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

Abstract

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.

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

“…The expression of structurally intact carboxysomes within a C 3 plant chloroplast is a critical and complex engineering milestone towards the longer-term goal of attaining a functional chloroplastic CCM in C 3 crop plants 4,5 . The structures reported here mimic the gross structure of carboxysomes from Cyanobium but lack specific components expected to be required for full functionality in an operating CCM (viz.…”

“…We hypothesize that inclusion of one or all these factors should enhance the observed Rubisco catalysis to that found for Cyanobium carboxysomes. Beyond the construction of a functional carboxysome, a working CCM is ultimately dependent upon transport proteins, which can boost stromal HCO 3 − concentrations to levels that enable carboxysome operation 5 . Recent advances in correct localization of inorganic carbon pumps to the chloroplast inner membrane 60,61 provide encouraging progress towards the achievement of this goal.…”

“…In combination, a high stromal HCO 3 − pool generated by active HCO 3 − transporters and a fully functional carboxysome where CO 2 can be elevated could improve C 3 plant CO 2 fixation and yield up to 60% 15,20 . This improvement would provide savings in energy costs for the plant and both nitrogen and carbon investment in the CO 2 fixation machinery 5 . Elimination of the native stromal CA and C 3 Rubisco to further improve the accumulation of HCO 3 − within the stroma is required to realize an optimal functioning chloroplastic CCM 4,6,16 .…”

“…Additionally, transient expression studies showed that carboxysome shell proteins could interact and form structures within chloroplasts 26 . However, these attempts could not generate structural carboxysomes nor encapsulate Rubisco, key requirements to generate CO 2 around Rubisco and for overall CCM functionality 5 . While carboxysomes have been heterologously expressed in bacterial systems 27,28 , there are currently no reports of α- or β-carboxysome biogenesis in eukaryotic systems.…”

Optimizing photorespiration for improved crop productivity

“…In pyrenoid formation, Rubisco recruitment is mediated through a-helices contained on the small subunit of the Form 1 Rubisco, that are predicted to interact with the linking protein present in the pyrenoid (Meyer et al 2012;Mackinder et al 2016). Engineering native smallsubunits through direct replacement of the two surface a-helices from Chlamydomonas reinhardtii results in Rubiscos that are catalytically competent and represent an ideal background to test candidates for new recruitment and linker proteins as they emerge (Atkinson et al 2017).…”