Enhancing 2-Pyrone Synthase Efficiency by High-Throughput Mass-Spectrometric Quantification and In Vitro/In Vivo Catalytic Performance Correlation
© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH..
Engineering efficient biocatalysts is essential for metabolic engineering to produce valuable bioproducts from renewable resources. However, due to the complexity of cellular metabolic networks, it is challenging to translate success in vitro into high performance in cells. To meet such a challenge, an accurate and efficient quantification method is necessary to screen a large set of mutants from complex cell culture and a careful correlation between the catalysis parameters in vitro and performance in cells is required. In this study, we employed a mass-spectrometry based high-throughput quantitative method to screen new mutants of 2-pyrone synthase (2PS) for triacetic acid lactone (TAL) biosynthesis through directed evolution in E. coli. From the process, we discovered two mutants with the highest improvement (46 fold) in titer and the fastest kcat (44 fold) over the wild type 2PS, respectively, among those reported in the literature. A careful examination of the correlation between intracellular substrate concentration, Michaelis-Menten parameters and TAL titer for these two mutants reveals that a fast reaction rate under limiting intracellular substrate concentrations is important for in-cell biocatalysis. Such properties can be tuned by protein engineering and synthetic biology to adopt these engineered proteins for the maximum activities in different intracellular environments.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:25 |
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| Enthalten in: |
Chembiochem : a European journal of chemical biology - 25(2024), 5 vom: 01. März, Seite e202300849 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Zhou, Yu [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 04.03.2024 Date Revised 26.03.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1002/cbic.202300849 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 500 | |a published: Print-Electronic | ||
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| 520 | |a © 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH. | ||
| 520 | |a Engineering efficient biocatalysts is essential for metabolic engineering to produce valuable bioproducts from renewable resources. However, due to the complexity of cellular metabolic networks, it is challenging to translate success in vitro into high performance in cells. To meet such a challenge, an accurate and efficient quantification method is necessary to screen a large set of mutants from complex cell culture and a careful correlation between the catalysis parameters in vitro and performance in cells is required. In this study, we employed a mass-spectrometry based high-throughput quantitative method to screen new mutants of 2-pyrone synthase (2PS) for triacetic acid lactone (TAL) biosynthesis through directed evolution in E. coli. From the process, we discovered two mutants with the highest improvement (46 fold) in titer and the fastest kcat (44 fold) over the wild type 2PS, respectively, among those reported in the literature. A careful examination of the correlation between intracellular substrate concentration, Michaelis-Menten parameters and TAL titer for these two mutants reveals that a fast reaction rate under limiting intracellular substrate concentrations is important for in-cell biocatalysis. Such properties can be tuned by protein engineering and synthetic biology to adopt these engineered proteins for the maximum activities in different intracellular environments | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, U.S. Gov't, Non-P.H.S. | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a Biocatalysis | |
| 650 | 4 | |a Directed evolution | |
| 650 | 4 | |a High-throughput method | |
| 650 | 4 | |a Polyketides | |
| 650 | 4 | |a Synthetic Biology | |
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| 700 | 1 | |a Lyons, Scott |e verfasserin |4 aut | |
| 700 | 1 | |a Sun, Haoran |e verfasserin |4 aut | |
| 700 | 1 | |a Sweedler, Jonathan V |e verfasserin |4 aut | |
| 700 | 1 | |a Lu, Yi |e verfasserin |4 aut | |
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