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.
- 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
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 CO2 at 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
-
DABs are inorganic carbon pumps found throughout prokaryotic phylaNature Microbiology 4:2204–2215.https://doi.org/10.1038/s41564-019-0520-8
-
Evolution of oxygenic photosynthesisAnnual Review of Earth and Planetary Sciences 44:647–683.https://doi.org/10.1146/annurev-earth-060313-054810
-
Prospects for engineering biophysical CO2 concentrating mechanisms into land plants to enhance yieldsAnnual Review of Plant Biology 71:461–485.https://doi.org/10.1146/annurev-arplant-081519-040100
-
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
Article and author information
Author details
Publication history
- 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
-
- 2,397
- views
-
- 181
- downloads
-
- 2
- 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)
Genetic Engineering: Increasing the uptake of carbon dioxide
eLife 9:e64380.
https://doi.org/10.7554/eLife.64380
Further reading
-
- Biochemistry and Chemical Biology
A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimer–tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.
-
- Biochemistry and Chemical Biology
- Cell Biology
Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.
