Genetic Engineering: Increasing the uptake of carbon dioxide

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

Dec 3, 2020

https://doi.org/10.7554/eLife.64380

Open access
Copyright information

Download
Cite
CommentOpen annotations (there are currently 0 annotations on this page).
Share

Department of Molecular Biology, Princeton University, United States

Look around: how many things do you see made of wood, cloth or plastic? These items may seem wildly different, but they all contain organic carbon and, therefore, they can only exist because plants, algae and certain bacteria are constantly using photosynthesis to turn sunlight, water and atmospheric carbon dioxide (CO₂) into most of our food, furniture and fuel (Fischer et al., 2016). However, this process has gotten more difficult over time. Modern CO₂ levels are less than 1% of what they were when photosynthetic organisms first evolved, making the work of Rubisco, the enzyme that converts CO₂ into organic molecules, more difficult. In turn, the slow rate of CO₂ uptake limits the growth of many plants, including crops such as rice and wheat (Long et al., 2006).

Some organisms, however, have evolved ways to concentrate CO₂ around Rubisco, allowing the enzyme to run faster (Hennacy and Jonikas, 2020). Introducing such carbon-concentrating mechanisms into crops could increase yields by 60% while reducing water and fertilizer requirements (McGrath and Long, 2014). The best understood carbon-concentrating mechanism is the one found in bacteria, which is based on a protein structure called the ‘carboxysome’ that contains Rubisco and other carbon fixation-related enzymes. These species actively import carbon in the form of bicarbonate (HCO₃^–), which diffuses into the carboxysome and is converted to CO₂. The resulting high CO₂ concentration achieved within the carboxysome maximizes the activity of Rubisco and therefore increases overall CO₂ uptake (Figure 1A).

Figure 1

Download asset Open asset

Engineering a carbon-concentrating mechanism into *E. coli*.

(A) *Halothiobacillus neapolitanus* has a carbon-concentrating mechanism that relies on structures called carboxysomes. The cell imports CO₂ as bicarbonate (HCO₃^–), which diffuses into the carboxysome (green hexagon) and is converted into concentrated CO₂. The elevated levels of CO₂ in the carboxysome allow the enzyme Rubisco (dark green) to convert it to biomass more efficiently. (B) Flamholz et al. engineered a strain of *E. coli* to be dependent on Rubisco activity for its growth. Rubisco runs slowly in this strain as it can only use CO₂ which diffuses (dotted line) into the cell from the atmosphere. (C) However, adding just 20 genes from *H. neapolitanus* and selecting for cells that can grow in low levels of CO₂ led to an *E. coli* strain with a reconstituted *H. neapolitanus* carbon-concentrating mechanism based on carboxysomes, which allows Rubisco to run much faster.

Figure created with BioRender.com.

Previous work managed to assemble carboxysome-like structures in the non-photosynthetic model bacterium Escherichia coli (Bonacci et al., 2012). However, these cells required high levels of CO₂ for growth, indicating that additional components were required to concentrate CO₂. Now, in eLife, David Savage, Ron Milo and colleagues – including Avi Flamholz as first author – report how they have engineered a functional carbon-concentrating mechanism into an organism that lacks one (Flamholz et al., 2020).

The team, which is based at the University of California, Berkeley, the Weizmann Institute of Science and the Max Planck Institute of Molecular Plant Physiology, chose the bacterium Halothiobacillus neapolitanus as the genetic donor for their experiment. Carboxysomes in this species are simple and well-studied: in particular, Savage and co-workers had previously identified 20 candidate genes likely needed for these structures to work properly (Desmarais et al., 2019).

As their recipient species, Flamholz et al. chose E. coli, which they genetically modified to rely on Rubisco’s activity for growth (Figure 1B). Without a carbon-concentrating mechanism, this strain could not grow in ambient air – it required supplementation with CO₂ levels about 100 times higher than those found in the atmosphere. Hoping to reconstitute a functional carbon-concentrating mechanism, the team transferred the 20 candidate genes from H. neapolitanus to their E. coli strain. Unsurprisingly, the strain was still unable to grow in ambient CO₂at first, as simply adding genes is often not enough to engineer a complex pathway into a new organism (Antonovsky et al., 2016).

