Toward a genome scale sequence specific dynamic model of cell-free protein synthesis in Escherichia coli
In this study, we developed a dynamic mathematical model of E. coli cell-free protein synthesis (CFPS). Model parameters were estimated from a dataset consisting of glucose, organic acids, energy species, amino acids, and protein product, chloramphenicol acetyltransferase (CAT) measurements. The mod...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2020-06-01
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| Series: | Metabolic Engineering Communications |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2214030118300452 |
| _version_ | 1828774809572474880 |
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| author | Nicholas Horvath Michael Vilkhovoy Joseph A. Wayman Kara Calhoun James Swartz Jeffrey D. Varner |
| author_facet | Nicholas Horvath Michael Vilkhovoy Joseph A. Wayman Kara Calhoun James Swartz Jeffrey D. Varner |
| author_sort | Nicholas Horvath |
| collection | DOAJ |
| description | In this study, we developed a dynamic mathematical model of E. coli cell-free protein synthesis (CFPS). Model parameters were estimated from a dataset consisting of glucose, organic acids, energy species, amino acids, and protein product, chloramphenicol acetyltransferase (CAT) measurements. The model was successfully trained to simulate these measurements, especially those of the central carbon metabolism. We then used the trained model to evaluate the performance, e.g., the yield and rates of protein production. CAT was produced with an energy efficiency of 12%, suggesting that the process could be further optimized. Reaction group knockouts showed that protein productivity was most sensitive to the oxidative phosphorylation and glycolysis/gluconeogenesis pathways. Amino acid biosynthesis was also important for productivity, while overflow metabolism and TCA cycle affected the overall system state. In addition, translation was more important to productivity than transcription. Finally, CAT production was robust to allosteric control, as were most of the predicted metabolite concentrations; the exceptions to this were the concentrations of succinate and malate, and to a lesser extent pyruvate and acetate, which varied from the measured values when allosteric control was removed. This study is the first to use kinetic modeling to predict dynamic protein production in a cell-free E. coli system, and could provide a foundation for genome scale, dynamic modeling of cell-free E. coli protein synthesis. |
| first_indexed | 2024-12-11T15:27:42Z |
| format | Article |
| id | doaj.art-3a9051a6d90f4559914fc1cabbf345cb |
| institution | Directory Open Access Journal |
| issn | 2214-0301 |
| language | English |
| last_indexed | 2024-12-11T15:27:42Z |
| publishDate | 2020-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Metabolic Engineering Communications |
| spelling | doaj.art-3a9051a6d90f4559914fc1cabbf345cb2022-12-22T01:00:09ZengElsevierMetabolic Engineering Communications2214-03012020-06-0110e00113Toward a genome scale sequence specific dynamic model of cell-free protein synthesis in Escherichia coliNicholas Horvath0Michael Vilkhovoy1Joseph A. Wayman2Kara Calhoun3James Swartz4Jeffrey D. Varner5Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USARobert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USASchool of Applied and Engineering Physics, Cornell University, Ithaca, NY, 14853, USASchool of Chemical Engineering, Stanford University, Stanford, CA, 94395, USASchool of Chemical Engineering, Stanford University, Stanford, CA, 94395, USARobert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA; Corresponding author.In this study, we developed a dynamic mathematical model of E. coli cell-free protein synthesis (CFPS). Model parameters were estimated from a dataset consisting of glucose, organic acids, energy species, amino acids, and protein product, chloramphenicol acetyltransferase (CAT) measurements. The model was successfully trained to simulate these measurements, especially those of the central carbon metabolism. We then used the trained model to evaluate the performance, e.g., the yield and rates of protein production. CAT was produced with an energy efficiency of 12%, suggesting that the process could be further optimized. Reaction group knockouts showed that protein productivity was most sensitive to the oxidative phosphorylation and glycolysis/gluconeogenesis pathways. Amino acid biosynthesis was also important for productivity, while overflow metabolism and TCA cycle affected the overall system state. In addition, translation was more important to productivity than transcription. Finally, CAT production was robust to allosteric control, as were most of the predicted metabolite concentrations; the exceptions to this were the concentrations of succinate and malate, and to a lesser extent pyruvate and acetate, which varied from the measured values when allosteric control was removed. This study is the first to use kinetic modeling to predict dynamic protein production in a cell-free E. coli system, and could provide a foundation for genome scale, dynamic modeling of cell-free E. coli protein synthesis.http://www.sciencedirect.com/science/article/pii/S2214030118300452Biochemical engineeringCell-free protein synthesisKinetic modeling |
| spellingShingle | Nicholas Horvath Michael Vilkhovoy Joseph A. Wayman Kara Calhoun James Swartz Jeffrey D. Varner Toward a genome scale sequence specific dynamic model of cell-free protein synthesis in Escherichia coli Metabolic Engineering Communications Biochemical engineering Cell-free protein synthesis Kinetic modeling |
| title | Toward a genome scale sequence specific dynamic model of cell-free protein synthesis in Escherichia coli |
| title_full | Toward a genome scale sequence specific dynamic model of cell-free protein synthesis in Escherichia coli |
| title_fullStr | Toward a genome scale sequence specific dynamic model of cell-free protein synthesis in Escherichia coli |
| title_full_unstemmed | Toward a genome scale sequence specific dynamic model of cell-free protein synthesis in Escherichia coli |
| title_short | Toward a genome scale sequence specific dynamic model of cell-free protein synthesis in Escherichia coli |
| title_sort | toward a genome scale sequence specific dynamic model of cell free protein synthesis in escherichia coli |
| topic | Biochemical engineering Cell-free protein synthesis Kinetic modeling |
| url | http://www.sciencedirect.com/science/article/pii/S2214030118300452 |
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