Engineering a tunable bicistronic TetR autoregulation expression system in Gluconobacter oxydans

Acetic acid bacteria are well-known for their ability to incompletely oxidize their carbon sources. Many of the products of these oxidations find industrial uses. Metabolic engineering of acetic acid bacteria would improve production efficiency and yield by allowing controllable gene expression. However, the molecular tools necessary for regulating gene expression have only recently started being explored. To this end the ability of the activation-dependent P_lux system and two constitutive repression P_tet systems were examined for their ability to modulate gene expression in Gluconobacter oxydans. The activation-dependent P_lux system increased gene expression approximately 5-fold regardless of the strength of the constitutive promoter used to express the luxR transcriptional activator. The P_tet system was tunable and had a nearly 20-fold induction when the tetR gene was expressed from the strong constitutive promoters P₀₁₆₉ and P₂₆₄, but only had a 4-fold induction when a weak constitutive promoter (P₄₅₂) was used for tetR expression. However, the P_tet system was somewhat leaky when uninduced. To mitigate this background activity, a bicistronic TetR expression system was constructed. Based on molecular modeling, this system is predicted to have low background activity when not induced with anhydrotetracycline. The bicistronic system was inducible up to >3,000-fold and was highly tunable with almost no background expression when uninduced, making this bicistronic system potentially useful for engineering G. oxydans and possibly other acetic acid bacteria. These expression systems add to the newly growing repertoire of suitable regulatable promoter systems in acetic acid bacteria.