Regulation of tylosin production in Streptomyces fradiae

The tylosin biosynthetic (tyl) gene cluster of S. fradiae contains at least five regulatory genes (tylP, Q, R, S, T). During promoter-probe analysis, TylP inhibited expression from tylPp in S. lividans, raising the possibility of autoregulation. TylP also negatively regulated the expression of tylQ in S. lividans, although tylQ was still switched off in a S. fradiae strain disrupted in tylP. The latter strain produced more tylosin and sporulated a day earlier, whereas over-expression of tylP resulted in reduced levels of antibiotic. Expression analysis by RT-PCR performed on the latter strain revealed barely detectable transcription from multiple genes, including tylS. This was probably a direct effect since TylP repressed tylSp in S. lividans. Meanwhile, expression analysis before and after the onset of tylosin production implied that TylQ is a key, negative regulator of tylosin production. The latter hypothesis was substantiated by expression analysis of a tylosin non-producing strain wherein tylQ was over-expressed. Moreover, expression analysis of a tylR-disrupted strain confirmed that TylQ controls tylosin production by repressing tylR. TylS, on the other hand, is essential for tylR activation and maximal expression of tylGIII-MIII, as shown by expression analysis of a strain disrupted in tylS. In the same and other genetically modified strains, expression analysis revealed that tylG and/or tylM genes are not necessarily co-transcribed or even co-regulated. This finding was supported by promoter probing in S. lividans and S. fradiae.;During an extensive portion of the empirical strain improvement program carried out at Lilly Laboratories, few changes of significance seem to have occurred within the tyl cluster. No mutations were found in the tyl promoters analyzed. Of regulatory genes, only tylQ had undergone a single point mutation that resulted in inactivation of its product. Enhancement of tylosin yields in empirically improved strains appears to have mainly occurred by mutations present outside the tyl cluster.