Prospects for Engineering Biophysical CO₂ Concentrating Mechanisms into Land Plants to Enhance Yields

Abstract

Although cyanobacteria and algae represent a small fraction of the biomass of all primary producers, their photosynthetic activity accounts for roughly half of the daily CO₂ fixation that occurs on Earth. These microorganisms are able to accomplish this feat by enhancing the activity of the CO₂-fixing enzyme Rubisco using biophysical CO₂ concentrating mechanisms (CCMs). Biophysical CCMs operate by concentrating bicarbonate and converting it into CO₂ in a compartment that houses Rubisco (in contrast with other CCMs which concentrate CO₂ via an organic intermediate, such as malate in the case of C₄ CCMs). This activity provides Rubisco with a high concentration of its substrate, thereby increasing its reaction rate. The genetic engineering of a biophysical CCM into land plants is being pursued as a strategy to increase crop yields. This review focuses on the progress toward understanding the molecular components of cyanobacterial and algal CCMs, as well as recent advances toward engineering these components into land plants.

INTRODUCTION

The CO₂-Fixing Activity of the Enzyme Rubisco Is Limited by Slow Catalytic Rate and a Competing Reaction with O₂

Carbon is an essential building block for life on Earth, and nearly all the organic carbon that is accessible to living organisms is thought to have passed through the enzyme Rubisco at some point in time. Rubisco, short for d-ribulose-1,5-bisphosphate carboxylase/oxygenase, captures inorganic CO₂ and catalyzes its addition to ribulose-1,5-bisphosphate (RuBP), generating two molecules of 3-phosphoglycerate (3-PGA) that continue through the Calvin Benson Bassham (CBB) cycle to produce a sugar precursor.

Rubisco is highly productive on a global scale, collectively fixing roughly 10¹¹ tons of carbon per year (22). However, the CO₂-fixing activity of Rubisco is limited by a slow catalytic rate and a competing reaction with O₂. Whereas the median turnover rate (k_cat) of central carbon metabolism enzymes is approximately 79 reactions per second (6a), more than 95% of characterized Rubiscos catalyze only 1 to 10 carboxylation reactions per second (24). Furthermore, when Rubisco binds to O₂ instead of CO₂, it catalyzes a counterproductive oxygenation reaction that generates 2-phosphoglycolate from RuBP, which results in the removal of carbon from the CBB cycle. 2-phosphoglycolate must be recycled through a process called photorespiration that wastes energy and liberates fixed CO₂ (68).

According to molecular dynamics simulations, Rubisco has an obvious preference for CO₂ over O₂ when both gases are present at equal concentrations (91). However, at atmospheric concentrations of 21% O₂ and 0.04% CO₂, Rubisco’s oxygenase activity occurs frequently, leading to substantial photorespiration. In C₃ plants, which include trees and many crops such as rice and wheat, photorespiration results in the loss of about a quarter of fixed carbon (94).

Some organisms have evolved Rubisco with a higher specificity for CO₂ (S_CO2/O2), but this seems to come at the expense of the enzyme’s turnover rate for CO₂ (k_cat,C). The examination of Rubisco orthologs from a variety of species has revealed an inverse correlation between Rubisco’s k_cat,C and its S_CO2/O2, suggesting a trade-off between the two parameters (81). A proposed explanation for this trade-off is that because CO₂ is a fairly featureless molecule, improving Rubisco’s S_CO2/O2 may only be possible through stabilizing the carboxyketone intermediate that forms while CO₂ is being added to RuBP (89). Tighter binding to the carboxyketone intermediate would slow conversion to the final product, resulting in a slower k_cat,C.

C₃ plants usually contain Rubiscos with a higher S_CO2/O2 but lower k_cat,C, and to compensate for Rubisco’s slow activity, these plants express abundant amounts of the enzyme, dedicating up to 25% of leaf nitrogen to do so (78).

CO₂ Concentrating Mechanisms Promote Rubisco’s Carboxylase Activity

Many organisms, including C₄ plants, cyanobacteria, and algae, overcome Rubisco’s limitations by using CO₂ concentrating mechanisms (CCMs). CCMs increase the CO₂/O₂ ratio around Rubisco, thereby speeding carboxylation while limiting the occurrence of oxygenation.

By creating an environment around Rubisco that is enriched with CO₂, organisms with CCMs are able to use a Rubisco with a lower S_CO2/O2 and higher k_cat,C. With a faster Rubisco, the same rate of carbon fixation can be achieved using less of the enzyme, which allows the organism to allocate fewer resources to producing Rubisco, improving the growth rate and amount of biomass produced per unit of nitrogen (28). Additionally, for land plants, CCMs allow capture of a greater fraction of the CO₂ that diffuses into leaves through stomata, allowing plants to have fewer and/or smaller stomata, which decreases water losses due to transpiration and thus improves growth in arid environments. These benefits come at an energetic cost, but this is a small price to pay in many environments where photosynthetic energy is available in excess.

CCMs increase the concentration of CO₂ in a compartment that houses Rubisco, but CO₂ cannot be directly transported because it easily diffuses across membranes. Therefore, CCMs operate by converting CO₂ to a charged intermediate that can be concentrated before being converted back to CO₂ at a site near Rubisco.

C₄ plants have biochemical CCMs, which rely on an enzyme temporarily fixing CO₂ into a four-carbon organic molecule that is shuttled to a compartment containing Rubisco, where the molecule is decarboxylated to produce concentrated CO₂. In most C₄ plants, the initial fixation and decarboxylation occur in different tissues, the mesophyll and the bundle sheath, respectively. These two tissues are arranged in characteristic concentric rings, known as Kranz anatomy (Figure 1a).

Figure 1.

C₄ plants, cyanobacteria, and algae concentrate CO₂ around the enzyme Rubisco using different strategies. (a) In the mesophyll cells of C₄ plants, HCO₃⁻ is added to the three-carbon molecule phosphoenolpyruvate (PEP) by the enzyme PEP carboxylase, generating the four-carbon molecule malate or aspartate. The malate or aspartate is then decarboxylated to form the three-carbon molecule pyruvate and release CO₂ in bundle sheath cells, where Rubisco is expressed. The biophysical CCMs of (b) cyanobacteria and (c) algae actively transport HCO₃⁻ into the cell, then convert it to CO₂ at a site of clustered Rubisco, which is either the (b) carboxysome or (c) pyrenoid

There are three known subtypes of the C₄ mechanism, but in general they operate by using the enzyme phosphoenolpyruvate (PEP) carboxylase to temporarily fix CO₂ in mesophyll cells. Dissolved CO₂ is converted into bicarbonate (HCO₃⁻ hereafter), and PEP carboxylase adds HCO₃⁻ to the three-carbon molecule PEP, generating the four-carbon product oxaloacetate. Oxaloacetate is eventually converted to malate or aspartate, which is transported into bundle sheath cells that surround the vascular tissues. Within the bundle sheath cells, the four-carbon malate or aspartate is decarboxylated to the three-carbon molecule pyruvate, releasing CO₂ in the vicinity of Rubisco. It is worth noting that some plants (18a, 82a) and some diatoms (78a) operate a single-celled C₄ mechanism where the metabolic pathways are compartmentalized within a single cell rather than partitioned between two cell types. A more detailed discussion of the C₄ pathway and efforts to engineer it into C₃ plants is beyond the scope of this review; we refer the reader to expert reviews by Schlüter and Weber in this volume (81a) and by others (5, 26, 82).

