FK506 biosynthesis is regulated by two positive regulatory elements in <it>Streptomyces tsukubaensis</it>

<p>Abstract</p> <p>Background</p> <p>FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various <it>Streptomyces</it> species. Considering the complex structure of FK506, it is reasonable to expect complex regulatory networks controlling its biosynthesis. Regulatory elements, present in gene clusters can have a profound influence on the final yield of target product and can play an important role in development of industrial bioprocesses.</p> <p>Results</p> <p>Three putative regulatory elements, namely <it>fkbR</it>, belonging to the LysR-type family, <it>fkbN</it>, a large ATP-binding regulator of the LuxR family (LAL-type) and <it>allN</it>, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from <it>Streptomyces tsukubaensis</it> NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of <it>fkbN</it> caused a complete disruption of FK506 biosynthesis, while inactivation of <it>fkbR</it> resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the <it>allN</it> gene. Using RT-PCR and a reporter system based on a chalcone synthase <it>rppA</it>, we demonstrated, that in the wild type as well as in <it>fkbN</it>- and <it>fkbR</it>-inactivated strains, <it>fkbR</it> is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas <it>fkbN</it> expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of <it>fkbN</it> (or <it>fkbR</it>) does not abolish the transcription of the genes in the FK506 gene cluster in general, but may reduce expression of some of the tested biosynthetic genes. Finally, introduction of a second copy of the <it>fkbR</it> or <it>fkbN</it> genes under the control of the strong <it>ermE</it>* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively.</p> <p>Conclusions</p> <p>Our results clearly demonstrate the positive regulatory role of <it>fkbR</it> and <it>fkbN</it> genes in FK506 biosynthesis in <it>S. tsukubaensis</it> NRRL 18488. We have shown that regulatory mechanisms can differ substantially from other, even apparently closely similar FK506-producing strains, reported in literature. Finally, we have demonstrated the potential of these genetically modified strains of <it>S. tsukubaensis</it> for improving the yield of fermentative processes for production of FK506.</p>