Engineering rhamnose-inducible T7 expression systems in <i>Bacillus subtilis</i> utilizing the <i>rhaEW</i> promoter and identifying critical residues in RhaR regulating this promoter

We constructed T7 expression systems in Bacillus subtilis, in which the rhamnose-inducible rhaEW promoter and its derivative were used to control the T7 RNA polymerase gene. This promoter is regulated by the RhaR repressor, which is derepressed by rhamnulose-1-phosphate derived from rhamnose. To prolong the induction, we deleted the chromosomal rhaEW gene, thereby accumulating the effector. A mutated green fluorescent protein (EGFP) was expressed using these systems. However, high basal activity of the rhaEW promoter caused loss of regulation and growth inhibition of the expression strains upon rhamnose addition. Two spontaneous mutant strains carrying single-point mutations in the rhaR gene (T103A and Q188L) alleviated rhamnose-dependent growth inhibition and enabled rhamnose-induced EGFP expression. The lacZ reporter analysis showed that both mutations reduced RhaR's sensitivity to the effector. Incorporation of each mutant rhaR allele into the T7 expression system, distinct from the spontaneous mutants, improved the induction ratio.