Such truncated proteins could potentially interfere with the function of intact FkbN protein, produced in the complementation experiment. All this shows, that FkbN

RG-7388 manufacturer is indispensable for FK506 production, which is in agreement with recently published results [28]. Clearly, fkbN also shows important potential for application in genetic/metabolic engineering of industrial FK506 producing strains. In the next step, an additional copy of the fkbR gene was introduced into S. tsukubaensis under the control of the ermE* and Streptomyces RBS [38]. Like in the case of fkbN, FK506-production Adavosertib nmr was increased demonstrating that fkbR also has

a positive regulatory role in S. tsukubaensis NRRL 18488. However, yield increase was moderate with FK506 production GDC-0068 in vivo approximately 30% higher than in the control strain (Figure 3). The fkbR gene-disrupted mutants (Figure 2B; Additional file 2) displayed a significant reduction in FK506 production and on average they retained only approximately 20% of the wild-type production level, clearly demonstrating a positive role of this regulatory protein. Unlike FkbN, the FkbR regulatory protein is not indispensable for FK506-production. Interestingly, the

ΔfkbR strains, complemented with the fkbR gene transcribed under the ermE* promoter showed recovery of FK506 production to wild-type levels (Figure 3). As expected, double mutant strains ΔfkbRΔfkbN were unable to produce FK506. Neither addition of a second copy of the allN gene transcribed under the ermE* promoter, nor the inactivation of allN, located on the left fringe of FK506 gene cluster, showed any influence on FK506 production or any other phenotypic characteristic (e.g. morphological), as the mutant strains retained wild-type values of FK506 yield. The result was the same when allM and allN were overexpressed together. Gene expression in FK506 gene cluster is not abolished ID-8 by inactivation of fkbN or fkbR In the next step we aimed to identify genes in the FK506 gene cluster, the transcription of which could possibly be regulated by FkbN and FkbR transcriptional regulators. We constructed reporter plasmids based on the rppA gene chalcone synthase from S. erythraea, described previously [20, 41]. For the purpose of this work, we selected six different approximately 500-bp long putative promoter regions, located upstream of start codons of representative CDSs of the FK506 gene cluster.