1. Biochemistry and Chemical Biology
  2. Microbiology and Infectious Disease
Download icon

Functional reconstitution of a bacterial CO2 concentrating mechanism in E. coli

  1. Avi I Flamholz
  2. Eli Dugan
  3. Cecilia Blikstad
  4. Shmuel Gleizer
  5. Roee Ben-Nissan
  6. Shira Amram
  7. Niv Antonovsky
  8. Sumedha Ravishankar
  9. Elad Noor
  10. Arren Bar-Even
  11. Ron Milo  Is a corresponding author
  12. David Savage  Is a corresponding author
  1. University of California, Berkeley, United States
  2. Weizmann Institute of Science, Israel
  3. University of California, San Diego, United States
  4. ETH Zurich, Switzerland
  5. Max Planck Institute of Molecular Plant Physiology, Germany
Research Article
  • Cited 16
  • Views 5,658
  • Annotations
Cite this article as: eLife 2020;9:e59882 doi: 10.7554/eLife.59882

Abstract

Many photosynthetic organisms employ a CO2 concentrating mechanism (CCM) to increase the rate of CO2 fixation via the Calvin cycle. CCMs catalyze ≈50% of global photosynthesis, yet it remains unclear which genes and proteins are required to produce this complex adaptation. We describe the construction of a functional CCM in a non-native host, achieved by expressing genes from an autotrophic bacterium in an E. coli strain engineered to depend on rubisco carboxylation for growth. Expression of 20 CCM genes enabled E. coli to grow by fixing CO2 from ambient air into biomass, with growth in ambient air depending on the components of the CCM. Bacterial CCMs are therefore genetically compact and readily transplanted, rationalizing their presence in diverse bacteria. Reconstitution enabled genetic experiments refining our understanding of the CCM, thereby laying the groundwork for deeper study and engineering of the cell biology supporting CO2 assimilation in diverse organisms.

Data availability

All source data for all figures is available in the linked github repository along with accompanying Jupyter notebooks generating the data-driven portions of all figures.

Article and author information

Author details

  1. Avi I Flamholz

    Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9278-5479

  2. Eli Dugan

    Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2400-5511

  3. Cecilia Blikstad

    Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5740-926X

  4. Shmuel Gleizer

    Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    No competing interests declared.

  5. Roee Ben-Nissan

    Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    No competing interests declared.

  6. Shira Amram

    Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    No competing interests declared.

  7. Niv Antonovsky

    Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    No competing interests declared.

  8. Sumedha Ravishankar

    Division of Biological Sciences, University of California, San Diego, San Diego, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4026-0742

  9. Elad Noor

    Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8776-4799

  10. Arren Bar-Even

    Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany
    Competing interests
    Arren Bar-Even, A.B.-E. is co-founder of b.fab, a company aiming to commercialize engineered C1-assimilation in microorganisms. The company was not involved in this work in any way..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1039-4328

  11. Ron Milo

    Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel
    For correspondence
    ron.milo@weizmann.ac.il
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1641-2299

  12. David Savage

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    savage@berkeley.edu
    Competing interests
    David Savage, D.F.S. is a co-founder of Scribe Therapeutics and a scientific advisory board member of Scribe Therapeutics and Mammoth Biosciences. These companies were not involved in this work in any way..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0042-2257

Funding

Energy Biosciences Institute (CW163755)

  • David Savage

US Department of Energy (DE-SC00016240)

  • David Savage

European Research Council (NOVCARBFIX 646827)

  • Ron Milo

National Science Foundation (MCB-1818377)

  • David Savage

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Manajit Hayer-Hartl, Max Planck Institute of Biochemistry, Germany

Publication history

  1. Received: June 11, 2020
  2. Accepted: October 20, 2020
  3. Accepted Manuscript published: October 21, 2020 (version 1)
  4. Version of Record published: December 3, 2020 (version 2)

Copyright

© 2020, Flamholz et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 5,658
    Page views
  • 809
    Downloads
  • 16
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Categories and tags

Research organism

    1. Related to

    Genetic Engineering: Increasing the uptake of carbon dioxide

    Eric Franklin, Martin Jonikas
  2. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease

    Genetic Engineering: Increasing the uptake of carbon dioxide

    Eric Franklin, Martin Jonikas
    Insight

    A mechanism for concentrating carbon dioxide has for the first time been successfully transferred into a species that lacks such a process.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions

    Aixin Song et al.
    Research Article Updated

    UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently, it was reported that UCH37 activity is stimulated by branched ubiquitin (Ub) chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and Nuclear Magnetic Resonance (NMR) structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal Ubs that emanate from a branched Ub. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear Ub chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome for the next round of substrate processing. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique Ub chain architecture is aided by a DUB.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Disentangling bias between Gq, GRK2, and arrestin3 recruitment to the M3 muscarinic acetylcholine receptor

    Anja Floeser et al.
    Research Article

    G protein-coupled receptors (GPCRs) transmit extracellular signals to the inside by activation of intracellular effector proteins. Different agonists can promote differential receptor-induced signaling responses – termed bias – potentially by eliciting different levels of recruitment of effector proteins. As activation and recruitment of effector proteins might influence each other, thorough analysis of bias is difficult. Here, we compared the efficacy of seven agonists to induce G protein, G protein-coupled receptor kinase 2 (GRK2), as well as arrestin3 binding to the muscarinic acetylcholine receptor M3 by utilizing FRET-based assays. In order to avoid interference between these interactions, we studied GRK2 binding in the presence of inhibitors of Gi and Gq proteins and analyzed arrestin3 binding to prestimulated M3 receptors to avoid differences in receptor phosphorylation influencing arrestin recruitment. We measured substantial differences in the agonist efficacies to induce M3R-arrestin3 versus M3R-GRK2 interaction. However, the rank order of the agonists for G protein- and GRK2-M3R interaction was the same, suggesting that G protein and GRK2 binding to M3R requires similar receptor conformations, whereas requirements for arrestin3 binding to M3R are distinct.