The DOI matches its Crossref record; 27 of the 27 references it cites were found in a registry or an index.
Identification of a rate-limiting step in a metabolic pathway using the kinetic model and in vitro experiment
Abstract
Identification of the rate-limiting step in a metabolic pathway is an important challenge in metabolic engineering for enhancing pathway flow. Although specific enzyme activities (V max) provide valuable clues for the identification, it is time-consuming and difficult to measure multiple enzymes in the pathway because different assay protocols are required for each enzyme. In the present study, we propose a method to simultaneously determine the V max values of multiple enzymes using a kinetic model with a time course of the intermediate concentrations through an in vitro experiment. To demonstrate this method, nine glycolysis reactions for converting glucose-6-phosphate (G6P) to pyruvate in Escherichia coli were considered. In a reaction mixture containing G6P and cofactors, glycolysis was initiated by adding a crude cell extract obtained from stationary phase cells. The V max values were optimized to minimize the difference between the measured and simulated time-courses using a kinetic model. Metabolic control analysis using the kinetic model with the estimated V max values revealed that fructose bisphosphate aldolase (FBA) was the rate-limiting step in the upper part of glycolysis. The addition of FBA in the reaction mixture successfully increased the glycolytic flux in vitro. Furthermore, in vivo, the specific glucose consumption rate of an FBA overexpression strain was 1.4 times higher than that of the control strain during the stationary phase. These results confirmed that FBA was the rate-limiting step in glycolysis under the stationary phase. This approach provides V max values of multiple enzymes in a pathway for metabolic control analysis with a kinetic model.
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“Previous studies have indicated the possibility of PYK, PFK, and GAPD being rate-limiting reactions of glycolysis (Iwami and Yamada, 1980; Boscá and Corredor, 1984); however, fructose-1,6-bisphosphate aldolase (FBA) appears to control the overall flux of glycolysis in Syn3A according to our simulations, which agrees with the findings of Kitamura et al. (2021).”
From Fundamental behaviors emerge from simulations of a living minimal cell (2022) · cites this paper as Kitamura et al. (2021)
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Cited by
Sorted by citations
Fundamental behaviors emerge from simulations of a living minimal cell
Thornburg, Z. R., Bianchi, D. M., Brier, T. A., Gilbert, B. R., Earnest, E. E., Melo, M. C. R., Safronova, N., Sáenz, J. P., Cook, A. T., Wise, K. S., et al. (2022) · Cell · doi:10.1016/j.cell.2021.12.025 · Cited by 165
“…GAPD being rate-limiting reactions of glycolysis (Iwami and Yamada, 1980; Boscá and Corredor, 1984); however, fructose-1,6-bisphosphate aldolase (FBA) appears to control the overall flux of glycolysis in Syn3A according to our simulations, which agrees with the findings of Kitamura et al. (2021).”
Zhao, Y., Jiao, F., Tang, H., Xu, H., Zhang, L., & Wu, H. (2021) · Plant Signaling & Behavior · doi:10.1080/15592324.2021.1938442 · Cited by 24
Engineering analysis of multienzyme cascade reactions for 3ʹ‐sialyllactose synthesis
Schelch, S., Eibinger, M., Belduma, S. G., Petschacher, B., Kuballa, J., & Nidetzky, B. (2021) · Biotechnology and Bioengineering · doi:10.1002/bit.27898 · Cited by 17
Soma, Y., Tominaga, S., Tokito, K., Imado, Y., Naka, K., Hanai, T., Takahashi, M., Izumi, Y., & Bamba, T. (2023) · Scientific Reports · doi:10.1038/s41598-023-44526-4 · Cited by 14
Pichia pastoris growth—coupled heme biosynthesis analysis using metabolic modelling
Pentjuss, A., Bolmanis, E., Šuleiko, A., Didrihsone, E., Šuleiko, A., Dubencovs, K., Liepiņš, J., Kazāks, A., & Vanags, J. (2023) · Scientific Reports · doi:10.1038/s41598-023-42865-w · Cited by 16
“7) and 66 downregulation/deletion (2.98% of total) reaction candidates (Supplementary Materials 6 Table S6.”
Influential
Metadata
- Authors
- Kitamura, S., Shimizu, H., & Toya, Y.
- Affiliations
- The University of Osaka (JP)
- Corresponding author
- Toya, Yoshihiro
- Venue
- Journal of Bioscience and Bioengineering
- Publisher
- Elsevier BV
- Published
- 2021-03
- Volume
- 131
- Issue
- 3
- Pages
- 271-276
- ISSN
- 1347-4421 (ISSN-L)
- 1389-1723
- OpenAlex
- W3097503126
- Cited by
- 18
- References
- 27
- Citations by year
- 2026: 3 · 2025: 6 · 2024: 2 · 2023: 3 · 2022: 1 · 2021: 3
- FWCI
- 0.57
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Bioscience and Bioengineering (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Japan Society for the Promotion of Science (18K04850)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Keywords
- rate-determining step, metabolic pathways, enzyme kinetics, kinetic modeling, metabolic control analysis, glycolysis, fructose-1,6-bisphosphate aldolase, Escherichia coli, glycolytic flux, stationary phase, glucose consumption rate, metabolic engineering
- MeSH terms
- Escherichia coli, Fructose-Bisphosphate Aldolase, Glucose, Glycolysis, Kinetics, Models, Biological, Metabolic Networks and Pathways
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