Development of a regulatable plasmid-based gene expression system for Clostridium thermocellum.

Clostridium thermocellum can rapidly solubilize cellulose and produces ethanol as an end product of its metabolism. As such, it is a candidate for bioethanol production from plant matter. In this study, we developed an inducible expression system for C. thermocellum based on its native celC operon. We enhanced expression over the native operon structure by placing the repressor gene, glyR3, immediately after the celC promoter, and expressing the target gene after glyR3. Upon the addition of the inducer substrate, laminaribiose, an approximately 40-fold increase in gene expression was obtained using the test gene spo0A. Furthermore, induction of the sporulation histidine kinase, clo1313_1942, increased sporulation frequency by approximately 10,000-fold relative to an uninduced control. We have also shown that the laminaribiose (β1-3-linked carbon source) utilization pathway is not catabolite repressed by cellobiose, a β1-4-linked carbon source frequently used for C. thermocellum cultivation in laboratory conditions. Selective expression of target genes has the potential to inform metabolic engineering strategies as well as increase fundamental understanding of C. thermocellum biology.