Engineering and Modulating Functional Cyanobacterial CO2-Fixing Organelles

Abstract

Bacterial microcompartments (BMCs) are proteinaceous organelles widespread among bacterial phyla and provide a means for compartmentalizing specific metabolic pathways. They sequester catalytic enzymes from the cytoplasm, using an icosahedral proteinaceous shell with selective permeability to metabolic molecules and substrates, to enhance metabolic efficiency. Carboxysomes were the first BMCs discovered and their unprecedented capacity of CO2 fixation allows cyanobacteria to make a significant contribution to global carbon fixation. There is an increasing interest in utilizing synthetic biology to construct synthetic carboxysomes in new hosts, i.e., higher plants, to enhance carbon fixation and productivity. Here, we report the construction of a synthetic operon of the β-carboxysome from the cyanobacterium Synechococcus elongatus PCC7942 to generate functional β-carboxysome-like structures in Escherichia coli. The protein expression, structure, assembly, and activity of synthetic β-carboxysomes were characterized in depth using confocal, electron and atomic force microscopy, proteomics, immunoblot analysis, and enzymatic assays. Furthermore, we examined the in vivo interchangeability of β-carboxysome building blocks with other BMC components. To our knowledge, this is the first production of functional β-carboxysome-like structures in heterologous organisms. It provides important information for the engineering of fully functional carboxysomes and CO2-fixing modules in higher plants. The study strengthens our synthetic biology toolbox for generating BMC-based organelles with tunable activities and new scaffolding biomaterials for metabolic improvement and molecule delivery.

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment

“…Precise quantification of the protein stoichiometry and organizational regulation of carboxysomes provides insight into their assembly principles, structure and function. In this work, we functionally Despite prior efforts on understanding carboxysome structure and function, the relative stoichiometry of functional carboxysome components in their native cell environment − key information required for reconstituting entire active carboxysome structures in synthetic biology (Fang et al, 2018), was still unclear. The major challenges have been the poor specificity of immunoblot and mass spectrometry, given the homology of carboxysome proteins and the lack of effective purification of intact carboxysomes from host cells, as well as the heterogeneity of carboxysome structures (Long et al, 2005).…”

“…Given their selfassembly, modularity and high efficiency in enhancing carbon fixation, carboxysomes have attracted tremendous interest to engineering this CO2-fixing organelle into other organisms, for example C3 plants, with the intent of increasing photosynthetic efficiency and crop production (Lin et al, 2014b;Lin et al, 2014a;Occhialini et al, 2016;Long et al, 2018). Recently, we have reported the engineering of functional β-carboxysome structures in E. coli -a step towards constructing functional βcarboxysomes in eukaryotic organisms (Fang et al, 2018). Our present study, by evaluating the actual protein stoichiometry and structural variability of native β-carboxysomes, sheds light on the molecular basis underlying the assembly, formation and regulation of functional carboxysomes.…”

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

“…The BMC shell, consisting of numerous protein homologs, is an ideal system for studying protein self-assembly and interactions. As a powerful technique for analyzing biomembrane organization, protein assembly, and physical interactions that are highly relevant to the physiological roles of biological systems [32,35,38,39], AFM has been exploited to visualize the organization and self-assembly dynamics of BMC shell proteins and the architectures and mechanical features of BMC structures [12,30,31,[40][41][42]. This work represents, to our knowledge, the first quantitative determination of the self-assembly dynamics of BMC shell proteins in the formation of two-dimensional sheets in response to environmental changes using AFM.…”

“…However, some key issues remain to be tackled in BMC bioengineering, for example, how stable the BMC structures are and how to manipulate and assess effectively the self-assembly and formation of BMC protein aggregates. Investigations of the structures and assembly of BMC shells and entire BMCs have been carried out using X-ray crystallography, electron microscopy (EM), fluorescence microscopy, and dynamic light scattering (DSL) [10,11,16,22,[27][28][29][30][31]. Recently, we have exploited high-speed AFM (HS-AFM) to conduct the first visualization of the dynamic self-assembly process of BMC-H proteins [12].…”

Occurrence and stability of hetero-hexamer associations formed by β-carboxysome CcmK shell components

“…processes by concentrating the enzymes and substrates in a limited volume, and also by sequestering toxic or volatile reaction intermediates. These properties, their natural diversity, the possibility to reprogram BMC contents by means of targeting peptides [4][5][6], the modularity evidenced by the fact that bricks from different BMC could be assembled together [7,8], as well as the possibility to reconstitute BMC in recombinant hosts by operon transfer [9][10][11][12], justify the strong interest for BMC as prototypes for engineering future nano-reactors for synthetic biology purposes.An intense effort has been devoted to the structural characterization of BMC [1]. Information for individual components was largely obtained by means of X-ray crystallography.…”