Cyanobacteria and algae have biophysical CCMs that operate by transporting HCO₃⁻ and converting it to CO₂ near a site of clustered Rubisco (Figure 1b,c). The negative charge on HCO₃⁻ impedes its diffusion across membranes, preventing it from escaping and allowing high concentrations of HCO₃⁻ to be maintained within the cell. HCO₃⁻ is then converted to CO₂ in the proximity of Rubisco.

Cyanobacteria and algae leverage the properties of HCO₃⁻ and CO₂ to enhance their CCMs. In this field, HCO₃⁻ and CO₂ are referred to as inorganic carbon (Ci) because they lack C–H bonds. These Ci species equilibrate with each other, and their equilibrium concentrations depend on pH. HCO₃⁻ is the most abundant Ci species between pH 6 and 9, while CO₂ is the most abundant below pH 6. Both algal (64) and cyanobacterial (54) CCMs leverage a high pH to capture CO₂ into HCO₃⁻. Algae appear to additionally convert HCO₃⁻ into CO₂ at a low pH, which favors the reaction and uses protons concentrated by the photosynthetic light reactions to create a sink for HCO₃⁻. Spontaneous equilibration between HCO₃⁻ and CO₂ occurs slowly, on the timescale of 10 seconds (32), but is accelerated dramatically by carbonic anhydrase enzymes, which are among the world’s fastest enzymes, able to perform up to 10⁶ reactions per second (38). Thus, by localizing the carbonic anhydrases to specific subcellular compartments, the cell is able to control where the conversion happens.

Engineering a CO₂ Concentrating Mechanism into Land Plants Has the Potential to Improve Yields

Genetically engineering a CCM into C₃ crops could improve yields by both increasing Rubisco’s carboxylation rate and reducing the energy lost to photorespiration. Free-air CO₂ enrichment experiments have shown that crops produce higher yields when grown with elevated levels of CO₂ (35). This supports the idea that engineering plants to concentrate CO₂ at the subcellular level would also lead to increased yields. The percentage increase in yield observed in free-air CO₂ enrichment experiments varies depending on the plant species. For example, cotton has an average of 42% increase in yield, whereas wheat and rice have a more modest yield increase of 15% (1). This discrepancy is attributed to differences in physiology; cotton leaves develop more rapidly, which may allow the plant to take advantage of enhanced photosynthesis starting at an earlier stage of its growth.

Limiting the occurrence of photorespiration is one of the key ways in which a CCM could improve plant growth. By elevating the CO₂/O₂ ratio around Rubisco, CCMs decrease both the energy and fixed CO₂ lost to photorespiration. Significant progress has already been made toward engineering an alternative pathway that reduces the energetic cost of processing 2-phosphoglycolate, although it still liberates fixed CO₂ (86). Transgenic tobacco plants engineered with the alternative pathway display a 24% increase in seasonal biomass as compared to wild-type plants. C₃ plants engineered to contain a CCM would be expected to achieve the same benefits and additional yield increases by not liberating fixed CO₂. Furthermore, plants with an engineered CCM are expected to have improved nitrogen and water use efficiency because they will require less Rubisco and less leaf gas exchange, as is the case for existing C₄ plants (28).

This review focuses on our current understanding of the molecular components of biophysical CCMs as well as recent progress toward engineering these components into other organisms. The first section of this review covers progress that has been made toward characterizing the cyanobacterial CCM. The second section of this review discusses what is known about the algal CCM from studies of the freshwater alga Chlamydomonas reinhardtii. Within each section, progress toward engineering these CCMs into land plants and important future directions are discussed.

THE CYANOBACTERIAL CO₂ CONCENTRATING MECHANISM

The concentration of Ci within a cyanobacterial cell begins with the active transport of HCO₃⁻ across the plasma membrane. Once concentrated within the cytosol, HCO₃⁻ diffuses into the carboxysome, which is composed of an icosahedral protein shell wrapped around Rubisco and a carbonic anhydrase. This carbonic anhydrase catalyzes the conversion of HCO₃⁻ into CO₂, feeding Rubisco’s carboxylase activity (Figure 1b).

Two types of cyanobacteria, α-cyanobacteria, which are found predominately in marine environments, and β-cyanobacteria, which favor freshwater, appear to have convergently evolved carboxysomes. Components of α-carboxysomes are encoded by the cso operon in α-cyanobacteria, and components of β-carboxysomes are encoded either by the ccm operon or other operons at separate genomic loci. This review focuses on the β-carboxysome, but many of the molecular components that make up the α-cyanobacterial CCM are conceptually similar and are described briefly. Table 1 lists likely functionally analogous proteins between the CCMs of α-cyanobacteria, β-cyanobacteria, and the eukaryotic alga C. reinhardtii. Several outstanding recent reviews provide more detailed comparisons of α- and β-carboxysomes (37, 75, 101).

Table 1.

The biophysical CO₂ concentrating mechanisms (CCMs) of α-cyanobacteria, β-cyanobacteria, and the alga Chlamydomonas reinhardtii have many analogous core components, although they evolved independently

Type of protein	α-Cyanobacteria	β-Cyanobacteria	Chlamydomonas reinhardtii
Rubisco large subunit	CbbL	RbcL	RbcL
Rubisco small subunit	CbbS	RbcS	RbcS
Rubisco linker	CsoS2 (CsoS2A and CsoS2B isoforms)	CcmM (M35 and M58 isoforms)	EPYC1
Carbonic anhydrases near the site of Rubisco	CsoS3 (CsoSCA)	CcmM58 N terminus, CcaA	CAH3
Shell proteins: hexameric, pentameric, pore-forming	Hexameric: CsoS1A, CsoS1B, CsoS1C Pentameric: CsoS4A, CsoS4B Pore-forming: CsoS1D, CsoS1E	Hexameric: CcmK2, CcmK3, CcmK4, CcmO Pentameric: CcmL Pore-forming: CcmP	The Chlamydomonas pyrenoid does not appear to have a shell.
Bicarbonate transporters	Plasma membrane: BicA, SbtA	Plasma membrane: BicA, SbtA, BCT1	Plasma membrane: HLA3 and LCI1 Chloroplast envelope: LCIA and CIA8 Thylakoid membrane: BST1, BST2, BST3
CO2 recapture	NDH-I4	NDH-I3, NDH-I4	LCIB/LCIC complex

Inorganic Carbon Is Concentrated in the Cell by HCO₃⁻ Transporters and Converted to CO₂ by a Carbonic Anhydrase

At the plasma membrane of β-cyanobacteria, the heteromultimeric ATP-binding cassette transporter BCT1 and the homomultimeric Na⁺/HCO₃⁻ symporters BicA and SbtA actively transport HCO₃⁻ into the cytosol (Figure 2) (69). Both BCT1 and SbtA have a high affinity for HCO₃⁻, while BicA has a low affinity. A previously published review has covered these C_i transporters in more detail (e.g., 69).

