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

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.

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

  • 11,306
    views
  • 1,485
    downloads
  • 91
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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)

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

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

  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
  12. David Savage
(2020)
Functional reconstitution of a bacterial CO2 concentrating mechanism in E. coli
eLife 9:e59882.
https://doi.org/10.7554/eLife.59882

Share this article

https://doi.org/10.7554/eLife.59882

Further reading

    1. Biochemistry and Chemical Biology
    2. Cancer Biology
    Flavie Coquel, Sing-Zong Ho ... Philippe Pasero
    Research Article

    Cancer cells display high levels of oncogene-induced replication stress (RS) and rely on DNA damage checkpoint for viability. This feature is exploited by cancer therapies to either increase RS to unbearable levels or inhibit checkpoint kinases involved in the DNA damage response. Thus far, treatments that combine these two strategies have shown promise but also have severe adverse effects. To identify novel, better-tolerated anticancer combinations, we screened a collection of plant extracts and found two natural compounds from the plant, Psoralea corylifolia, that synergistically inhibit cancer cell proliferation. Bakuchiol inhibited DNA replication and activated the checkpoint kinase CHK1 by targeting DNA polymerases. Isobavachalcone interfered with DNA double-strand break repair by inhibiting the checkpoint kinase CHK2 and DNA end resection. The combination of bakuchiol and isobavachalcone synergistically inhibited cancer cell proliferation in vitro. Importantly, it also prevented tumor development in xenografted NOD/SCID mice. The synergistic effect of inhibiting DNA replication and CHK2 signaling identifies a vulnerability of cancer cells that might be exploited by using clinically approved inhibitors in novel combination therapies.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Santi Mestre-Fos, Lucas Ferguson ... Jamie HD Cate
    Research Article

    Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3’ untranslated region (3’-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3’-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3’-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling, but not with translational efficiency. The results presented here show that eIF3 engages with 3’-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.