However, Flamholz et al. were able to leverage an important feature of their genetically engineered E. coli strain – its growth rate is proportional to Rubisco’s activity. This allowed the team to use a natural selection experiment to spot mutations that make the carbon-concentrating mechanism work, and therefore increase Rubisco activity. The experiment revealed a mutant that could grow at ambient CO₂ levels, apparently by adjusting the expression levels of the proteins taking part in the carbon-concentrating process.

This result suggested that a carbon-concentrating mechanism based on H. neapolitanus carboxysomes had successfully been reconstituted in their E. coli strain (Figure 1C). To further support this conclusion, electron microscopy was used to observe the carboxysome-like structures within the engineered E. coli strain. To make sure these structures were functional, they individually knocked out several genes known to be essential for carboxysome function in the native host. These mutations had the same effect in E. coli as in H. neapolitanus – the cells no longer grew at ambient CO₂ levels – confirming that the carboxysome was working the same way in the engineered strain as in the native host.

These results from Flamholz et al. indicate that a carboxysome-based carbon-concentrating mechanism can be transferred and function in another organism, providing a blueprint that paves the way toward engineering plants with increased CO₂ uptake and thus greater yields.

References

1. Antonovsky N
2. Gleizer S
3. Noor E
4. Zohar Y
5. Herz E
6. Barenholz U
7. Zelcbuch L
8. Amram S
9. Wides A
10. Tepper N
11. Davidi D
12. Bar-On Y
13. Bareia T
14. Wernick DG
15. Shani I
16. Malitsky S
17. Jona G
18. Bar-Even A
19. Milo R
(2016) Sugar synthesis from CO₂ in Escherichia coli
Cell 166:115–125.

https://doi.org/10.1016/j.cell.2016.05.064
- PubMed
- Google Scholar
1. Bonacci W
2. Teng PK
3. Afonso B
4. Niederholtmeyer H
5. Grob P
6. Silver PA
7. Savage DF
(2012) Modularity of a carbon-fixing protein organelle
PNAS 109:478–483.

https://doi.org/10.1073/pnas.1108557109
- PubMed
- Google Scholar
1. Desmarais JJ
2. Flamholz AI
3. Blikstad C
4. Dugan EJ
5. Laughlin TG
6. Oltrogge LM
7. Chen AW
8. Wetmore K
9. Diamond S
10. Wang JY
11. Savage DF
(2019) DABs are inorganic carbon pumps found throughout prokaryotic phyla
Nature Microbiology 4:2204–2215.

https://doi.org/10.1038/s41564-019-0520-8
- PubMed
- Google Scholar
(2016) Evolution of oxygenic photosynthesis
Annual Review of Earth and Planetary Sciences 44:647–683.

https://doi.org/10.1146/annurev-earth-060313-054810
- Google Scholar
1. Flamholz AI
2. Dugan E
3. Blikstad C
4. Gleizer S
5. Ben-Nissan R
6. Amram S
7. Antonovsky N
8. Ravishankar S
9. Noor E
10. Bar-Even A
11. Milo R
12. Savage DF
(2020) Functional reconstitution of a bacterial CO₂ concentrating mechanism in Escherichia coli
eLife 9:e59882.

https://doi.org/10.7554/eLife.59882
- Google Scholar
1. Hennacy JH
2. Jonikas MC
(2020) Prospects for engineering biophysical CO₂ concentrating mechanisms into land plants to enhance yields
Annual Review of Plant Biology 71:461–485.

https://doi.org/10.1146/annurev-arplant-081519-040100
- PubMed
- Google Scholar
1. Long SP
2. Zhu X
3. Naidu SL
4. Ort DR
(2006) Can improvement in photosynthesis increase crop yields?
Plant, Cell and Environment 29:315–330.

https://doi.org/10.1111/j.1365-3040.2005.01493.x
- Google Scholar
1. McGrath JM
2. Long SP
(2014) Can the cyanobacterial carbon-concentrating mechanism increase photosynthesis in crop species? A theoretical analysis
Plant Physiology 164:2247–2261.

https://doi.org/10.1104/pp.113.232611
- PubMed
- Google Scholar

Article and author information

Author details

Eric Franklin

Eric Franklin is in the Department of Molecular Biology, Princeton University, Princeton, United States

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0001-7365-6447
Martin Jonikas

Martin Jonikas is in the Department of Molecular Biology, Princeton University, Princeton, United States

For correspondence
mjonikas@princeton.edu

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0002-9519-6055

Publication history

Version of Record published: December 3, 2020

Copyright

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