Figure 2.

HCO₃⁻ transport in a β-cyanobacterial cell starts at the plasma membrane, where the transporters BicA, SbtA, and BCT1 are located. After being concentrated in the cytoplasm, HCO₃⁻ diffuses into the carboxysome, likely via pores in the shell protein CcmK4. Inside the carboxysome, carbonic anhydrases convert HCO₃⁻ into CO₂, which can then be fixed by Rubisco clustered there. CO₂ that diffuses into the cytoplasm or leaks out of the carboxysome is converted to HCO₃⁻ by the NDH-1₃ and NDH-1₄ complexes.

CO₂ that diffuses across the plasma membrane or that leaks out of the carboxysome is captured in the cytosol and converted into HCO₃⁻ by the NDH-1₃ and NDH-1₄ complexes (Figure 2). β-cyanobacteria express both complexes, whereas α-cyanobacteria only encode NDH-1₄ (6). In β-cyanobacteria, the expression of the higher-affinity NDH-1₃ is induced when C_i is limited, whereas the lower-affinity NDH-1₄ is constitutively expressed (69).

Both of the NDH-1 complexes are membrane-bound, but whether they reside on the plasma or thylakoid membrane is controversial and may vary between species (6, 8). Little is known about the precise mechanism through which the NDH-1₃ and NDH-1₄ complexes facilitate CO₂ uptake, but it is thought that they operate by using energy from electron transport to drive the unidirectional hydration of CO₂ into HCO₃⁻ (5a).

Ultimately, the coordinated action of these C_i uptake systems leads to a state where the cytosolic HCO₃⁻ concentration is about 30 times the concentration found in water with pH 7 at 25°C (93). Maintaining this high concentration of HCO₃⁻ within the cytosol is important for the CCM, as it drives HCO₃⁻ diffusion into the carboxysome where HCO₃⁻ is converted into concentrated CO₂ to feed Rubisco (72). The absence of carbonic anhydrase activity in the cytosol is critical for maintaining CCM function. When a carbonic anhydrase from humans was heterologously expressed in the cyanobacterial cytosol, cells required high CO₂ to grow, indicating that their CCM was impaired (70). This is presumably because the presence of a carbonic anhydrase in the cytosol drives the conversion of HCO₃⁻ into CO₂, allowing CO₂ to diffuse out of the cell before it reaches Rubisco within the carboxysome.

Engineering a HCO₃⁻ Transport System Is an Important Step in Transferring a Cyanobacterial CO₂ Concentrating Mechanism into Land Plants

Installing a cyanobacterial HCO₃⁻ transporter at the chloroplast inner envelope membrane (IEM) of a land plant has been suggested as a simple strategy for modestly boosting CO₂ flux to Rubisco in the chloroplast without needing to build a carboxysome (71). The imported HCO₃⁻ could be converted to CO₂ by β-carbonic anhydrases that are natively expressed in plant chloroplasts (16). One computational model predicted that incorporating a single HCO₃⁻ transporter could increase CO₂ uptake by 9% and that expressing multiple transporters could lead to a 16% increase (58). However, this model had assumed ideal growth conditions, and a more detailed model using field data for crop plants (102) predicts that a full cyanobacterial CCM is needed to see crop improvement.

Because cyanobacteria are prokaryotes, they have a different cellular organization from plants, which adds a challenge for properly localizing engineered components. Cyanobacteria are the evolutionary relative and topological equivalent of plant chloroplasts. Therefore, to be targeted to their site of function, components from cyanobacteria need to either be directly transformed into the chloroplast genome or transformed into the nuclear genome fused to an exogenous chloroplast targeting sequence (also called transit sequence). Chloroplast transformation is straightforward in the dicotyledonous reference plant tobacco, which facilitates proof-of-concept studies. However, chloroplast genome transformation is ineffective in monocotyledonous plants, which include major CCM target crops such as rice and wheat, because a lack of selectable markers makes it difficult to obtain plants where each chloroplast expresses the transgenes (30). Therefore, practical efforts toward achieving a CCM in these target crops are likely to ultimately require expressing components from the nuclear genome with targeting sequences to localize them to the desired chloroplast sub-compartment.

An initial attempt was made to express BicA in tobacco leaf chloroplasts using biolistic transformation to insert the foreign DNA directly into the chloroplast genome (67). While BicA could be expressed without hindering the plant’s growth, only about 25% of the protein localized to the chloroplast envelope while 75% was found in the thylakoid membranes instead.

Experiments performed using nuclear transformation of BicA and SbtA fused with chloroplast transit peptides were more successful, both in Nicotiana benthamiana (79) and in Arabidopsis (90). Rolland et al. (79) identified N-terminal sequences in large Arabidopsis transmembrane proteins that could reliably redirect BicA and SbtA to the chloroplast envelope of N. benthamiana. Uehara et al. (90) were able to specifically direct BicA and SbtA to the IEM of Arabidopsis by fusing these HCO₃⁻ transporters to both a chloroplast transit peptide and a mature portion of a protein that is natively located in the IEM. In their study, BicA could stay embedded in the IEM even after removal of the transit peptide. This represents an encouraging step toward engineering functional prokaryotic transporters into the chloroplast membrane.

To identify the HCO₃⁻ transporter homologs that are most likely to function in other organisms, researchers expressed HCO₃⁻ transporters from various cyanobacterial species in Escherichia coli and tested them for functionality through examining cellular uptake of NaH¹⁴CO₃ (17). This screen identified six active SbtA homologs, but none of the BicA or BCT1 homologs tested were able to transport HCO₃⁻. Identifying additional factors necessary for BicA or BCT1 functionality could be important for reconstituting a cyanobacterial CCM in a land plant.

Going forward, a major goal for the field will be demonstrating that HCO₃⁻ is concentrated in the chloroplasts of engineered plants.

