Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coli
Utilizing anaerobic metabolisms for the production of biotechnologically relevant products presents potential advantages, such as increased yields and reduced energy dissipation. However, lower energy dissipation may indicate that certain reactions are operating closer to their thermodynamic equilib...
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| Format: | Article |
| Language: | English |
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KeAi Communications Co., Ltd.
2023-12-01
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| Series: | Synthetic and Systems Biotechnology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405805X23000790 |
| _version_ | 1797372940942049280 |
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| author | Karel Olavarria Marco V. Becker Diana Z. Sousa Mark C.M. van Loosdrecht S. Aljoscha Wahl |
| author_facet | Karel Olavarria Marco V. Becker Diana Z. Sousa Mark C.M. van Loosdrecht S. Aljoscha Wahl |
| author_sort | Karel Olavarria |
| collection | DOAJ |
| description | Utilizing anaerobic metabolisms for the production of biotechnologically relevant products presents potential advantages, such as increased yields and reduced energy dissipation. However, lower energy dissipation may indicate that certain reactions are operating closer to their thermodynamic equilibrium. While stoichiometric analyses and genetic modifications are frequently employed in metabolic engineering, the use of thermodynamic tools to evaluate the feasibility of planned interventions is less documented. In this study, we propose a novel metabolic engineering strategy to achieve an efficient anaerobic production of poly-(R)-3-hydroxybutyrate (PHB) in the model organism Escherichia coli. Our approach involves re-routing of two-thirds of the glycolytic flux through non-oxidative glycolysis and coupling PHB synthesis with NADH re-oxidation. We complemented our stoichiometric analysis with various thermodynamic approaches to assess the feasibility and the bottlenecks in the proposed engineered pathway. According to our calculations, the main thermodynamic bottleneck are the reactions catalyzed by the acetoacetyl-CoA β-ketothiolase (EC 2.3.1.9) and the acetoacetyl-CoA reductase (EC 1.1.1.36). Furthermore, we calculated thermodynamically consistent sets of kinetic parameters to determine the enzyme amounts required for sustaining the conversion fluxes. In the case of the engineered conversion route, the protein pool necessary to sustain the desired fluxes could account for 20% of the whole cell dry weight. |
| first_indexed | 2024-03-08T18:43:06Z |
| format | Article |
| id | doaj.art-8c93bb8f0b5546ce9c848669f4f450e7 |
| institution | Directory Open Access Journal |
| issn | 2405-805X |
| language | English |
| last_indexed | 2024-03-08T18:43:06Z |
| publishDate | 2023-12-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Synthetic and Systems Biotechnology |
| spelling | doaj.art-8c93bb8f0b5546ce9c848669f4f450e72023-12-29T04:45:37ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2023-12-0184629639Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coliKarel Olavarria0Marco V. Becker1Diana Z. Sousa2Mark C.M. van Loosdrecht3S. Aljoscha Wahl4Laboratory of Microbiology, Wageningen University and Research, Stippenenweg 4, 6708 WE, Wageningen, The Netherlands; Centre for Living Technologies, Eindhoven-Wageningen-Utrecht Alliance, Princetonlaan 6, 3584 CB, Utrecht, The Netherlands; Corresponding author. Laboratory of Microbiology, Wageningen University and Research, Stippenenweg 4, 6708 WE, Wageningen, the Netherlands.Department of Biotechnology, Applied Sciences Faculty, Delft University of Technology, van der Maasweg 9, 2629 HZ, Delft, The NetherlandsLaboratory of Microbiology, Wageningen University and Research, Stippenenweg 4, 6708 WE, Wageningen, The Netherlands; Centre for Living Technologies, Eindhoven-Wageningen-Utrecht Alliance, Princetonlaan 6, 3584 CB, Utrecht, The NetherlandsDepartment of Biotechnology, Applied Sciences Faculty, Delft University of Technology, van der Maasweg 9, 2629 HZ, Delft, The NetherlandsLehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität, Paul-Gordan-Strasse 3, 91052, Erlangen, GermanyUtilizing anaerobic metabolisms for the production of biotechnologically relevant products presents potential advantages, such as increased yields and reduced energy dissipation. However, lower energy dissipation may indicate that certain reactions are operating closer to their thermodynamic equilibrium. While stoichiometric analyses and genetic modifications are frequently employed in metabolic engineering, the use of thermodynamic tools to evaluate the feasibility of planned interventions is less documented. In this study, we propose a novel metabolic engineering strategy to achieve an efficient anaerobic production of poly-(R)-3-hydroxybutyrate (PHB) in the model organism Escherichia coli. Our approach involves re-routing of two-thirds of the glycolytic flux through non-oxidative glycolysis and coupling PHB synthesis with NADH re-oxidation. We complemented our stoichiometric analysis with various thermodynamic approaches to assess the feasibility and the bottlenecks in the proposed engineered pathway. According to our calculations, the main thermodynamic bottleneck are the reactions catalyzed by the acetoacetyl-CoA β-ketothiolase (EC 2.3.1.9) and the acetoacetyl-CoA reductase (EC 1.1.1.36). Furthermore, we calculated thermodynamically consistent sets of kinetic parameters to determine the enzyme amounts required for sustaining the conversion fluxes. In the case of the engineered conversion route, the protein pool necessary to sustain the desired fluxes could account for 20% of the whole cell dry weight.http://www.sciencedirect.com/science/article/pii/S2405805X23000790Pathway feasibility analysisProtein costAnaerobic metabolismEngineered pathwaysMetabolite concentrations |
| spellingShingle | Karel Olavarria Marco V. Becker Diana Z. Sousa Mark C.M. van Loosdrecht S. Aljoscha Wahl Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coli Synthetic and Systems Biotechnology Pathway feasibility analysis Protein cost Anaerobic metabolism Engineered pathways Metabolite concentrations |
| title | Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coli |
| title_full | Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coli |
| title_fullStr | Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coli |
| title_full_unstemmed | Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coli |
| title_short | Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coli |
| title_sort | design and thermodynamic analysis of a pathway enabling anaerobic production of poly 3 hydroxybutyrate in escherichia coli |
| topic | Pathway feasibility analysis Protein cost Anaerobic metabolism Engineered pathways Metabolite concentrations |
| url | http://www.sciencedirect.com/science/article/pii/S2405805X23000790 |
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