Developing Riboswitch-Mediated Gene Regulatory Controls in Thermophilic Bacteria

Thermophilic
bacteria are attractive hosts to produce bio-based
chemicals. While various genetic manipulations have been employed
in the metabolic engineering of thermophiles, a robust means to regulate
gene expression in these bacteria (∼55 °C) is missing.
Our bioinformatic search for various riboswitches in thermophilic
bacteria revealed that major classes of riboswitches are present,
suggesting riboswitches’ regulatory roles in these bacteria.
By building synthetic constructs incorporating natural and engineered
purine riboswitch sequences originated from foreign species, we quantified
respective riboswitches activities in repressing and up-regulating
gene expression in Geobacillus thermoglucosidasius using a green fluorescence protein. The elicited regulatory response
was ligand-concentration-dependent. We further demonstrated that riboswitch-mediated
gene expression of adhE (responsible for ethanol
production) in Clostridium thermocellum can modulate
ethanol production, redirect metabolites, and control cell growth
in the adhE knockout mutant. This work has made tunable
gene expression feasible across different thermophiles for broad applications
including biofuels production and gene-to-trait mapping.