Shell Proteins Allow Selective Diffusion of Charged Molecules Between the Cytosol and the Carboxysome Interior

The protein shell of the carboxysome encapsulates densely packed Rubisco and is thought to selectively allow the channeling of HCO₃⁻ and RuBP into the carboxysome and 3-PGA out. Just three monomeric proteins are sufficient for building the icosahedral shell structure; these are CcmK2, CcmO, and CcmL (Figure 3a,b) (74). CcmK2 forms a hexamer that takes on a flattened hexagonal shape and forms the large sheets that make up the faces of the carboxysome icosahedron. CcmO forms trimers that have a hexagonal shape, and it likely bends to form the edges of the icosahedron. Finally, CcmL is a pentamer that creates the vertices of the shell. In α-cyanobacteria, analogous roles are performed by the hexameric proteins CsoS1A, CsoS1B, and CsoS1C, which make up the bulk of the carboxysome shell faces, and the pentameric proteins CsoS4A and CsoS4B, which form the vertices (Table 1) (40, 88).

Figure 3.

Carboxysomes are made up of an icosahedral protein shell that surrounds clustered Rubisco. (a) There are several carboxysomes per cyanobacterial cell, and they are evenly distributed across the longitudinal axis. (b) Assembly of the icosahedral carboxysome shell relies on the hexameric CcmK2 that makes up the faces of the icosahedron, the trimeric CcmO at the edges, and pentameric CcmL at the vertices. The hexameric protein CcmK4 has a small ionic pore through which HCO₃⁻ could diffuse, and CcmP has a larger pore that could allow an exchange of Calvin cycle intermediates. The CcmK3/K4 hexamer may act as a cap to block the flow of HCO₃⁻ through CcmK4 when needed. (c) The interior of the carboxysome is made up of Rubisco that is clustered by the CcmM isoform M35. The CcmM isoform M58 also binds Rubisco and is thought to reside at the edges, where CcmN links it to the shell.

Although CcmK2, CcmO, and CcmL are sufficient to build the shell’s structure, the oligomers CcmP, CcmK3, and CcmK4 are thought to be necessary for mediating diffusion of metabolites across the shell, thereby enabling the CCM to function properly (Figure 3b). CcmP is a hexamer and forms a double layer of hexagonal shapes (42). The double layer allows CcmP to have an open and a closed conformation. In its open conformation, CcmP has a central pore large enough to allow the diffusion of CBB cycle metabolites such as the Rubisco substrate RuBP and product 3-PGA.

CcmK4 can independently form a hexamer containing a central pore that is predicted to be permeable to anions such as HCO₃⁻ (53). The CcmK4 hexamer is thought to be dispersed throughout the faces of the carboxysome and to control the influx of HCO₃⁻.

CcmK3 cannot form large oligomers on its own, but it is able to form a heterohexamer with CcmK4 that has a ratio of four CcmK4 monomers to two CcmK3 monomers (85). Certain residues on CcmK3 make it unlikely to fit favorably alongside CcmK2 in the carboxysome shell. However, the CcmK3/K4 heterohexamer can form a dodecamer by fitting on the top of CcmK4, suggesting a model in which the heterohexamer may be used as a cap to limit the flux of HCO₃⁻ under certain conditions that would induce CcmK3 expression (Figure 3b).

CcmK3 and CcmK4 are encoded at a separate locus from the rest of the ccm operon, indicating that they could be under different transcriptional control (84). While CcmK4 is necessary for growth, CcmK3 is not (85).

Linker Proteins Bind and Cluster Rubisco Inside the Carboxysome

In β-cyanobacteria, carboxysome assembly begins with the clustering of Rubisco into an electron-dense body called the procarboxysome (13). This clustering is carried out by M35, a truncated isoform of the protein CcmM that arises by translation initiated at an internal ribosome entry site in the ccmM transcript (Figure 3c) (48). M35 is necessary for procarboxysome formation (49) and sufficient for clustering Rubisco into a procarboxysome-like structure in cyanobacterial mutants lacking the ccm operon (13).

The amino acid sequence of M35 consists of three repeated Rubisco small subunit-like (SSUL) domains, each of which binds to the Rubisco large subunit, allowing its clustering to form a matrix within the carboxysome. The sequence similarity between the SSUL domains of CcmM and the Rubisco small subunit led Price et al. (72a) to propose a model where each SSUL domain of CcmM binds to the Rubisco large subunit by displacing the Rubisco small subunit. Intriguingly, recent work has shown that in vitro the SSUL domain can bind at an equatorial region between the Rubisco large subunit dimers, suggesting that CcmM could link Rubiscos without displacing the Rubisco small subunit (80, 95). However, it remains possible that the original hypothesis is correct, and the interactions observed in vitro are representative of an intermediate complex that forms before a chaperone replaces the Rubisco small subunit with the SSUL domain.

The full-length 58-kDa isoform of CcmM, M58, has an N-terminal γ-carbonic anhydrase domain in addition to the three SSUL domain repeats (49). In some species, this N-terminal domain functions as a carbonic anhydrase to convert HCO₃⁻ into CO₂ within the carboxysome, and in other species, there is a separate carbonic anhydrase called CcaA that is recruited to the carboxysome by the M58 N-terminal domain (46, 59).

M58 is expressed less abundantly than M35, supporting a model in which M35 clusters Rubisco into a matrix at the carboxysome center while M58 resides at the periphery and mediates interactions between the Rubisco matrix and the shell (Figure 3c) (48). Interactions between the shell and the carboxysome interior are thought to be facilitated by the protein CcmN. The N terminus of CcmN interacts with M58, while the C terminus interacts with the abundant hexagonal shell protein CcmK2 (41). CcmK2 fails to localize to the procarboxysome in mutants lacking CcmN, demonstrating that CcmN is necessary to recruit CcmK2 to the procarboxysome (13).

In α-cyanobacteria, Rubisco clustering and carboxysome shell assembly are thought to occur simultaneously (33). The protein CsoS2 plays a similar role to the β-cyanobacterial protein CcmM, acting as a Rubisco linker (12). Like CcmM, CsoS2 has two isoforms that arise through posttranscriptional mechanisms: the full-length isoform CsoS2B, and a truncated isoform, CsoS2A (15). However, while the truncated M35 form of CcmM is produced through an internal ribosomal entry site, CsoS2A is produced from programmed ribosomal frameshifting.

While both CcmM and CsoS2 cluster Rubisco into a dense matrix, they are different in several structural and functional aspects. For example, CcmM contains SSUL domains and binds to the Rubisco large subunit, but CsoS2 is intrinsically disordered and interacts with the Rubisco small subunit (45). While both isoforms of CcmM are necessary to assemble a β-carboxysome, only the full-length CsoS2B isoform is necessary to assemble an α-carboxysome, and the role of CsoS2A is unknown (15).

