Abstract
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.
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Acknowledgments
We would like to thank Dr. Adam Guss for his suggestions and input on the manuscript.
This research was supported by a grant from the BioEnergy Science Center (BESC), Oak Ridge National Laboratory, a US Department of Energy (DOE) BioEnergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.
Portions of this research were performed during an internship at the Mascoma Corporation. We would like to thank the Mascoma Corporation for their generous gift of strain M1726 and for their support during the duration of this work.
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Mearls, E.B., Olson, D.G., Herring, C.D. et al. Development of a regulatable plasmid-based gene expression system for Clostridium thermocellum . Appl Microbiol Biotechnol 99, 7589–7599 (2015). https://doi.org/10.1007/s00253-015-6610-5
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DOI: https://doi.org/10.1007/s00253-015-6610-5