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

Abstract

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.

Self-Assembly Stability and Variability of Bacterial Microcompartment Shell Proteins in Response to the Environmental Change

“…Given the self-assembly of BMC structures, there is a significant interest in engineering BMCs and design of new BMC-based nanobioreactors, molecular scaffolds, and biomaterials in biotechnology applications, for example, enhancing cell metabolism, enzyme encapsulation, molecular delivery, and therapy. Advanced knowledge about the structural resilience and variability of BMCs in response to environmental changes will not only inform strategies for producing robust BMC-based nanostructures in heterologous hosts, i.e., E. coli or plants [31,53,54], but also pave the way for modulating the formation of 2D nanomaterials as well as the opening and closure of BMC shell-based protein cages, thereby facilitating the functional regulation and targeted molecular delivery. Previously, we have demonstrated the feasibility of using genetic modification approach to manipulate the specific contacts at the interfaces of shell proteins and their self-assembly behaviors [12].…”

Electrical Energy Storage with Engineered Biological Systems

“…Given these limitations, other carbon fixation cycles found in nature could be attractive ( Table 3). It is conceivable, given recent advances in compartmentalization in synthetic biology [115,116] that highly efficient pathways like the Wood-Ljungdahl pathway that require high CO2 concentrations could be implemented under atmospheric CO2 concentrations in rewired carbon fixation organisms using synthetic carbon concentrating compartments or heterologously expressed carboxysomes [117].…”

Engineering of Metabolic Pathways Using Synthetic Enzyme Complexes

“…YFP, tagged with a small targeting peptide from the carboxysome-organizing protein, CcmN, was incorporated into the shells. In a recent breakthrough, a minimal functional carboxysome was expressed in tobacco chloroplasts (Long et al, 2018). This result was achieved by introducing two a-carboxysome coat proteins and the large and small subunits from the cyanobacterium Cyanobium.…”