Steps Have Been Taken to Build Synthetic Carboxysomes

To achieve the goal of engineering a cyanobacterial CCM into land plants, researchers are pursuing efforts to reconstitute carboxysomes in heterologous systems in parallel with efforts to target cyanobacterial HCO₃⁻ transporters to plant membranes (Figure 4). Both β- and α-carboxysome-like structures have been reconstituted in heterologous systems including E. coli and tobacco. While tobacco is a good reference for the crop plants that will ultimately be the target for engineering, E. coli is a useful chassis organism for rapidly identifying the minimal gene set for assembling a carboxysome.

Figure 4.

The goals of localizing HCO₃⁻ transporters to the proper plant membranes and building a carboxysome have been pursued in parallel. Once HCO₃⁻ transporters and carboxysomes can independently function in a plant cell, combining them into a single plant will be the next step toward reconstituting a cyanobacterial CO₂ concentrating mechanism (CCM) in land plants.

Simple β-carboxysome-like shells could be assembled in E. coli using a synthetic operon consisting of only four genes from the cyanobacterium Halothece sp. PCC 7418 (11). These genes encode the hexameric proteins that make up the faces of the carboxysome icosahedron, CcmK2 and K1; a K2 homolog found in a subset of β-cyanobacteria; the trimeric protein CcmO that forms the edges; and the pentameric vertex CcmL.

Fang et al. (20) expanded on this work by creating and expressing an IPTG-inducible synthetic ccm operon using genes found in the β-cyanobacterial reference organism Synechococcus elongatus PCC 7942. This synthetic operon combined 12 genes normally found at five different chromosomal loci into a single operon. These genes encode the shell-associated proteins CcmK2, CcmO, CcmL, CcmK3, CcmK4, CcmP, and CcmN, as well as the Rubisco linker CcmM, the Rubisco large and small subunits, the Rubisco chaperone RbcX, and the carbonic anhydrase CcaA. When expressed in E. coli, the synthetic operon generated carboxysome-like structures that were slightly larger and more irregularly shaped than native carboxysomes, indicating that the ratio of these carboxysome components may need to be optimized in future experiments. Nevertheless, the packing of Rubisco within the synthetic carboxysome lumen was similar to the native packing density. Furthermore, ¹⁴C could be fixed by isolated carboxysomes treated with NaH¹⁴CO₃, indicating that the carboxysomes had an active carbonic anhydrase and Rubisco.

An α-carboxysome has been assembled in E. coli using the cso operon from Halothiobacillus neapolitanus, a chemolithotrophic proteobacteria that also packages Rubisco into a microcompartment, demonstrating that all the proteins necessary to build an α-carboxysome are encoded in the cso operon (9). Furthermore, α-carboxysome-like structures could be observed in tobacco chloroplasts expressing simply the α-cyanobacterial Rubisco large and small subunits alongside the Rubisco linker CsoS2 and the shell protein CsoS1A (47).

Given the promising results in E. coli, a major near-term frontier is the production of carboxysomes in plant chloroplasts. Replacing a plant’s endogenous Rubisco with the ortholog from cyanobacteria is a critical step toward ensuring that the enzyme can be packaged into the carboxysome, as the linker protein interacts with specific sites in the cyanobacterial ortholog. Lin et al. (44) have been able to replace the native Rubisco large subunit gene in tobacco transplastomic lines with the β-cyanobacterial large and small subunits, and Long et al. (47) accomplished this with the α-cyanobacterial Rubisco. In both cases, the transgenic plants are able to grow autotrophically in high CO₂, indicating that the cyanobacterial Rubisco is functional in the plant.

Excitingly, minimal carboxysome-like structures have been reconstituted in tobacco using either β- or α-carboxysome components. Lin et al. (44) observed procarboxysome-like structures when the β-cyanobacterial Rubisco was expressed along with the M35 isoform of CcmM in tobacco. Long et al. (47) reconstituted minimal α-carboxysome-like structures in tobacco using a minimal gene set containing Rubisco, the linker CsoS2, and the shell component CsoS1A. Going forward, the next challenge for both systems is incorporating the remaining components necessary for a fully functional carboxysome, including other shell proteins and a carbonic anhydrase. Once this is accomplished, a functional carboxysome can be combined with a HCO₃⁻ transport system to reconstitute a full CCM (Figure 4).

THE CO₂ CONCENTRATING MECHANISM IN EUKARYOTIC ALGAE

Much of our molecular understanding of the algal CCM has come from studies of the reference freshwater alga Chlamydomonas reinhardtii (Chlamydomonas hereafter), which is the focus of this section of the review. The CCM in Chlamydomonas relies on HCO₃⁻ transport across several different membranes to concentrate Ci near Rubisco, which is housed in a non-membrane-bound organelle within the chloroplast called the pyrenoid (Figure 5). The core of the pyrenoid is a region known as the matrix, which consists of densely packed Rubisco. Starch granules form a sheath around the matrix. Membrane tubules traverse the matrix, exit through gaps in the starch sheath, and connect to the photosynthetic thylakoid membranes in the stroma. The core model of the algal CCM is as follows: HCO₃⁻ is concentrated in the lumen of the pyrenoid tubules through the action of HCO₃⁻ transporters. There, an acidic environment and a carbonic anhydrase cause the conversion of HCO₃⁻ to CO₂. This CO₂ is then able to diffuse across the pyrenoid tubule membranes out to the Rubisco in the matrix.

Figure 5.

In the green alga Chlamydomonas reinhardtii, HCO₃⁻ transport across a series of membranes leads to concentrated HCO₃⁻ in the pyrenoid tubules, which are continuous with the thylakoid membranes. In the tubules, a carbonic anhydrase (CAH3) converts HCO₃⁻ to CO₂, which then diffuses out to reach Rubisco in the matrix. CO₂ that leaks out of the pyrenoid is converted back into HCO₃⁻ by the LCIB/LCIC complex and then recycled through the pyrenoid. The pH of various subcellular compartments is important for determining the net direction of CO₂/HCO₃⁻ conversion. The neutral to slightly basic pH of the periplasm and chloroplast stroma supports conversion of CO₂ into HCO₃⁻, and the acidic pH of the thylakoid lumen supports conversion into CO₂.

HCO₃⁻ Transport Must Occur at the Plasma Membrane, Chloroplast Envelope, and Thylakoid Membrane

The flux of Ci through a Chlamydomonas cell begins in the periplasm, where the carbonic anhydrases CAH1 (Cre04.g223100) and CAH2 (Cre04.g223050) are located (Figure 5, Table S1). The pH of the periplasm is thought to match the pH of the extracellular environment. Active transport at the plasma membrane removes HCO₃⁻ from the periplasm, so CAH1 and CAH2 likely function to replenish HCO₃⁻ by converting CO₂ into HCO₃⁻ (21).

