Exploring alternative pathways for the in vitro establishment of the HOPAC cycle for synthetic CO <sub>2</sub> fixation

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Abstract

Nature has evolved eight different pathways for the capture and conversion of CO ₂, including the Calvin-Benson-Bassham cycle of photosynthesis. Yet, these pathways underlie constrains and only represent a fraction of the thousands of theoretically possible solutions. To overcome the limitations of natural evolution, we introduce the HydrOxyPropionyl-CoA/Acrylyl-CoA (HOPAC) cycle, a new-to-nature CO ₂-fixation pathway that was designed through metabolic retrosynthesis around the reductive carboxylation of acrylyl-CoA, a highly efficient principle of CO ₂ fixation. We realized the HOPAC cycle in a step-wise fashion and used rational engineering approaches and machine learning–guided workflows to further optimize its output by more than one order of magnitude. Version 4.0 of the HOPAC cycle encompasses 11 enzymes from six different organisms, converting ~3.0 mM CO ₂ into glycolate within 2 hours. Our work moves the hypothetical HOPAC cycle from a theoretical design into an established in vitro system that forms the basis for different potential applications.

Abstract

Natural CO ₂-fixation pathways are expanded by a human-made alternative.

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Tissue sulfhydryl groups

George L. Ellman (1959)

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A synthetic pathway for the fixation of carbon dioxide in vitro.

Thomas Schwander, Lennart von Borzyskowski, Simon Burgener … (2016)

Carbon dioxide (CO2) is an important carbon feedstock for a future green economy. This requires the development of efficient strategies for its conversion into multicarbon compounds. We describe a synthetic cycle for the continuous fixation of CO2 in vitro. The crotonyl-coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle is a reaction network of 17 enzymes that converts CO2 into organic molecules at a rate of 5 nanomoles of CO2 per minute per milligram of protein. The CETCH cycle was drafted by metabolic retrosynthesis, established with enzymes originating from nine different organisms of all three domains of life, and optimized in several rounds by enzyme engineering and metabolic proofreading. The CETCH cycle adds a seventh, synthetic alternative to the six naturally evolved CO2 fixation pathways, thereby opening the way for in vitro and in vivo applications.

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Design and analysis of synthetic carbon fixation pathways.

Arren Bar-Even, Elad Noor, Nathan E. Lewis … (2010)

Carbon fixation is the process by which CO(2) is incorporated into organic compounds. In modern agriculture in which water, light, and nutrients can be abundant, carbon fixation could become a significant growth-limiting factor. Hence, increasing the fixation rate is of major importance in the road toward sustainability in food and energy production. There have been recent attempts to improve the rate and specificity of Rubisco, the carboxylating enzyme operating in the Calvin-Benson cycle; however, they have achieved only limited success. Nature employs several alternative carbon fixation pathways, which prompted us to ask whether more efficient novel synthetic cycles could be devised. Using the entire repertoire of approximately 5,000 metabolic enzymes known to occur in nature, we computationally identified alternative carbon fixation pathways that combine existing metabolic building blocks from various organisms. We compared the natural and synthetic pathways based on physicochemical criteria that include kinetics, energetics, and topology. Our study suggests that some of the proposed synthetic pathways could have significant quantitative advantages over their natural counterparts, such as the overall kinetic rate. One such cycle, which is predicted to be two to three times faster than the Calvin-Benson cycle, employs the most effective carboxylating enzyme, phosphoenolpyruvate carboxylase, using the core of the naturally evolved C4 cycle. Although implementing such alternative cycles presents daunting challenges related to expression levels, activity, stability, localization, and regulation, we believe our findings suggest exciting avenues of exploration in the grand challenge of enhancing food and renewable fuel production via metabolic engineering and synthetic biology.

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Author and article information

Contributors

Richard McLean:

ORCID: https://orcid.org/0000-0002-3237-3288

Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing - original draftRole: Writing - review & editing

Thomas Schwander:

ORCID: https://orcid.org/0000-0001-9201-3017

Role: ConceptualizationRole: InvestigationRole: MethodologyRole: Writing - review & editing

Christoph Diehl:

ORCID: https://orcid.org/0000-0002-8768-9044

Role: MethodologyRole: ResourcesRole: Software

Niña Socorro Cortina:

ORCID: https://orcid.org/0000-0002-0930-660X

Role: Data curationRole: Formal analysisRole: InvestigationRole: Software

Nicole Paczia:

ORCID: https://orcid.org/0000-0003-3859-8186

Role: Formal analysisRole: MethodologyRole: ValidationRole: Writing - review & editing

Jan Zarzycki:

ORCID: https://orcid.org/0000-0002-6307-6497

Role: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing - review & editing

Tobias J. Erb:

ORCID: https://orcid.org/0000-0003-3685-0894

Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: SupervisionRole: Writing - original draftRole: Writing - review & editing

Journal

Journal ID (nlm-ta): Sci Adv

Journal ID (iso-abbrev): Sci Adv

Journal ID (publisher-id): sciadv

Journal ID (hwp): advances

Title: Science Advances

Publisher: American Association for the Advancement of Science

ISSN (Electronic): 2375-2548

Publication date Collection: June 2023

Publication date (Electronic, pub): 14 June 2023

Volume: 9

Issue: 24

Electronic Location Identifier: eadh4299

Affiliations

[ ¹ ]Department of Biochemistry and Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.

[ ² ]Core Facility for Metabolomics and Small Molecule Mass Spectrometry, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.

[ ³ ]SYNMIKRO Center of Synthetic Microbiology, Marburg, Germany.

Author notes

[* ]Corresponding author. Email: toerb@ 123456mpi-marburg.mpg.de

[†]

Present address: ZHAW School of Life Sciences and Facility Management, Zurich, Switzerland.

[‡]

Present address: LiVeritas Biosciences Inc., 432N Canal St.; Ste. 20, South San Francisco, CA 94080, USA.

Author information

Richard McLean https://orcid.org/0000-0002-3237-3288

Thomas Schwander https://orcid.org/0000-0001-9201-3017

Christoph Diehl https://orcid.org/0000-0002-8768-9044

Niña Socorro Cortina https://orcid.org/0000-0002-0930-660X

Nicole Paczia https://orcid.org/0000-0003-3859-8186

Jan Zarzycki https://orcid.org/0000-0002-6307-6497

Tobias J. Erb https://orcid.org/0000-0003-3685-0894

Article

Publisher ID: adh4299

DOI: 10.1126/sciadv.adh4299

PMC ID: 10266731

PubMed ID: 37315145

SO-VID: fe6d4d75-1b75-4450-a0b0-af984fb9676c

Copyright © Copyright © 2023 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

History

Date received : 02 March 2023

Date accepted : 08 May 2023

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Copyeditor Judith Urtula

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