Autotrophic growth of Escherichia coli is achieved by a small number of genetic changes

  1. Roee Ben Nissan
  2. Eliya Milshtein
  3. Vanessa Pahl
  4. Benoit de Pins
  5. Ghil Jona
  6. Dikla Levi
  7. Hadas Yung
  8. Noga Nir
  9. Dolev Ezra
  10. Shmuel Gleizer
  11. Hannes Link
  12. Elad Noor
  13. Ron Milo  Is a corresponding author
  1. Department of Plant and Environmental Sciences, Weizmann Institute of Science, Israel
  2. Interfaculty Institute for Microbiology and Infection Medicine Tübingen, University of Tübingen, Germany
  3. Department of Life Sciences Core Facilities, Weizmann Institute of Science, Israel

Peer review process

Version of Record: This is the final version of the article.

Read more about eLife's peer review process.

Editors

Senior Editor
  1. Aleksandra M Walczak
  2. École Normale Supérieure - PSL, France
Reviewing Editor
  1. Joseph T Wade
  2. New York State Department of Health, United States

Joint Public Review:

The authors previously showed that expressing formate dehydrogenase, rubisco, carbonic anhydrase, and phosphoribulokinase in Escherichia coli, followed by experimental evolution, led to the generation of strains that can metabolise CO2. Using two rounds of experimental evolution, the authors identify mutations in three genes - pgi, rpoB, and crp - that allow cells to metabolise CO2 in their engineered strain background. The authors make a strong case that mutations in pgi are loss-of-function mutations that prevent metabolic efflux from the reductive pentose phosphate autocatalytic cycle. The authors also use proteomic analysis to probe the role of the mutations in crp and rpoB. While they do not reach strong conclusions about how these mutations promote autotrophic growth, they provide some clues, leading to valuable speculation.

Comments on revised version:

The authors have thoroughly addressed the reviewers' comments. The major addition to the paper is the proteomic analysis of single and double mutants of crp and rpoB. These new data provide clues as to the role of the crp and rpoB mutations in promoting autotrophic growth, which the authors discuss. The authors acknowledge that it will require additional experiments to determine whether the speculated mechanisms are correct. Nonetheless, the new data provide valuable new insight into the role of the crp and rpoB mutations. The authors have also expanded their description of the crp and rpoB mutations, making it clearer that the effects of these mutations are likely to be distinct, albeit with potential for overlap in function.

https://doi.org/10.7554/eLife.88793.4.sa1

Author response

The following is the authors’ response to the previous reviews.

Recommendations for the authors:

The single-mutant and double-mutant crp/rpoB strains were made by co-transduction with a nearby gene deletion (kanR-marked). I couldn't tell from the methods section whether these mutants, e.g., crp-H22N delta-chiA, were compared to wild-type cells or deletion mutants, e.g., delta chiA, in the proteomics experiments. I encourage the authors to explain this more clearly in the methods section, and to briefly mention in the Results section and relevant figure legends that the crp/rpoB mutant strains (and possibly the "wild-type" strains) also have gene deletions. If the comparison "wild-type" strains are fully wild-type (i.e., not deleted for chiA/yjaH), it is especially important to mention this in the Results section and the figure legends since the phenotypic changes could be due to the gene deletions rather than the mutations in crp/rpoB

We appreciate and agree with the editor's suggestion to clarify this point.

Accordingly, we have made the following changes to the text:

p11 L30-34 in the main text:

"The second experiment similarly compared an engineered BW25113 (BW) strain, containing the two regulatory mutations from the compact set (i.e., crp H22N and rpoB A1245V) together with the deletions used to insert them (see methods and DataS1 file), to a “wild type” BW strain (a corresponding knockout strain without the mutations, see methods)."

p28 under Chemostat proteomics experiment L13-16 in methods:

"The starting volume of each bioreactor was 150 ml M9 media supplemented with either30 mM and 10mM D-xylose for the evolved and ancestor samples or only 10mMD-xylose for BW including compact set mutations and/or the deletions used for their insertions (DataS1 file). The minimal media also included trace elements and vitamin B1 was omitted."

https://doi.org/10.7554/eLife.88793.4.sa2

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. Roee Ben Nissan
  2. Eliya Milshtein
  3. Vanessa Pahl
  4. Benoit de Pins
  5. Ghil Jona
  6. Dikla Levi
  7. Hadas Yung
  8. Noga Nir
  9. Dolev Ezra
  10. Shmuel Gleizer
  11. Hannes Link
  12. Elad Noor
  13. Ron Milo
(2024)
Autotrophic growth of Escherichia coli is achieved by a small number of genetic changes
eLife 12:RP88793.
https://doi.org/10.7554/eLife.88793.4

Share this article

https://doi.org/10.7554/eLife.88793