At the plasma membrane, HCO₃⁻ uptake is mediated by LCI1 (low CO₂ inducible protein 1, Cre03.g162800) (66) and HLA3 (high light activated protein 3, Cre02.g097800) (18), which form a complex (51). Overexpression of LCI1 in high CO₂ conditions, when the rest of the CCM is not induced, leads to higher accumulation of C_i within the cell (66). The sequence of LCI1 does not have any recognizable structural domains, so its specific role in C_i uptake is still unknown (99), and HLA3 is a putative ATP-binding cassette HCO₃⁻ transporter (18). The ATP-dependent HCO₃⁻ transport activity of HLA3 has been demonstrated in a heterologous expression system, indicating that it can function independently from other Chlamydomonas-specific factors to actively pump HCO₃⁻ into the cell (57).

Once in the cytoplasm, HCO₃⁻ must then pass through the chloroplast envelope to become concentrated in the chloroplast stroma. LCIA (limiting CO₂ inducible A, Cre06.g309000), a formate/nitrite transporter homolog, localizes to the chloroplast envelope and has been implicated in bicarbonate transport there (99), although it is unclear whether LCIA mediates active or passive transport. Photosynthesis is inhibited in lcia mutants at pH 9, when most C_i is expected to be HCO₃⁻, demonstrating that LCIA likely has a role in HCO₃⁻ uptake (97). Furthermore, heterologous expression of LCIA in Xenopus oocytes led to a twofold increase in HCO₃⁻ accumulation (55).

Inside the stroma, according to the prevailing model, HCO₃⁻ crosses one final membrane to enter the thylakoid lumen (63). The genes BST1 (Cre16.g662600), BST2 (Cre16.gg663400), and BST3 (Cre16.g663450) each encode putative bestrophins (65), which are a family of chloride channels that are in some cases permeable to HCO₃⁻ (73). BST1, BST2, and BST3 each localize to the thylakoid membrane, suggesting that one or more of these proteins could facilitate the shuttling of HCO₃⁻ into the thylakoid lumen (51). The hypothesis that BST1-3 play an important role in the CCM is further supported by the observation that a bst1-3 triple knockdown generated by RNAi grows more slowly at air levels of CO₂ than wildtype cells (65).

Proton pumping during the light reactions of photosynthesis creates an acidic environment within the thylakoid lumen, which promotes the conversion of HCO₃⁻ to CO₂ (2, 77). This conversion is likely catalyzed by the carbonic anhydrase CAH3 (Cre09.g415700), which localizes to the thylakoid lumen (63). Concentrated CO₂ produced in the thylakoid lumen is thought to diffuse across the pyrenoid tubules to feed the Rubisco in the matrix.

The proteins LCIB (Cre10.g452800) and LCIC (Cre06.g307500) form a complex that is thought to recapture unfixed CO₂ that leaks out of the matrix, converting this CO₂ back into HCO₃⁻ (100). The structure of LCIB resembles functional β-carbonic anhydrases, and homologs of LCIB have carbonic anhydrase activity (36). LCIB and LCIC bind to some of the bestrophins on the thylakoid membrane (51), suggesting that the HCO₃⁻ recaptured by LCIB and LCIC can rapidly access the thylakoid lumen for another opportunity to be fixed by Rubisco.

CIA8 (Ci accumulation 8, Cre09.g395700), a putative member of the sodium bile acid symporter family, has recently been implicated in Ci uptake, although both its function and localization within the chloroplast are yet unresolved (50). cia8 mutants have impaired growth at air levels of CO₂ and reduced Ci accumulation both at pH 7.3 and at pH 9. This is notable because many of the proteins involved in Ci uptake have overlapping functions, so a growth defect is usually not observed from a single gene knockout. Continuing to study the function of CIA8 will lead to a more complete picture of Ci uptake in Chlamydomonas.

Rubisco, Linked by EPYC1, Is Clustered in the Liquid-Like Pyrenoid Matrix

When the CCM is induced, an estimated 90% of the cell’s Rubisco clusters in the pyrenoid matrix (10). This clustering of Rubisco ensures that the enzyme is supplied with concentrated CO₂, which is thought to arrive via the membranous pyrenoid tubules that traverse the matrix.

Assembly of the Chlamydomonas pyrenoid matrix relies on the Rubisco linker EPYC1 (essential pyrenoid component 1, Cre10.g436550) (Figure 6a), which is one of the most abundant proteins in the matrix (52). The amino acid sequence of EPYC1 consists primarily of four nearly identical repeats, each with a predicted α-helical region followed by a region predicted to be highly disordered, suggesting that EPYC1 has at least four binding sites for Rubisco. Aggregation of Rubisco into the pyrenoid requires two solvent-facing α-helices found on the algal Rubisco small subunit, making this region a potential site for EPYC1 binding (60, 4a).

Figure 6.

A pyrenoid forms at the center of the Chlamydomonas cell’s cup-shaped chloroplast. (a) Rubisco is clustered within the pyrenoid matrix by the linker protein EPYC1. (b) Before the Rubisco matrix is fully encapsulated by a starch sheath, Rubisco can directly exchange its substrate ribulose-1,5-bisphosphate (RuBP) and its product 3-phosphoglycerate (3-PGA) with the other Calvin Benson Bassham (CBB) cycle enzymes in the chloroplast stroma. However, when a starch sheath is in place, this exchange may depend on diffusion through minitubules embedded within the pyrenoid tubules. These minitubules are continuous with the stroma and pyrenoid matrix.

Observation of fluorescently tagged matrix components within Chlamydomonas has revealed that the matrix displays liquid-like properties, such as internal mixing and formation of spherical droplets that can fuse (25). Pyrenoids are also inherited by daughter cells through fission of the mother cell’s pyrenoid, although a portion of the matrix rapidly disperses into the stroma at the end of each cell division cycle. The rapid dispersal of the pyrenoid during cell division could be important for ensuring that both daughter cells have enough starting material to form a pyrenoid de novo in case fission fails. In addition, the phase-separated nature of the pyrenoid matrix could allow it to form rapidly as a response to conditions that induce the CCM, such as low CO₂ (10).

Purified Rubisco does not form liquid-like droplets on its own in vitro, but the addition of EPYC1 is sufficient to drive demixing into liquid droplets (98). The internal mixing dynamics of these in vitro droplets occur on a similar time scale to the pyrenoid, suggesting that EPYC1 and Rubisco are sufficient to reconstitute the Chlamydomonas pyrenoid matrix.

The Pyrenoid Starch Sheath May Serve as a Diffusion Barrier to Slow CO₂ Escape

Both green algae and plants store some of the energy they capture from photosynthesis as starch, a polymer of glucose that forms large lens-shaped granules in the chloroplast stroma. Many green algal species also assemble a subset of their starch granules in a shell around the pyrenoid, forming a structure called the starch sheath. Unlike globular stromal starch, this pyrenoid starch has a curved morphology, and is made up of distinct plates that wrap around the pyrenoid and appear to form a seal interrupted by gaps to allow the passage of pyrenoid tubules (61).

