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

For regulatable target gene expression in the acetic acid bacterium (AAB) 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 and its target promoters P , P , and P could also be used in for regulatable target gene expression. Interestingly, in contrast to the responsiveness in , in RhaS increased the expression from P in the absence of l-rhamnose and repressed P in the presence of l-rhamnose. Inserting an additional RhaS binding site directly downstream from the -10 region generating promoter variant P almost doubled the apparent RhaS-dependent promoter strength. Plasmid-based P and P activity could be reduced up to 90% by RhaS and l-rhamnose, while a genomic copy of P 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 and the RhaS-P systems represent the first heterologous repressible expression systems for . In contrast to P , the promoter P was almost inactive in the absence of RhaS. In the presence of RhaS, the P 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 system could be suitable for tunable low-level expression of difficult enzymes or membrane proteins in . The insertion of an additional RhaS binding site directly downstream from the P -10 region increased the non-induced expression strength and reversed the regulation by RhaS and l-rhamnose from inducible to repressible. The P 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 with its target promoters P , P , P and variants thereof provide new opportunities for regulatable gene expression in 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.