Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts.

The ATP-driven bicarbonate transporter 1 (BCT1), a four-component complex in the cyanobacterial CO2-concentrating mechanism, could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis.

A carboxysome-based CO2 concentrating mechanism for C3 crop chloroplasts: advances and the road ahead.

The introduction of the carboxysome-based CO2 concentrating mechanism (CCM) into crop plants has been modelled to significantly increase crop yields. This projection serves as motivation for pursuing this strategy to contribute to global food security. The successful implementation of this engineering challenge is reliant upon the transfer of a microcompartment that encapsulates cyanobacterial Rubisco, known as the carboxysome, alongside active bicarbonate transporters. To date, significant progress has been achieved with respect to understanding various aspects of the cyanobacterial CCM, and more recently, different components of the carboxysome have been successfully introduced into plant chloroplasts. In this Perspective piece, we summarise recent findings and offer new research avenues that will accelerate research in this field to ultimately and successfully introduce the carboxysome into crop plants for increased crop yields.

Plant-based carboxysomes: another step toward increased crop yields.

Synthetically reconstructed carboxysomes form the basis of CO2-concentrating mechanisms (CCMs) that could enhance the photosynthetic efficiency of crops and improve yield. Recently, Chen et al. revealed another step toward the reconstruction of bacterial carboxysomes in plants, reporting the formation of almost-complete carboxysomes in the chloroplast of Nicotiana tabacum.

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.