart, J., 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
- 2010-08-15
- Volume
- 76
- Issue
- 16
- Pages
- 5383-5389
- ISSN
- 0099-2240 (ISSN-L)
- 1098-5336
- PMC
- PMC2918957
- OpenAlex
- W2103349249
- Cited by
- 49
- References
- 45
- Citations by year
- 2026: 2 · 2025: 1 · 2023: 2 · 2022: 2 · 2020: 5 · 2019: 6 · 2018: 5 · 2017: 4 · 2016: 1 · 2015: 3 · 2014: 5 · 2013: 4 · 2012: 3
- FWCI
- 1.69
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Applied and Environmental Microbiology (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
- Research Repository (Delft University of Technology) (submittedVersion)
- Zenodo (CERN European Organization for Nuclear Research) (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
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial metabolism and enzyme function
- Keywords
- phosphoenolpyruvate carboxykinase, anaplerosis, pyruvate carboxylase, pyruvate kinase, Saccharomyces cerevisiae, succinic acid, Actinobacillus succinogenes, evolutionary engineering, ATP yield, gluconeogenesis, thermodynamic equilibrium
- MeSH terms
- Actinobacillus, Carbon, Carbon Dioxide, DNA Mutational Analysis, Glucose, Phosphoenolpyruvate, Pyruvate Carboxylase, Pyruvate Kinase, Saccharomyces cerevisiae, Gene Deletion, Point Mutation, Phosphoenolpyruvate Carboxykinase (ATP), Mutant Proteins
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
