Towards engineering a hybrid carboxysome.

Carboxysomes are bacterial microcompartments, whose structural features enable the encapsulated Rubisco holoenzyme to operate in a high-CO2 environment. Consequently, Rubiscos housed within these compartments possess higher catalytic turnover rates relative to their plant counterparts. This particular enzymatic property has made the carboxysome, along with associated transporters, an attractive prospect to incorporate into plant chloroplasts to increase future crop yields. To date, two carboxysome types have been characterized, the α-type that has fewer shell components and the ß-type that houses a faster Rubisco. While research is underway to construct a native carboxysome in planta, work investigating the internal arrangement of carboxysomes has identified conserved Rubisco amino acid residues between the two carboxysome types which could be engineered to produce a new, hybrid carboxysome. In theory, this hybrid carboxysome would benefit from the simpler α-carboxysome shell architecture while simultaneously exploiting the higher Rubisco turnover rates in ß-carboxysomes. Here, we demonstrate in an Escherichia coli expression system, that the Thermosynechococcus elongatus Form IB Rubisco can be imperfectly incorporated into simplified Cyanobium α-carboxysome-like structures. While encapsulation of non-native cargo can be achieved, T. elongatus Form IB Rubisco does not interact with the Cyanobium carbonic anhydrase, a core requirement for proper carboxysome functionality. Together, these results suggest a way forward to hybrid carboxysome formation.

Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts.

A long-term strategy to enhance global crop photosynthesis and yield involves the introduction of cyanobacterial CO2-concentrating mechanisms (CCMs) into plant chloroplasts. Cyanobacterial CCMs enable relatively rapid CO2 fixation by elevating intracellular inorganic carbon as bicarbonate, then concentrating it as CO2 around the enzyme Rubisco in specialized protein micro-compartments called carboxysomes. To date, chloroplastic expression of carboxysomes has been elusive, requiring coordinated expression of almost a dozen proteins. Here we successfully produce simplified carboxysomes, isometric with those of the source organism Cyanobium, within tobacco chloroplasts. We replace the endogenous Rubisco large subunit gene with cyanobacterial Form-1A Rubisco large and small subunit genes, along with genes for two key α-carboxysome structural proteins. This minimal gene set produces carboxysomes, which encapsulate the introduced Rubisco and enable autotrophic growth at elevated CO2. This result demonstrates the formation of α-carboxysomes from a reduced gene set, informing the step-wise construction of fully functional α-carboxysomes in chloroplasts.

Progress and challenges of engineering a biophysical CO2-concentrating mechanism into higher plants.

Growth and productivity in important crop plants is limited by the inefficiencies of the C3 photosynthetic pathway. Introducing CO2-concentrating mechanisms (CCMs) into C3 plants could overcome these limitations and lead to increased yields. Many unicellular microautotrophs, such as cyanobacteria and green algae, possess highly efficient biophysical CCMs that increase CO2 concentrations around the primary carboxylase enzyme, Rubisco, to enhance CO2 assimilation rates. Algal and cyanobacterial CCMs utilize distinct molecular components, but share several functional commonalities. Here we outline the recent progress and current challenges of engineering biophysical CCMs into C3 plants. We review the predicted requirements for a functional biophysical CCM based on current knowledge of cyanobacterial and algal CCMs, the molecular engineering tools and research pipelines required to translate our theoretical knowledge into practice, and the current challenges to achieving these goals.

Cyanobacterial CO2-concentrating mechanism components: function and prospects for plant metabolic engineering.

Global population growth is projected to outpace plant-breeding improvements in major crop yields within decades. To ensure future food security, multiple creative efforts seek to overcome limitations to crop yield. Perhaps the greatest limitation to increased crop yield is photosynthetic inefficiency, particularly in C3 crop plants. Recently, great strides have been made toward crop improvement by researchers seeking to introduce the cyanobacterial CO2-concentrating mechanism (CCM) into plant chloroplasts. This strategy recognises the C3 chloroplast as lacking a CCM, and being a primordial cyanobacterium at its essence. Hence the collection of solute transporters, enzymes, and physical structures that make cyanobacterial CO2-fixation so efficient are viewed as a natural source of genetic material for C3 chloroplast improvement. Also we highlight recent outstanding research aimed toward the goal of introducing a cyanobacterial CCM into C3 chloroplasts and consider future research directions.

