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.
  1. Eric Franklin
  2. Martin Jonikas  Is a corresponding author
  1. 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).

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 COat 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

Article and author information

Author details

  1. Eric Franklin

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

    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7365-6447
  2. 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
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9519-6055

Publication history

  1. Version of Record published: December 3, 2020 (version 1)

Copyright

© 2020, Franklin and Jonikas

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.

Metrics

  • 1,493
    Page views
  • 123
    Downloads
  • 1
    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)

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. Eric Franklin
  2. Martin Jonikas
(2020)
Genetic Engineering: Increasing the uptake of carbon dioxide
eLife 9:e64380.
https://doi.org/10.7554/eLife.64380
  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Dirk H Siepe, Lukas T Henneberg ... Kenan Christopher Garcia
    Tools and Resources

    Secreted proteins, which include cytokines, hormones and growth factors, are extracellular ligands that control key signaling pathways mediating cell-cell communication within and between tissues and organs. Many drugs target secreted ligands and their cell-surface receptors. Still, there are hundreds of secreted human proteins that either have no identified receptors ('orphans') and are likely to act through cell surface receptors that have not yet been characterized. Discovery of secreted ligand-receptor interactions by high-throughput screening has been problematic, because the most commonly used high-throughput methods for protein-protein interaction (PPI) screening do not work well for extracellular interactions. Cell-based screening is a promising technology for definition of new ligand-receptor interactions, because multimerized ligands can enrich for cells expressing low affinity cell-surface receptors, and such methods do not require purification of receptor extracellular domains. Here, we present a proteo-genomic cell-based CRISPR activation (CRISPRa) enrichment screening platform employing customized pooled cell surface receptor sgRNA libraries in combination with a magnetic bead selection-based enrichment workflow for rapid, parallel ligand-receptor deorphanization. We curated 80 potentially high value orphan secreted proteins and ultimately screened 20 secreted ligands against two cell sgRNA libraries with targeted expression of all single-pass (TM1) or multi-pass (TM2+) receptors by CRISPRa. We identified previously unknown interactions in 12 of these screens, and validated several of them using surface plasmon resonance and/or cell binding. The newly deorphanized ligands include three receptor tyrosine phosphatase (RPTP) ligands and a chemokine like protein that binds to killer cell inhibitory receptors (KIR's). These new interactions provide a resource for future investigations of interactions between the human secreted and membrane proteomes.

    1. Biochemistry and Chemical Biology
    Meiling Wu, Anda Zhao ... Dongyun Shi
    Research Article

    Antioxidant intervention is considered to inhibit reactive oxygen species (ROS) and alleviates hyperglycemia. Paradoxically, moderate exercise can produce ROS to improve diabetes. The exact redox mechanism of these two different approaches remains largely unclear. Here, by comparing exercise and antioxidants intervention on type 2 diabetic rats, we found moderate exercise upregulated compensatory antioxidant capability and reached a higher level of redox balance in the liver. In contrast, antioxidant intervention achieved a low-level redox balance by inhibiting oxidative stress. Both of these two interventions could promote glucose catabolism and inhibit gluconeogenesis through activation of hepatic AMPK signaling, therefore ameliorating diabetes. During exercise, different levels of ROS generated by exercise have differential regulations on the activity and expression of hepatic AMPK. Moderate exercise-derived ROS promoted hepatic AMPK glutathionylation activation. However, excessive exercise increased oxidative damage and inhibited the activity and expression of AMPK. Overall, our results illustrate that both exercise and antioxidant intervention improve blood glucose in diabetes by promoting redox balance, despite different levels of redox balance. These results indicate that the AMPK signaling activation, combined with oxidative damage markers, could act as a sensitive biomarker, reflecting the threshold of redox balance defining effective treatment in diabetes. These findings provide theoretical evidence for the precise treatment of diabetes by antioxidants and exercise.