Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways
Copyright © 2020 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved..
Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic part function, gene expression patterns, and biosynthetic pathway performance in vitro before implementing designs in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 ± 24 μg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map aerobic cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2020 |
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| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:62 |
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| Enthalten in: |
Metabolic engineering - 62(2020) vom: 05. Nov., Seite 95-105 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Krüger, Antje [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 26.10.2021 Date Revised 26.10.2021 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.ymben.2020.06.004 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a Copyright © 2020 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic part function, gene expression patterns, and biosynthetic pathway performance in vitro before implementing designs in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 ± 24 μg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map aerobic cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a Research Support, U.S. Gov't, Non-P.H.S. | |
| 650 | 4 | |a Cell-free gene expression | |
| 650 | 4 | |a Cell-free metabolic engineering | |
| 650 | 4 | |a Cell-free protein synthesis | |
| 650 | 4 | |a Clostridia | |
| 650 | 4 | |a Prototyping genetic parts | |
| 650 | 4 | |a Synthetic Biology | |
| 700 | 1 | |a Mueller, Alexander P |e verfasserin |4 aut | |
| 700 | 1 | |a Rybnicky, Grant A |e verfasserin |4 aut | |
| 700 | 1 | |a Engle, Nancy L |e verfasserin |4 aut | |
| 700 | 1 | |a Yang, Zamin K |e verfasserin |4 aut | |
| 700 | 1 | |a Tschaplinski, Timothy J |e verfasserin |4 aut | |
| 700 | 1 | |a Simpson, Sean D |e verfasserin |4 aut | |
| 700 | 1 | |a Köpke, Michael |e verfasserin |4 aut | |
| 700 | 1 | |a Jewett, Michael C |e verfasserin |4 aut | |
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