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

Abstract

Microbes of the order Sulfolobales are potentially next generation platforms for cellulose degradation and utilization. However, the mechanisms of cellulose degradation and utilization remain unclear. In this study, we analyzed the enzymatic activity, localization, transcriptional regulation, and interplay of three endogenous cellulases Cel1, Cel2A, and LacS, and the association of transcription with cellular processes in the model thermophilic archaeon Saccharolobus islandicus REY15A. Overexpression strains based on a vector developed in this study as well as single and double deletion mutants of the three cellulase genes were constructed and analyzed. We reveal that Cel1 and Cel2A are membrane-associated, and a higher proportion of Cel2A is secreted into the medium than Cel1. The expression of all the three cellulases is induced by carboxymethylcellulose sodium (CMC). Furthermore, we found that the transcriptional levels of cel1, cel2A, and lacS are interdependent and cel1 and cel2A levels apparently increased with lacS deletion and overexpression of an ABC transporter, suggesting of an intracellular oligosaccharide-dependent regulatory mechanism. We showed that a cell cycle transcription factor aCcr1 binds to the promoter of cel1 in vitro. Additionally, we accidentally found that the cellulase genes expression increases in the presence of uracil synthesis pathway and deletion of the cellulase genes facilitates cell growth in CMC-containing medium. This study reveals an ingenious and intricated mechanism for cellulose utilization to adapt to extreme environment in archaea and provide insights for engineering Sulfolobales archaea as biomass-degrading and utilization platforms.