Bacillus subtilis GntR regulation modified to devise artificial transient induction systems.

We modified GntR regulation in Bacillus subtilis to devise transient induction systems. GntR is the repressor antagonized by gluconate to induce transcription of the gntRKPZ operon for gluconate catabolism. On the other hand, the gnt operon is repressed by glucose via carbon catabolite repression involving CcpA/P-ser-HPr, which binds to two cre sites: one located in the gnt promoter region and the other within the gntR coding region. We initiated gntKPZ encoding of enzymes for gluconate catabolism expressed independently from the operon; this allowed constitutive degradation of gluconate. Both cre sites were mutated to abolish catabolite repression. The mutated gnt promoter was set up to drive the expression of the lacZ reporter under the control of GntR. Even in the presence of glucose, lacZ was induced upon the addition of gluconate and shut down again as gluconate was consumed. Thus, modified GntR regulation enables artificial transient induction. This will allow us to design a flexible metabolic engineering system with genes expressed only temporarily as desired.