Development of a thermophilic l-arabinose-inducible system in Acetivibrio thermocellus (Clostridium thermocellum)

Inducible genetic operation systems constitute essential tools in microbial synthetic biology and metabolic engineering. However, inducible systems in non-model microbes, particularly thermophiles, are rarely reported. Acetivibrio thermocellus (previously termed Clostridium thermocellum), a representative strain of thermophilic non-model microbes, currently serves as a promising chassis organism in biorefinery. Although various genetic tools are available for A. thermocellus, superior thermophilic inducible systems are in high demand. In this study, we developed a thermostable l-arabinose-inducible system (ThermoARAi) in A. thermocellus by utilizing the inducible promoter P_abnE and repressor AraR from Geobacillus stearothermophilus T-6. Through systematic promoter engineering and optimization of induction conditions using a thermostable β-glucuronidase as reporter, the system exhibited dynamic range improvement from a 5.4-fold induction to a 175-fold induction with negligible leakage. Furthermore, the ThermoARAi system was appropriate for use in metabolic engineering, as validated by its applications in whole-cell saccharification of cellulosic substrates and degradation of amorphous polyethylene terephthalate films. The ThermoARAi system significantly expands the genetic toolkit for precise gene expression modulation, metabolic engineering, and biotechnological applications in A. thermocellus. Importantly, this approach may also serve as a foundation for developing genetic tools in other Clostridia that play key roles in diverse ecosystems, including the gut.