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Title: Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

Abstract

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 in this paper, 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 regulatorymore » elements in non-model, industrially relevant microbes.« less

Authors:
 [1];  [2]; ORCiD logo [1];  [3];  [3];  [3];  [2];  [2]; ORCiD logo [4]
  1. Northwestern Univ., Evanston, IL (United States). Chemistry of Life Processes Institute, Center for Synthetic Biology
  2. LanzaTech Inc., Skokie, IL (United States)
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  4. Northwestern Univ., Evanston, IL (United States). Chemistry of Life Processes Institute, Center for Synthetic Biology; Northwestern Univ., Chicago, IL (United States). Robert H. Lurie Comprehensive Cancer Center, Simpson Querrey Institute
Publication Date:
Research Org.:
Northwestern Univ., Evanston, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division; National Science Foundation (NSF); David and Lucille Packard Foundation; Camille-Dreyfus Teacher-Scholar Program
OSTI Identifier:
1660457
Alternate Identifier(s):
OSTI ID: 1658634
Grant/Contract Number:  
SC0018249; DGE-1842165; AC02-05CH11231; AC05-00OR22725; FWP ERKP903
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 62; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
Clostridia; Cell-free gene expression; Cell-free protein synthesis; Cell-free metabolic engineering; Synthetic Biology; Prototyping genetic parts

Citation Formats

Krüger, Antje, Mueller, Alexander P., Rybnicky, Grant A., Engle, Nancy L., Yang, Zamin K., Tschaplinski, Timothy J., Simpson, Sean D., Köpke, Michael, and Jewett, Michael C. Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways. United States: N. p., 2020. Web. doi:10.1016/j.ymben.2020.06.004.
Krüger, Antje, Mueller, Alexander P., Rybnicky, Grant A., Engle, Nancy L., Yang, Zamin K., Tschaplinski, Timothy J., Simpson, Sean D., Köpke, Michael, & Jewett, Michael C. Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways. United States. doi:10.1016/j.ymben.2020.06.004.
Krüger, Antje, Mueller, Alexander P., Rybnicky, Grant A., Engle, Nancy L., Yang, Zamin K., Tschaplinski, Timothy J., Simpson, Sean D., Köpke, Michael, and Jewett, Michael C. Sun . "Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways". United States. doi:10.1016/j.ymben.2020.06.004.
@article{osti_1660457,
title = {Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways},
author = {Krüger, Antje and Mueller, Alexander P. and Rybnicky, Grant A. and Engle, Nancy L. and Yang, Zamin K. and Tschaplinski, Timothy J. and Simpson, Sean D. and Köpke, Michael and Jewett, Michael C.},
abstractNote = {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 in this paper, 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.},
doi = {10.1016/j.ymben.2020.06.004},
journal = {Metabolic Engineering},
issn = {1096-7176},
number = ,
volume = 62,
place = {United States},
year = {2020},
month = {11}
}

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