Abstract
Compartmentalization is a ubiquitous building principle in cells, which permits segregation of biological elements and reactions. The carboxysome is a specialized bacterial organelle that encapsulates enzymes into a virus-like protein shell and plays essential roles in photosynthetic carbon fixation. The naturally designed architecture, semi-permeability, and catalytic improvement of carboxysomes have inspired rational design and engineering of new nanomaterials to incorporate desired enzymes into the protein shell for enhanced catalytic performance. Here, we build large, intact carboxysome shells (over 90 nm in diameter) in the industrial microorganism Escherichia coli by expressing a set of carboxysome protein-encoding genes. We develop strategies for enzyme activation, shell self-assembly, and cargo encapsulation to construct a robust nanoreactor that incorporates catalytically active [FeFe]-hydrogenases and functional partners within the empty shell for the production of hydrogen. We show that shell encapsulation and the internal microenvironment of the new catalyst facilitate hydrogen production of the encapsulated oxygen-sensitive hydrogenases. The study provides insights into the assembly and formation of carboxysomes and paves the way for engineering carboxysome shell-based nanoreactors to recruit specific enzymes for diverse catalytic reactions.
Highlights
Compartmentalization is a ubiquitous building principle in cells, which permits segregation of biological elements and reactions
Distinct from the ‘inside out’ de novo assembly of βcarboxysomes[31,32,33], the assembly of α-carboxysomes appears to start from shell formation[34] or a simultaneous shell-interior assembly[35], highlighting the possibility of generating and reprogramming entire α-carboxysome shells
The α-carboxysome proteins of the chemoautotrophic bacterium Halothiobacillus neapolitanus are mostly encoded by genes that are located in a single cso operon, including cbbL and cbbS that encode the large and small subunits of Rubisco, respectively, and genes that encode shell proteins (Fig. 1b)
Summary
Generation of an entire synthetic α-carboxysome shell. The α-carboxysome proteins of the chemoautotrophic bacterium Halothiobacillus neapolitanus are mostly encoded by genes that are located in a single cso operon, including cbbL and cbbS that encode the large and small subunits of Rubisco, respectively, and genes that encode shell proteins (Fig. 1b). We generated the synthetic cso-1 and cso-2 operons, which were modified based on the native H. neapolitanus cso operon, to heterologously express α-carboxysome shell proteins in E. coli BL21(DE3) (Fig. 1b). The cso-1 operon contains the genes encoding carboxysome shell proteins (csoS2, csoS4AB, csoS1CAB, csoS1D), as well as csoSCA that encodes β-carbonic anhydrase (CA)[38]. The cso-2 operon comprises only the shell protein-encoding genes (csoS2, csoS4AB, csoS1CAB, and csoS1D), without csoSCA. Mass spectrometry further showed the presence of all seven shell proteins and CA encoded by the cso-1 operon and seven shell proteins encoded by the cso-2 operon in the 20% sucrose fraction (Supplementary Tables 1 and 2), confirming the self-assembly of expressed carboxysome shell proteins to form shell supramolecular structures
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