Humans have long utilized cellulose II, known as regenerated cellulose, for fibers like rayon and Cupra and films like cellophane. While cellulose I, found in nature, consists of parallel molecular chains, cellulose II is characterized by the stable arrangement of molecules in an antiparallel orientation. Enzymatic synthesis of cellulose in vitro also affords cellulose II with various morphologies, from monolayer lamellae crystals to gels, but its formation mechanism remains obscure. Here, we demonstrate that cellodextrin phosphorylase (CDP) catalyzes the synthesis and orchestrates the antiparallel self-assembly of cellulose II nanocrystals, exceeding the paradigm of spontaneous crystallization. High-resolution structural analysis reveals CDP's key role in dictating crystal size and alignment, bridging the gap between enzymatic catalysis and biodirected material architecture. Our research unveils a unique protein-templated assembly process for advanced cellulose materials, paving the way for enzyme-guided construction of next-generation functional nanostructures.

Cellodextrin phosphorylase (CDP) plays a key role in energy-efficient cellulose metabolism of anaerobic bacteria by catalyzing phosphorolysis of cellodextrin to produce cellobiose and glucose 1-phosphate, which can be utilized for glycolysis without consumption of additional ATP. As the enzymatic phosphorolysis reaction is reversible, CDP is also employed to produce cellulosic materials in vitro. However, the enzyme is rapidly inactivated by oxidation, which hinders in vitro utilization in aerobic environments. It has been suggested that the cysteine residues of CDP, which do not form disulfide bonds, are responsible for the loss of activity, and the aim of the present work was to test this idea. For this purpose, we replaced all 11 free cysteine residues of CDP from Acetivibrio thermocellus (formerly known as Clostridium thermocellum) with serine, which structurally resembles cysteine in our previous work. Herein, we show that the resulting CDP variant, named CDP-CS, has comparable activity to the wild-type enzyme, but shows increased stability to oxidation during long-term storage. X-Ray crystallography indicated that the mutations did not markedly alter the overall structure of the enzyme. Ensemble refinement of the crystal structures of CDP and CDP-CS indicated that the C372S and C625S mutations reduce structural fluctuations in the protein main chain, which may contribute to the increased stability of CDP-CS to oxidation.

We have developed passive devices for conducting two-step enzyme reactions with a time lag. They are "n-paraffin separator device" and "osmotic timer device." We evaluated the feasibility of these devices using two reactions, cellulose synthesis and cellulose decomposition, catalyzed by different enzymes. We have verified that the n-paraffin separator device works well for the cellulose synthesis in space and the osmotic pressure timer device releases the cellulase with a time lag. This paper further reports that the devices functioned autonomously and carried out the two-step enzyme reactions in the International Space Station.

We previously reported in vitro synthesis of highly ordered crystalline cellulose II by reverse reaction of cellodextrin phosphorylase from the cellulolytic bacterium Clostridium (Hungateiclostridium) thermocellum (CtCDP), but the formation mechanism of the cellulose crystals and highly ordered structure has long been unclear. Considering the specific density of cellulose versus water, the formation of crystalline and highly ordered structure in an aqueous solution should be affected by gravity. Thus, we synthesized cellulose with CtCDP stable variant at the International Space Station, where sedimentation and convection due to gravity are negligible. Optical microscopic observation suggested that cellulose in space has a gel-like appearance without apparent aggregation, in contrast to cellulose synthesized on the ground. Small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) indicated that cellulose synthesized in space has a more uniform particle distribution in the ~ 100 nm scale region than cellulose synthesized on the ground. Scanning electron microscopy (SEM) showed that both celluloses have a micrometer scale network structure, whereas a fine fiber network was constructed only under microgravity. These results indicate that gravity plays a role in cellulose II crystal sedimentation and the building of network structure, and synthesis in space could play a role in designing unique materials.