Fig. 3: Construction of a hydrogenase-containing nanoreactor based on the carboxysome shell.

a Generation of the hyd operon to produce [FeFe]-hydrogenase HydA fused with ferredoxin (Fd) from the green alga Chlamydomonas, ferredoxin:NADP⁺-oxidoreductase (FNR) from E. coli, and the HydA maturases HydE, HydF, and HydG. The hyd and cso-2 plasmids were co-expressed to form a hydrogenase-containing nanoreactor based on the carboxysome shell (Shell-HydA). b Schematic of the Shell-HydA nanoreactor encapsulating Fd-HydA and FNR. The nanoreactors were tested for hydrogen production activity using endogenous NADPH in cells as the electron donor for in vivo assays, and for in vitro assays, using methyl viologen (MV⁺) as an electron donor, which were chemically reduced by sodium dithionite. c Western blot of E. coli expressing the hyd operon alone or Shell-HydA confirms the presence of Fd-HydA, FNR, and shell proteins in the samples purified from sucrose gradient ultracentrifugation, indicating the assembly of Shell-HydA. d Transmission EM of empty shells (left) and Shell-HydA (right). Yellow arrows indicate cargo proteins (Fd-HydA, FNR) in the luminal side of the shell. e In vivo hydrogenase activity assays. H₂ production (nmol L⁻¹ h⁻¹) of E. coli cells expressing free HydA or Shell-HydA grown under anaerobic (left) or aerobic (right) conditions was measured using gas chromatography. Values represent mean ± standard deviations (s.d.), n = 3 biologically independent experiments. *p = 0.0352 (left), **p = 0.0018 (right) (two-tailed unpaired t-test). Source data underlying c–e are provided as a Source data file.