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

  • 2,271
    Page views
  • 171
    Downloads
  • 2
    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

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Ivan Corbeski, Pablo Andrés Vargas-Rosales ... Amedeo Caflisch
    Research Article

    The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

    1. Biochemistry and Chemical Biology
    2. Developmental Biology
    Zhi Li, Yuedi Wang ... Zeyang Zhou
    Research Article

    Imidacloprid is a global health threat that severely poisons the economically and ecologically important honeybee pollinator, Apis mellifera. However, its effects on developing bee larvae remain largely unexplored. Our pilot study showed that imidacloprid causes developmental delay in bee larvae, but the underlying toxicological mechanisms remain incompletely understood. In this study, we exposed bee larvae to imidacloprid at environmentally relevant concentrations of 0.7, 1.2, 3.1, and 377 ppb. There was a marked dose-dependent delay in larval development, characterized by reductions in body mass, width, and growth index. However, imidacloprid did not affect on larval survival and food consumption. The primary toxicological effects induced by elevated concentrations of imidacloprid (377 ppb) included inhibition of neural transmission gene expression, induction of oxidative stress, gut structural damage, and apoptosis, inhibition of developmental regulatory hormones and genes, suppression of gene expression levels involved in proteolysis, amino acid transport, protein synthesis, carbohydrate catabolism, oxidative phosphorylation, and glycolysis energy production. In addition, we found that the larvae may use antioxidant defenses and P450 detoxification mechanisms to mitigate the effects of imidacloprid. Ultimately, this study provides the first evidence that environmentally exposed imidacloprid can affect the growth and development of bee larvae by disrupting molting regulation and limiting the metabolism and utilization of dietary nutrients and energy. These findings have broader implications for studies assessing pesticide hazards in other juvenile animals.