Functions, compositions, and evolution of the two types of carboxysomes: polyhedral microcompartments that facilitate CO2 fixation in cyanobacteria and some proteobacteria.

Cyanobacteria are the globally dominant photoautotrophic lineage. Their success is dependent on a set of adaptations collectively termed the CO2-concentrating mechanism (CCM). The purpose of the CCM is to support effective CO2 fixation by enhancing the chemical conditions in the vicinity of the primary CO2-fixing enzyme, D-ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), to promote the carboxylase reaction and suppress the oxygenase reaction. In cyanobacteria and some proteobacteria, this is achieved by encapsulation of RubisCO within carboxysomes, which are examples of a group of proteinaceous bodies called bacterial microcompartments. Carboxysomes encapsulate the CO2-fixing enzyme within the selectively permeable protein shell and simultaneously encapsulate a carbonic anhydrase enzyme for CO2 supply from a cytoplasmic bicarbonate pool. These bodies appear to have arisen twice and undergone a process of convergent evolution. While the gross structures of all known carboxysomes are ostensibly very similar, with shared gross features such as a selectively permeable shell layer, each type of carboxysome encapsulates a phyletically distinct form of RubisCO enzyme. Furthermore, the specific proteins forming structures such as the protein shell or the inner RubisCO matrix are not identical between carboxysome types. Each type has evolutionarily distinct forms of the same proteins, as well as proteins that are entirely unrelated to one another. In light of recent developments in the study of carboxysome structure and function, we present this review to summarize the knowledge of the structure and function of both types of carboxysome. We also endeavor to cast light on differing evolutionary trajectories which may have led to the differences observed in extant carboxysomes.

Cyanobacterial carboxysomes: microcompartments that facilitate CO2 fixation.

Carboxysomes are extraordinarily efficient proteinaceous microcompartments that encapsulate the primary CO2-fixing enzyme (ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBisCO) in cyanobacteria and some proteobacteria. These microbodies form part of a CO2-concentrating mechanism (CCM), operating together with active CO2 and HCO3(-) uptake transporters which accumulate HCO3(-) in the cytoplasm of the cell. Cyanobacteria (also known as blue-green algae) are highly productive on a global scale, especially those species from open-ocean niches, which collectively contribute nearly 30% of global net primary fixation. This productivity would not be possible without a CCM which is dependent on carboxysomes. Two evolutionarily distinct forms of carboxysome are evident that encapsulate proteobacterial RuBisCO form-1A or higher-plant RuBisCO form- 1B, respectively. Based partly on RuBisCO phylogeny, the two carboxysome types are known either as α-carboxysomes, found in predominantly oceanic cyanobacteria (α-cyanobacteria) and some proteobacteria, or as ß-carboxysomes, found mainly in freshwater/estuarine cyanobacteria (ß-cyanobacteria). Both carboxysome types are believed to have evolved in parallel as a consequence of fluctuating atmospheric CO2 levels and evolutionary pressure acting via the poor enzymatic kinetics of RuBisCO. The three-dimensional structures and protein components of each carboxysome type reflect distinct evolutionarily strategies to the same major functions: subcellular compartmentalization and RuBisCO encapsulation, oxygen exclusion, and CO2 concentration and fixation.

Structural determinants of the outer shell of ß-carboxysomes in Synechococcus elongatus PCC 7942: roles for CcmK2, K3-K4, CcmO, and CcmL.

