(188bf) Thermostable Laci for Inducible Expression in Geobacilli

Geobacilli are a genus of thermophilic bacteria that are able to grow at 45-70 °C. Isolated from diverse geothermal environments, many of them have demonstrated great potential for high-temperature bioprocessing, such as biofuel production, bioremediation, and biodegradation. Although the genome sequences of many Geobacillus strains with attractive features are available, the study and development of Geobacillus strains are significantly hindered due to the lack of genetic tools, such as thermostable inducible expression systems. The use of regulators of sugar metabolism from Geobacilli has been reported but it inevitably would interfere with the cellular metabolic network. The commonly used IPTG-LacI inducible system is not applicable in Geobacillus due to the deactivation of LacI at high temperatures. A mutant of LacI replacing the positively charged lysine (K84) to hydrophobic residues demonstrates enhanced thermostability in vitro, but its functionality has not been examined. In this study, we compared the function of three mutants of LacI with the wildtype LacI in both Geobacillus thermoglucosidasius and Escherichia coli using a thermostable sfGFP as the reporter. Our results show that these LacI mutants can be used as regulators for inducible expression in thermophilic bacteria and provides important tools for the research and engineering of thermophilic bacteria.