In Chlamydomonas, the conditions that induce the CCM also induce the starch sheath to form (76). The total amount of starch accumulated is coordinated with the diurnal cycle, but whether starch is primarily in the stroma or surrounding the pyrenoid depends on whether the CCM is active. Nitrogen deprivation, which slows down photosynthetic activity, causes cells to accumulate more stromal starch rather than pyrenoid starch (23). The apparent coordination between CCM induction and pyrenoid starch formation suggests that the starch sheath may have a role in the CCM.

It has been suggested that the starch sheath could act as a barrier to slow the CO₂ efflux from the pyrenoid (76). Starch is composed of alternating amorphous layers of the unbranched polymer amylose and crystalline layers of the branched polymer amylopectin (103), the latter of which is thought to be impermeable to gases, including CO₂ and O₂ (27). Furthermore, the curved morphology of pyrenoid starch granules and the seal they form around the pyrenoid would appear to support this function.

Despite the proposed role of the starch sheath in preventing CO₂ efflux, mutants that are unable to synthesize starch still appear to have a fully functional CCM at air levels of CO₂, suggesting that the starch sheath may not actually be necessary for the CCM after all (92). This conundrum could be resolved if the starch sheath were found to be important under slightly different growth conditions than those tested in the laboratory, or if it were found to confer only a small advantage that is still relevant evolutionarily.

Several proteins that contain starch-binding domains localize to the periphery of the pyrenoid and form different localization patterns there (51). The granule-bound starch synthase STA2 (Cre17.g721500) and the starch branching enzyme SBE3 (Cre10.g444700) appear to localize to the starch plates, whereas the protein LCI9 (Cre02.g130700) localizes to a mesh pattern, possibly filling the gaps between starch plates. LCI9 contains two starch-binding domains and is homologous to glucan 1,4-α-glucosidases, enzymes that typically function to liberate glucose monomers from glucan chains. This homology suggests that LCI9 could degrade starch at the gaps between starch plates, possibly ensuring a close fit for adjacent starch plates.

Two mutants have been described with interesting phenotypes related to the pyrenoid starch. Cells lacking a protein that localizes to the starch sheath, named SAGA1 (starch granules abnormal 1, Cre11.g467712), have thin and elongated pyrenoid starch granules (34). Cells lacking another protein called BSG1 (bimodal starch granule 1, Cre02.g091750) have enlarged pyrenoid starch granules under nitrogen-limiting conditions that would normally favor the accumulation of stromal starch, indicating that BSG1 may be involved in controlling the transition from pyrenoid to stroma starch (23). Continued studies of the pyrenoid starch sheath could provide insights into why starch granules across the green lineage have specific morphologies and how these morphologies are produced.

Pyrenoid Tubules Are Thought to Deliver Concentrated CO₂ and May Provide a Path for Diffusion of Calvin Benson Bassham Cycle Metabolites

The pyrenoid tubules, which are continuous with the thylakoid membranes, penetrate into the pyrenoid matrix through distinct gaps in the starch sheath and fuse into a reticulated network at the center (19). When a starch sheath is present, the tubules are thought to be the primary route for entry of inorganic carbon into the pyrenoid: HCO₃⁻ enters the thylakoid lumen via transporters outside the pyrenoid, and diffuses along the inside of the tubules before being converted to CO₂ in the portion of the tubules that traverses the matrix (Figure 5).

Formation of the starch sheath occurs more slowly than formation of the other components of the pyrenoid, so for a short period of time after the CCM is induced, the starch sheath does not fully enclose the pyrenoid matrix (76). During this time, Rubisco’s substrate RuBP and its product 3-PGA can directly diffuse between Rubisco in the pyrenoid and the other CBB cycle enzymes, which are located in the chloroplast stroma (87) (Figure 6b).

Cryo-electron tomography images of the pyrenoid tubules have shown that there are several minitubules embedded within each tubule (Figure 6b) (19). The lumens of these minitubules are continuous with both the pyrenoid and the chloroplast stroma, and are wide enough for the passage of RuBP and 3-PGA. Therefore, the minitubules may serve as a conduit for the diffusion of metabolites between Rubisco in the matrix and other CBB cycle enzymes in the stroma along their respective concentration gradients when the starch sheath is fully formed.

Mutants that lack a pyrenoid matrix still form a pyrenoid tubule network at the canonical location within the chloroplast (14). This observation suggests that the process of building the pyrenoid tubule network occurs separately from the assembly of the rest of the pyrenoid, and that these tubules contain the information for where a pyrenoid should be placed. Therefore, the pyrenoid tubules may actually localize the matrix and the starch sheath.

Although the pyrenoid tubules play a crucial role in the algal CCM, not much is known about their biogenesis. The topology of the transition zone between the thylakoid membranes that exist as stacked sheets in the chloroplast and the more cylindrical tubules that traverse the pyrenoid is complex (19), and how its formation is mediated molecularly is unknown. The protein PSAH (Cre07.g330250), associated with photosystem I, is enriched in the pyrenoid tubules (51) and may have a function there. Identifying and characterizing more proteins involved in constructing the pyrenoid tubules will be important for future attempts to install an algal CCM in land plants.

Early Progress Suggests Promising Prospects of Transferring an Algal CO₂ Concentrating Mechanism into Land Plants

Several important components of the Chlamydomonas CCM have been fused to green fluorescent protein (GFP) and expressed in Arabidopsis and tobacco leaves to examine their localization (3). Most of these algal proteins localized to the correct subcellular compartment in the plant cell without requiring changes to their protein sequence. For example, the periplasmic carbonic anhydrase CAH1 and the plasma membrane HCO₃⁻ transporters LCI1 and HLA3 all localized to the cell periphery. The putative CO₂ recapture complex proteins LCIB and LCIC localized to the chloroplast stroma, while the putative HCO₃⁻ channel LCIA was targeted to the chloroplast envelope. These results are encouraging because they suggest that expressing algal CCM components from the nucleus of a land plant will require very few modifications at the protein sequence level, and the key challenge is determining which genes need to be transferred.

As in the case of cyanobacterial transporters, it is not clear whether algal HCO₃⁻ transporters are active, and an important goal for the future will be to test their activity. Furthermore, the full algal CCM likely requires HCO₃⁻ transport across the thylakoid membranes. A voltage-dependent chloride channel that has homology to the bestrophin-like proteins on the Chlamydomonas thylakoids already resides on Arabidopsis thaliana thylakoids (31). It will be interesting to learn whether this channel is sufficient for CCM activity, or whether it is necessary to express the putative algal HCO₃⁻ transporters BST1, BST2 and BST3.

