PT - JOURNAL ARTICLE AU - Anthony Lanahan AU - Kamila Zakowicz AU - Liang Tian AU - Daniel G. Olson AU - Lee R. Lynd TI - A single nucleotide change in the <em>polC</em> DNA polymerase III in <em>Clostridium thermocellum</em> is sufficient to create a hypermutator phenotype AID - 10.1101/2021.07.30.454558 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.07.30.454558 4099 - http://biorxiv.org/content/early/2021/07/31/2021.07.30.454558.short 4100 - http://biorxiv.org/content/early/2021/07/31/2021.07.30.454558.full AB - Clostridium thermocellum is a thermophilic, anaerobic, bacterium that natively ferments cellulose to ethanol, and is a candidate for cellulosic biofuel production. Recently, we identified a hypermutator strain of C. thermocellum with a C669Y mutation in the polC gene, which encodes a DNA polymerase III enzyme. Here we reintroduce this mutation using recently-developed CRISPR tools to demonstrate that this mutation is sufficient to recreate the hypermutator phenotype. The resulting strain shows an approximately 30-fold increase in the mutation rate. This mutation appears to function by interfering with metal ion coordination in the PHP domain responsible for proofreading. The ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments.Importance Cellulosic biofuels are a promising approach to decarbonize the heavy duty transportation sector. A longstanding barrier to cost-effective cellulosic biofuel production is the recalcitrance of the material to solubilization. Native cellulose-consuming organisms, such as Clostridium thermocellum, are promising candidates for cellulosic biofuel production, however they often need to be genetically modified to improve product formation. One approach is adaptive laboratory evolution. Our findings demonstrate a way to increase the mutation rate in this industrially-relevant organism, which can reduce the time needed for adaptive evolution experiments.