Roles and transcriptional regulation of the endogenous cellulases in association with substrate and cellular processes in a thermoacidophilic archaeon.

Microbes of the order Sulfolobales represent promising next-generation platforms for cellulose degradation and utilization. However, the mechanism of cellulose degradation and utilization in these microorganisms remains unclear. In this study, the enzymatic activity, localization, transcriptional regulation, and interactions of three endogenous cellulases, Cel1, Cel2A, and LacS, along with the association between transcription and cellular processes, were analyzed in the model thermophilic archaeon Saccharolobus islandicus REY15A. Overexpression strains using a vector developed in this study, along with single and double deletion mutants of the three cellulase genes, were constructed and characterized. Our findings demonstrate that Cel1 and Cel2A are membrane-associated, with a higher proportion of Cel2A being secreted into the culture medium compared to Cel1. The expression of all three cellulases was induced by carboxymethylcellulose sodium (CMC). Additionally, we determined that the transcriptional levels of cel1, cel2A, and lacS are interdependent, with cel1 and cel2A levels being significantly elevated upon lacS deletion and overexpression of an ABC transporter, indicating an intracellular oligosaccharide-dependent regulatory mechanism. We demonstrated that the cell cycle transcription factor aCcr1 binds to the cel1 promoter in vitro. Furthermore, we observed that cellulase gene expression is upregulated in the presence of the uracil synthesis pathway, while deletion of the cellulase genes promotes cell growth in CMC-containing medium. This study elucidates an ingenious and intricate mechanism of cellulose utilization for adapting to extreme environments in archaea and provides insights for engineering Sulfolobales archaea as biomass-degrading and utilization platforms.