On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases

Fricke, Philipp Moritz; Klemm, Angelika; Bott, Michael; Polen, Tino

doi:10.1007/s00253-021-11269-z

Cited by 7 publications

(3 citation statements)

References 227 publications

(314 reference statements)

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“…For target gene expression in G. oxydans , only constitutive promoters were used in the past due to the lack of a regulatable promoter. For expression, derivatives of the pBBR1MCS plasmid family obtained from the endogenous plasmid pBBR1 from Bordetella bronchiseptica were the most successful shuttle and expression vectors used (reviewed in Fricke et al, 2021a ). Since pBBR1MCS-2 conferring kanamycine resistance typically results in an abnormal cell morphology of G. oxydans in the presence of kanamycin and potentially also in reduced expression performance, pBBR1MCS-5 and the use of gentamicin is advantageous ( Fricke et al, 2021b ).…”

Section: Introductionmentioning

confidence: 99%

The l-rhamnose-dependent regulator RhaS and its target promoters from Escherichia coli expand the genetic toolkit for regulatable gene expression in the acetic acid bacterium Gluconobacter oxydans

et al. 2022

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For regulatable target gene expression in the acetic acid bacterium (AAB) Gluconobacter oxydans only recently the first plasmids became available. These systems solely enable AraC- and TetR-dependent induction. In this study we showed that the l-rhamnose-dependent regulator RhaS from Escherichia coli and its target promoters PrhaBAD, PrhaT, and PrhaSR could also be used in G. oxydans for regulatable target gene expression. Interestingly, in contrast to the responsiveness in E. coli, in G. oxydans RhaS increased the expression from PrhaBAD in the absence of l-rhamnose and repressed PrhaBAD in the presence of l-rhamnose. Inserting an additional RhaS binding site directly downstream from the −10 region generating promoter variant PrhaBAD(+RhaS-BS) almost doubled the apparent RhaS-dependent promoter strength. Plasmid-based PrhaBAD and PrhaBAD(+RhaS-BS) activity could be reduced up to 90% by RhaS and l-rhamnose, while a genomic copy of PrhaBAD(+RhaS-BS) appeared fully repressed. The RhaS-dependent repression was largely tunable by l-rhamnose concentrations between 0% and only 0.3% (w/v). The RhaS-PrhaBAD and the RhaS-PrhaBAD(+RhaS-BS) systems represent the first heterologous repressible expression systems for G. oxydans. In contrast to PrhaBAD, the E. coli promoter PrhaT was almost inactive in the absence of RhaS. In the presence of RhaS, the PrhaT activity in the absence of l-rhamnose was weak, but could be induced up to 10-fold by addition of l-rhamnose, resulting in a moderate expression level. Therefore, the RhaS-PrhaT system could be suitable for tunable low-level expression of difficult enzymes or membrane proteins in G. oxydans. The insertion of an additional RhaS binding site directly downstream from the E. coli PrhaT −10 region increased the non-induced expression strength and reversed the regulation by RhaS and l-rhamnose from inducible to repressible. The PrhaSR promoter appeared to be positively auto-regulated by RhaS and this activation was increased by l-rhamnose. In summary, the interplay of the l-rhamnose-binding RhaS transcriptional regulator from E. coli with its target promoters PrhaBAD, PrhaT, PrhaSR and variants thereof provide new opportunities for regulatable gene expression in G. oxydans and possibly also for simultaneous l-rhamnose-triggered repression and activation of target genes, which is a highly interesting possibility in metabolic engineering approaches requiring redirection of carbon fluxes.

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Section: Introductionmentioning

confidence: 99%

The l-rhamnose-dependent regulator RhaS and its target promoters from Escherichia coli expand the genetic toolkit for regulatable gene expression in the acetic acid bacterium Gluconobacter oxydans

et al. 2022

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show abstract

Section: Introductionmentioning

confidence: 99%

“…For the expression of target genes in G. oxydans , so far only constitutive promoters have been used due to the lack of a regulatable promoter demonstrated to be functional and tunable in G. oxydans (reviewed in Fricke et al 2021 ). Only recently the first tight system became available for tunable induction of gene expression in G. oxydans .…”

Section: Introductionmentioning

confidence: 99%

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

Fricke

Lürkens

Hünnefeld

et al. 2021

Appl Microbiol Biotechnol

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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 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.

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Exploiting Aerobic Carboxydotrophic Bacteria for Industrial Biotechnology

Siebert

Eikmanns

Blombach

2021

One-Carbon Feedstocks for Sustainable Bioproduction

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On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases

Cited by 7 publications

References 227 publications

The l-rhamnose-dependent regulator RhaS and its target promoters from Escherichia coli expand the genetic toolkit for regulatable gene expression in the acetic acid bacterium Gluconobacter oxydans

The l-rhamnose-dependent regulator RhaS and its target promoters from Escherichia coli expand the genetic toolkit for regulatable gene expression in the acetic acid bacterium Gluconobacter oxydans

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

Exploiting Aerobic Carboxydotrophic Bacteria for Industrial Biotechnology

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