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For the acetic acid bacterium (AAB) Gluconobacter oxydans only recently the first tight system for regulatable target gene expression became available based on the heterologous repressor-activator protein AraC from Escherichia coli and the target promoter P araBAD . In this study, we tested pure repressor-based TetR- and LacI-dependent target gene expression in G. oxydans by applying the same plasmid backbone and construction principles that we have used successfully for the araC-P araBAD system. When using a pBBR1MCS-5-based plasmid, the non-induced basal expression of the Tn10-based TetR-dependent expression system was extremely low. This allowed calculated induction ratios of up to more than 3500-fold with the fluorescence reporter protein mNeonGreen (mNG). The induction was highly homogeneous and tunable by varying the anhydrotetracycline concentration from 10 to 200 ng/mL. The already strong reporter gene expression could be doubled by inserting the ribosome binding site AGGAGA into the 3' region of the P tet sequence upstream from mNG. Alternative plasmid constructs used as controls revealed a strong influence of transcription terminators and antibiotics resistance gene of the plasmid backbone on the resulting expression performance. In contrast to the TetR-P tet -system, pBBR1MCS-5-based LacI-dependent expression from P lacUV5 always exhibited some non-induced basal reporter expression and was therefore tunable only up to 40-fold induction by IPTG. The leakiness of P lacUV5 when not induced was independent of potential read-through from the lacI promoter. Protein-DNA binding simulations for pH 7, 6, 5, and 4 by computational modeling of LacI, TetR, and AraC with DNA suggested a decreased DNA binding of LacI when pH is below 6, the latter possibly causing the leakiness of LacI-dependent systems hitherto tested in AAB. In summary, the expression performance of the pBBR1MCS-5-based TetR-P tet system makes this system highly suitable for applications in G. oxydans and possibly in other AAB. KEY POINTS: • A pBBR1MCS-5-based TetR-P tet system was tunable up to more than 3500-fold induction. • A pBBR1MCS-5-based LacI-P lacUV5 system was leaky and tunable only up to 40-fold. • Modeling of protein-DNA binding suggested decreased DNA binding of LacI at pH &lt; 6.

Highly tunable TetR-dependent target gene expression in the acetic acid bacterium Gluconobacter oxydans