Cyanobacterial CO(2)-fixation is supported by a CO(2)-concentrating mechanism which improves photosynthesis by saturating the primary carboxylating enzyme, ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), with its preferred substrate CO(2). The site of CO(2)-concentration is a protein bound micro-compartment called the carboxysome which contains most, if not all, of the cellular RuBisCO. The shell of ß-type carboxysomes is thought to be composed of two functional layers, with the inner layer involved in RuBisCO scaffolding and bicarbonate dehydration, and the outer layer in selective permeability to dissolved solutes. Here, four genes (ccmK2-4, ccmO), whose products were predicted to function in the outer shell layer of ß-carboxysomes from Synechococcus elongatus PCC 7942, were investigated by analysis of defined genetic mutants. Deletion of the ccmK2 and ccmO genes resulted in severe high-CO(2)-requiring mutants with aberrant carboxysomes, whilst deletion of ccmK3 or ccmK4 resulted in cells with wild-type physiology and normal ultrastructure. However, a tandem deletion of ccmK3-4 resulted in cells with wild-type carboxysome structure, but physiologically deficient at low CO(2) conditions. These results revealed the minimum structural determinants of the outer shell of ß-carboxysomes from this strain: CcmK2, CcmO and CcmL. An accessory set of proteins was required to refine the function of the pre-existing shell: CcmK3 and CcmK4. These data suggested a model for the facet structure of ß-carboxysomes with CcmL forming the vertices, CcmK2 forming the bulk facet, and CcmO, a "zipper protein," interfacing the edges of carboxysome facets.

Over-expression of the ß-carboxysomal CcmM protein in Synechococcus PCC7942 reveals a tight co-regulation of carboxysomal carbonic anhydrase (CcaA) and M58 content.

Carboxysomes, containing the cell's complement of RuBisCO surrounded by a specialized protein shell, are a central component of the cyanobacterial CO(2)-concentrating mechanism. The ratio of two forms of the ß-carboxysomal protein CcmM (M58 and M35) may affect the carboxysomal carbonic anhydrase (CcaA) content. We have over-expressed both M35 and M58 in the ß-cyanobacterium Synechococcus PCC7942. Over-expression of M58 resulted in a marked increase in the amount of this protein in carboxysomes at the expense of M35, with a concomitant increase in the observed CcaA content of carboxysomes. Conversely, M35 over-expression diminished M58 content of carboxysomes and led to a decrease in CcaA content. Carboxysomes of air-grown wild-type cells contained slightly elevated CcaA and M58 content and slightly lower M35 content compared to their 2% CO(2)-grown counterparts. Over a range of CcmM expression levels, there was a strong correlation between M58 and CcaA content, indicating a constant carboxysomal M58:CcaA stoichiometry. These results also confirm a role for M58 in the recruitment of CcaA into the carboxysome and suggest a tight regulation of M35 and M58 translation is required to produce carboxysomes with an appropriate CA content. Analysis of carboxysomal protein ratios, resulting from the afore-mentioned over-expression studies, revealed that ß-carboxysomal protein stoichiometries are relatively flexible. Determination of absolute protein quantities supports the hypothesis that M35 is distributed throughout the ß-carboxysome. A modified ß-carboxysome packing model is presented.

The CO2-concentrating mechanism of Synechococcus WH5701 is composed of native and horizontally-acquired components.

The cyanobacterial CO(2)-concentrating mechanism (CCM) is an effective adaptation that increases the carbon dioxide (CO(2)) concentration around the primary photosynthetic enzyme Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO). α-Cyanobacteria (those containing Form1-A RuBisCO within cso-type α-carboxysomes) have a limited CCM composed of a small number of Ci-transporters whereas ß-cyanobacteria (those species containing Form-1B RuBisCO within ccm-type ß-carboxysomes) exhibit a more diverse CCM with a greater variety in Ci-transporter complement and regulation. In the coastal species Synechococcus sp. WH5701 (α-cyanobacteria), the minimal α-cyanobacterial CCM has been supplemented with ß-cyanobacterial Ci transporters through the process of horizontal gene transfer (HGT). These transporters are transcriptionally regulated in response to external Ci-depletion however this change in transcript abundance is not correlated with a physiological induction. WH5701 exhibits identical physiological responses grown at 4% CO(2) (K (1/2) ≈ 31 µM Ci) and after induction with 0.04% CO(2) (K (1/2) ≈ 29 µM Ci). Insensitivity to external Ci concentration is an unusual characteristic of the WH5701 CCM which is a result of evolution by HGT. Our bioinformatic and physiological data support the hypothesis that WH5701 represents a clade of α-cyanobacterial species in transition from the marine/oligotrophic environment to a coastal/freshwater environment.