Improving genetic tools in Clostridium thermocellum

Clostridium thermocellum is a model organism for consolidated bioprocessing (CBP) because of its anaerobic, cellulolytic, ethanol producing capabilities. However, it produces ethanol at lower titers and yields than are required for industrial application. In order to optimize ethanol production, targeted genetic engineering must be done to direct carbon flux solely to ethanol. While some genetic engineering has been done successfully in C. thermocellum DSM 1313, the efficiency is still extremely low.  E. coli dam methylation has previously been shown to improve transformation efficiency for C. thermocellum DSM 1313 substantially, and we hypothesize that another Restriction-Methylase system is present and causing restriction of foreign DNA. We are using a systematic approach to determine the methylation patterns native to the organism, then, to overcome restriction, we have deleted two of the restriction enzyme genes, and we have expressed the methyltransferase genes in E. coli for in vivo DNA methylation. We believe that these strategies will significantly improve transformation efficiency. We are using the same strategies in the strain C. thermocellum 27405 which has previously proven much more challenging to transform than DSM 1313. The methodologies applied here will provide insight into many challenging industrial organisms.