Another important step toward reconstituting a pyrenoid in a plant is to engineer the plant Rubisco small subunit to contain specific residues on the two solvent-facing α-helices present on the Chlamydomonas ortholog, which may be required for binding to the Rubisco linker EPYC1 (60). Atkinson et al. (4) were able to construct a functional hybrid small subunit which replaced the native Arabidopsis small subunit α-helices with those of Chlamydomonas. This hybrid small subunit was functional, as demonstrated by its ability to rescue an Arabidopsis double mutant that grew slowly due to impairment of two out of four homologous small subunit genes. Modifying these α-helices did not appear to significantly impact the catalytic properties of the Rubisco holoenzyme.

Several stages for engineering an algal system into plants can be pursued in parallel; these include reconstituting a pyrenoid matrix in a land plant, continuing to identify and transfer HCO₃⁻ transporters to plants, and studying the mechanisms of pyrenoid tubule biogenesis. After these stages are achieved, an important goal will be to orient a pyrenoid matrix around pyrenoid tubules and finally to combine this matrix/tubule system with the HCO₃⁻ transporters (Figure 7).

Figure 7.

Several milestones toward engineering an algal CO₂ concentrating mechanism (CCM) into land plants can be pursued in parallel.

Characterizing and Engineering the Algal CO₂ Concentrating Mechanism Will Benefit from New Resources

Much of the progress that has been made toward understanding the genes involved in the algal CCM has relied on forward genetics studies, wherein mutants generated by chemical mutagenesis or random insertion mutagenesis are screened for a CCM phenotype (86a, 96). The classic CCM phenotype is impaired photoautotrophic growth at air levels of CO₂ that is rescued by elevated CO₂. It will be important to be able to approach saturation in such screens to produce a comprehensive list of factors that need to be transferred to plants. To this end, an insertional mutant library has recently been created, covering 83% of Chlamydomonas nuclear genes (43). Many new genes with roles in photosynthesis have been identified through pooled screening of this library, and some may include novel CCM factors. Additionally, this library now enables the reverse genetic characterization of mutants in genes that become CCM candidates as a result of their sequence or presence in other high-throughput data sets. Furthermore, recent advances in RNA interference (39) and CRISPR-Cas9 gene editing (29) in Chlamydomonas provide the complementary ability to study genes that are not represented in the library or homologous genes that may be partially functionally redundant and thus may not show a phenotype in a single-gene knockout mutant.

Systematic characterization of protein localization and protein-protein interactions is synergistic with studies of mutant phenotypes because it identifies new genes of interest and greatly accelerates the process of understanding gene function. A high-throughput fluorescence protein-tagging effort in Chlamydomonas has provided data about the localizations of 135 candidate CCM proteins, 89 of which localize to at least six distinct patterns within the pyrenoid (51). Moreover, the interactions of 38 core CCM proteins were identified through affinity purification and mass spectrometry. A parallel effort characterized the Chlamydomonas pyrenoid proteome (104), providing independent evidence of pyrenoid localization of proteins that were also identified in the protein-tagging effort and additionally identifying other candidate pyrenoid components.

CONCLUSIONS AND OUTLOOK

Genetically engineered crops that are able to grow more quickly while using fewer resources will be important tools for meeting global agricultural demands, which are expected to rise significantly in the near future (89a). One promising target for engineering is the improvement of the function of the carbon-fixing enzyme Rubisco through installing a CCM, as this would promote Rubisco’s productive carboxylase activity while minimizing its unwanted oxygenase activity. Progress is being made toward understanding a variety of types of CCMs, such as the C₄ pathway found in many plant species and the biophysical CCMs found in single-celled cyanobacteria and algae. Efforts to reconstitute these CCMs into land plants are yielding encouraging results. Each system has its own set of opportunities and challenges, some of which remain unknown (Table 2), so continuing the pursuit of characterizing and engineering C₄, α-cyanobacterial, β-cyanobacterial, and algal CCMs in parallel will maximize the chance of at least one approach being successful. Exploring hybrid approaches that combine elements from different CCMs could also be advantageous. The availability of genetic tools that can now be used to both study the components of CCMs and begin to engineer them into plants is expected to lead to exciting advances in the near future.

Table 2.

C₄, cyanobacterial, and algal CO₂ concentrating mechanisms (CCMs) each provide different opportunities and challenges for engineering into land plants

Type of CCM	Advantages	Challenges
C4 plant	Most closely related evolutionarily to C3 plants	Requires engineering tissue development to give leaves Kranz anatomy
Both cyanobacteria and algae	Operates within a single cell, therefore no need to engineer cell differentiation	Requires replacing Rubisco, because Rubisco linkers appear to bind only Rubisco from their host organisms
Cyanobacteria	Components are well characterized	Most evolutionarily distant from plants Components are not natively encoded in a eukaryotic nucleus and targeted to a chloroplast
Algae	Components are natively encoded in a eukaryotic nucleus and targeted to a chloroplast	Components are poorly characterized

SUMMARY POINTS.

1. The enzyme Rubisco is crucial for converting CO₂ into biomass but has limitations, including a relatively slow reaction rate and a competing reaction with O₂.

2. CO₂ concentrating mechanisms (CCMs) enhance Rubisco’s CO₂-fixing activity by feeding the enzyme with concentrated CO₂ and increasing the CO₂/O₂ ratio so that carboxylation is favored over oxygenation.

3. The CCMs in both cyanobacteria and green algae operate by concentrating HCO₃⁻ through transport and converting the molecule into CO₂ at a site near clustered Rubisco.

4. Rubisco is clustered by linker proteins to form a subcellular structure: the carboxysome in cyanobacteria and the pyrenoid in algae.

5. In recent progress toward the goal of engineering functional cyanobacterial and algal CCMs into plants, researchers have targeted HCO₃⁻ transporters from both types of organisms to plant membranes.

6. Carboxysome-like structures have been synthesized in chassis organisms and land plants, and a pyrenoid matrix has been reconstituted in vitro.

Glossary

Rubisco

d-ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate (RuBP)

Rubisco’s substrate

3-Phosphoglycerate (3-PGA)

Rubisco’s product

CBB cycle

Calvin Benson Bassham cycle

k_cat

catalytic rate

S_CO2/O2

Rubisco’s specificity for CO₂ over O₂

k_cat,C

Rubisco’s turnover rate for CO₂

CCM

CO₂ concentrating mechanism

Biochemical CCM

A CO₂ concentrating mechanism that uses intermediate organic molecule that is decarboxylated to release CO₂ near Rubisco.

Kranz anatomy

the arrangement of mesophyll and bundle sheath tissues in a C₄ leaf

Biophysical CCM

A CO₂ concentrating mechanism that operates by directly transporting inorganic carbon (in the form of bicarbonate) and dehydrating it to CO₂ near Rubisco

Inorganic carbon (C_i)

Defined in this field as carbon that is not bound directly to hydrogen

IEM

chloroplast inner envelope membrane