Transformable Facultative Thermophile Geobacillus Stearothermophilus NUB3621 As a Host Strain for Metabolic Engineering

Protein engineering requires reliable expression of the protein by host cells. Engineering can be impeded by the misfolding of heterologously expressed proteins. Geobacillus stearothermophilus NUB3621 is a readily transformable facultative thermophile. It could be used to express and properly fold proteins derived from its many mesophilic or thermophilic Bacillaceae relatives or to direct the evolution of thermophilic variants of mesophilic proteins. Moreover, its capacity for high-temperature growth should accelerate chemical transformation rates in accordance with the Arrhenius equation and reduce the risks of microbial contamination. Its tendency to sporulate in response to nutrient depletion lowers the costs of storage and transportation. We have developed a draft genome sequence of G. stearothermophilus NUB3621 as well as inducible and constitutive expression plasmids that function in this organism. Utilizing these tools, we plan to evolve aminoglycoside phosphotransferase enzymes to be thermostable. We will then determine whether the thermostable variants are better able to adapt to a novel substrate, amikacin, than their mesophilic counterparts.