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

Antje Krüger; Alexander P. Mueller; Grant A. Rybnicky; Nancy L. Engle; Zamin K. Yang; Timothy J. Tschaplinski; Sean D. Simpson; Michael Köpke; Michael C. Jewett

doi:10.1016/j.ymben.2020.06.004

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

Antje Krüger, Alexander P. Mueller, Grant A. Rybnicky, Nancy L. Engle, Zamin K. Yang, Timothy J. Tschaplinski, Sean D. Simpson, Michael Köpke, Michael C. Jewett

Research output: Contribution to journal › Article › peer-review

33 Scopus citations

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, 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.

Original language	English
Pages (from-to)	95-105
Number of pages	11
Journal	Metabolic Engineering
Volume	62
DOIs	https://doi.org/10.1016/j.ymben.2020.06.004
State	Published - Nov 2020

Funding

The authors thank Ashty Karim (ORCID # 0000-0002-5789-7715), Lauren Clark, and Ben De-Soye for helpful comments on the manuscript and Ava Rhule-Smith, Jim Daleiden, Monica MacDonald, and Steven Glasker for their help growing the C. autoethanogenum cultures and Robert Nogle for help with subcloning the promoter regions. This work was supported by the U.S. Department of Energy, Office of Biological and Environmental Research in the DOE Office of Science under Award Number DE-SC0018249 and FWP ERKP903. G.A.R was supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1842165. M.C.J. also thanks the David and Lucille Packard Foundation and the Camille-Dreyfus Teacher-Scholar Program for their generous support. We also thank the Joint Genome Institute Community Science Program Project 503280. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. This manuscript has been co-authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. We also thank the following investors in LanzaTech's technology: BASF, CICC Growth Capital Fund I, CITIC Capital, Indian Oil Company, K1W1, Khosla Ventures, the Malaysian Life Sciences, Capital Fund, L. P. Mitsui, the New Zealand Superannuation Fund, Novo Holdings A/S, Petronas Technology Ventures, Primetals, Qiming Venture Partners, Softbank China, and Suncor. The authors thank Ashty Karim (ORCID # 0000-0002-5789-7715), Lauren Clark, and Ben De-Soye for helpful comments on the manuscript and Ava Rhule-Smith, Jim Daleiden, Monica MacDonald, and Steven Glasker for their help growing the C. autoethanogenum cultures and Robert Nogle for help with subcloning the promoter regions. This work was supported by the U.S. Department of Energy, Office of Biological and Environmental Research in the DOE Office of Science under Award Number DE-SC0018249 and FWP ERKP903 . G.A.R was supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1842165 . M.C.J. also thanks the David and Lucille Packard Foundation and the Camille-Dreyfus Teacher-Scholar Program for their generous support. We also thank the Joint Genome Institute Community Science Program Project 503280. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility , is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231 . This manuscript has been co-authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. We also thank the following investors in LanzaTech's technology: BASF, CICC Growth Capital Fund I, CITIC Capital, Indian Oil Company, K1W1, Khosla Ventures, the Malaysian Life Sciences, Capital Fund, L. P., Mitsui, the New Zealand Superannuation Fund, Novo Holdings A/S, Petronas Technology Ventures, Primetals, Qiming Venture Partners, Softbank China, and Suncor.

Funders	Funder number
CICC	K1W1
Lauren Clark
Malaysian Life Sciences, Capital Fund
Petronas Technology Ventures
U.S. Department of Energy Joint Genome Institute
National Science Foundation	DGE-1842165
David and Lucile Packard Foundation	503280
U.S. Department of Energy
BASF
Office of Science	FWP ERKP903, DE-AC02-05CH11231, DE-SC0018249
Biological and Environmental Research
Suncor Energy Incorporated
UT-Battelle	DE-AC05-00OR22725
Khosla Ventures

Keywords

Cell-free gene expression
Cell-free metabolic engineering
Cell-free protein synthesis
Clostridia
Prototyping genetic parts
Synthetic Biology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.ymben.2020.06.004

Cite this

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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, 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.",

keywords = "Cell-free gene expression, Cell-free metabolic engineering, Cell-free protein synthesis, Clostridia, Prototyping genetic parts, Synthetic Biology",

author = "Antje Kr{\"u}ger 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 Michael K{\"o}pke and Jewett, {Michael C.}",

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AU - Krüger, Antje

AU - Mueller, Alexander P.

AU - Rybnicky, Grant A.

AU - Engle, Nancy L.

AU - Yang, Zamin K.

AU - Tschaplinski, Timothy J.

AU - Simpson, Sean D.

AU - Köpke, Michael

AU - Jewett, Michael C.

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AB - 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.

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

Abstract

Funding

Keywords

UN SDGs

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