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

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 P(rhaBAD), P(rhaT), and P(rhaSR) 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 P(rhaBAD) in the absence of l-rhamnose and repressed P(rhaBAD) in the presence of l-rhamnose. Inserting an additional RhaS binding site directly downstream from the −10 region generating promoter variant P(rhaBAD(+RhaS-BS)) almost doubled the apparent RhaS-dependent promoter strength. Plasmid-based P(rhaBAD) and P(rhaBAD(+RhaS-BS)) activity could be reduced up to 90% by RhaS and l-rhamnose, while a genomic copy of P(rhaBAD(+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-P(rhaBAD) and the RhaS-P(rhaBAD(+RhaS-BS)) systems represent the first heterologous repressible expression systems for G. oxydans. In contrast to P(rhaBAD), the E. coli promoter P(rhaT) was almost inactive in the absence of RhaS. In the presence of RhaS, the P(rhaT) 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-P(rhaT) 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 P(rhaT) −10 region increased the non-induced expression strength and reversed the regulation by RhaS and l-rhamnose from inducible to repressible. The P(rhaSR) 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 P(rhaBAD), P(rhaT), P(rhaSR) 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.