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The thermophilic bacterium Thermus thermophilus (Tth) is an obligate aerobe with many important biotechnological applications and products. Thermostable proteins isolated from Thermus species have proven important in understanding adaptation to thermophilic environments. Thermophilic adaptations of respiratory electron transport chains (ETC) are not well characterized. Conventional homology-based, genetic, and biochemical techniques are limited in their ability to identify thermophilic Tth homologues of mesophilic respiratory ETC proteins due to the lack of a mutable facultative platform. We are attempting to generate this platform by increasing glycolytic flux through metabolic engineering techniques. Based on physiological studies, Tth HB27 demonstrates a low affinity for monosaccharide substrates, which limits glycolytic flux. We identify growth conditions suitable to increasing metabolic flux, as well as limitations to our current method for engineering a Tth mutable facultative platform. We also propose future direction in order to generate anaerobic derivatives of Tth HB27. 3

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