Abstract
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 ParaBAD. 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-ParaBAD 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 Ptet 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-Ptet-system, pBBR1MCS-5-based LacI-dependent expression from PlacUV5 always exhibited some non-induced basal reporter expression and was therefore tunable only up to 40-fold induction by IPTG. The leakiness of PlacUV5 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-Ptet system makes this system highly suitable for applications in G. oxydans and possibly in other AAB.Key Points• A pBBR1MCS-5-based TetR-Ptet system was tunable up to more than 3500-fold induction.• A pBBR1MCS-5-based LacI-PlacUV5 system was leaky and tunable only up to 40-fold.• Modeling of protein-DNA binding suggested decreased DNA binding of LacI at pH < 6.
Highlights
The acetic acid bacterium (AAB) Gluconobacter oxydans harbors the beneficial ability of regio- and stereoselective incomplete oxidation of a variety of substrates in the periplasm by membrane-bound dehydrogenases and release of resulting products into the cultivation medium (Mamlouk and Gullo 2013; Mientus et al 2017; Pappenberger and Hohmann 2014)
We found with a pBBR1MCS-5-based TetRPtet system that inducible target gene expression based solely on de-repression of the heterologous target promoter can perform extremely well in G. oxydans
With the pBBR1MCS-5-based plasmid constructed here, the anhydrotetracycline (ATc)-inducible promoter P tet derived from the E. coli transposon Tn10 exhibited excellently tunable expression performance in an inducer concentrationdependent manner with maximal induction ratios up to more than 3500-fold
Summary
The acetic acid bacterium (AAB) Gluconobacter oxydans harbors the beneficial ability of regio- and stereoselective incomplete oxidation of a variety of substrates (e.g., sugars and sugar alcohols) in the periplasm by membrane-bound dehydrogenases (mDHs) and release of resulting products into the cultivation medium (Mamlouk and Gullo 2013; Mientus et al 2017; Pappenberger and Hohmann 2014). Recently the first tight system became available for tunable induction of gene expression in G. oxydans This system is based on AraC-ParaBAD and the induction by l-arabinose binding to the regulator protein AraC (Fricke et al 2020). For another LacI-based system, expression was found to be very leaky in G. oxydans (Condon et al 1991)
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