Sugar-inducible promoters for manipulation of core metabolic pathways in the thermophilic acetogen Thermoanaerobacter kivui.

UNLABELLED: Acetogenic bacteria form an essential part of the global carbon cycle and show promise for green chemicals production thanks to their energy-efficient carbon fixation via the Wood-Ljungdahl pathway. Energy conservation in acetogens is dependent on one of two ion-pumping membrane complexes, Rnf or Ech. While the energetics of Rnf acetogens are relatively well understood, Ech-dependent metabolism remains unclear. Like many Ech acetogens, Thermoanaerobacter kivui (Topt = 66°C) encodes two separate Ech complexes and is genetically tractable, allowing it to serve as a model of Ech-dependent metabolism. While the role of the Ech2 complex has been determined biochemically and by gene knockout, Ech1 has eluded biochemical characterization, and knockout attempts have been unsuccessful, suggesting it is essential. This work identifies two sugar-inducible promoters with very low expression under non-inducing conditions and uses them to replace the native Ech1 promoter. The resulting strains exhibit reduced growth in non-inducing "knockdown" conditions, confirming the importance of Ech1 for metabolism. Additionally, knockdown of the gene encoding formyl-THF synthetase (Fhs)-which consumes the metabolic intermediate formate-resulted in significantly more formate accumulating in the culture medium. Together, these results confirm that the characterized promoters can be used to elucidate the function of essential genes not amenable to knockout.

IMPORTANCE: Acetogenic bacteria are industrially relevant for conversion of synthesis gas (syngas, a mixture of hydrogen, H2; carbon monoxide, CO; and carbon dioxide, CO2) to products. Thermophilic bacteria have long been of interest as industrial strains due to their resistance to contamination. Thermoanaerobacter kivui is among the most thermophilic acetogens (Topt = 66°C), with very short doubling times on H2+CO2 (< 2 h), and is therefore a prime candidate for synthesis gas conversion at high temperatures. Here, we expanded T. kivui's genetic toolkit by establishing a reporter gene operating at its temperature optimum, which was used to characterize sugar-inducible promoters. The identified promoters were used to control and engineer the metabolism of T. kivui, and may now be applied to elucidate remaining mysteries about the energetics of acetogenic metabolism.