Improvement of arabinoxylan degradation in Clostridium saccharobutylicum DSM 13864T fermentations by heterologous glycoside hydrolase supplementation and expression

Solventogenic Clostridium species can efficiently produce n-butanol and other valuable chemicals via acetone-butanol-ethanol (ABE) fermentation from plant-based feedstocks. For economic and ecological sustainability, cheap and abundant substrates such as lignocellulosic and hemicellulosic residues from agricultural or forestry side streams are preferable. Cereal brans, rich in hemicellulose, represent a promising substrate. However, for direct fermentation of this material, only low product titers are reported. In this study, we characterized the utilization of arabinoxylan, the main polysaccharide component of cereal bran, by the industrial ABE producer Clostridium saccharobutylicum DSM 13864^T and report inefficient degradation of the substrate. Supplementation with hemicellulolytic enzyme mixtures derived from the thermophilic organism Thermoclostridium stercorarium subsp. stercorarium DSM 8532^T significantly enhanced substrate utilization. The best improvement was achieved by the addition of the arabinofuranosidase Axh43A, which reduced the residual sugar content in the fermentation broth from 48.2 to 17.8%. Analysis of the remaining oligosaccharides after growth on arabinoxylan showed that C. saccharobutylicum cannot remove O-2 and O-3 α-L-arabinofuranosyl groups from double-substituted xyloses, creating a key bottleneck in arabinoxylan degradation that is overcome by Axh43A addition. Plasmid-based expression of Axh43A in C. saccharobutylicum DSM 13864^T replicated the enzymatic supplementation effects, confirming the enzyme's role in overcoming this limitation. This underscores the potential of genetic engineering to enhance the valorization of lignocellulosic biomass in biotechnological fermentation processes. KEY POINTS : C. saccharobutylicum DSM 13864^T insufficiently utilizes cereal arabinoxylan. Inability to cleave double-arabinosylated xylose moieties limits degradation. Heterologous expression of Axh43A strongly increases arabinoxylan utilization.