Abstract
BackgroundFK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces 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.ResultsThree putative regulatory elements, namely fkbR, belonging to the LysR-type family, fkbN, a large ATP-binding regulator of the LuxR family (LAL-type) and allN, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of fkbN caused a complete disruption of FK506 biosynthesis, while inactivation of fkbR resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the allN gene. Using RT-PCR and a reporter system based on a chalcone synthase rppA, we demonstrated, that in the wild type as well as in fkbN- and fkbR-inactivated strains, fkbR is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas fkbN expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of fkbN (or fkbR) 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 fkbR or fkbN genes under the control of the strong ermE* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively.ConclusionsOur results clearly demonstrate the positive regulatory role of fkbR and fkbN genes in FK506 biosynthesis in S. tsukubaensis 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 S. tsukubaensis for improving the yield of fermentative processes for production of FK506.
Highlights
FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species
The fkbN gene encodes a putative transcriptional regulator belonging to the Large ATP-binding regulators of the LuxR family (LAL) family [16,24] and fkbR encodes a putative transcriptional regulator belonging to the LysR-type transcriptional regulators (LTTR) family and seems to represent the right limit of the FK506 gene cluster (Figure 1B)
These regulators are relatively large in size (872-1159 aa) compared to the betterstudied Streptomyces antibiotic regulatory protein (SARP) (277-665 aa) [15] and they have been identified in several macrolide antibiotic pathways, including FkbN from Streptomyces hygroscopicus var. ascomyceticus in FK520 biosynthesis [21], PikD from Streptomyces venezuelae for pikromycin [46], RapH from Streptomyces hygroscopicus for rapamycin [20,24,47], NysRI/RIII from Streptomyces noursei for nystatin [48] and GdmRI and GdmRII from Streptomyces hygroscopicus 17997 for geldanamycin biosynthesis [49]
Summary
FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species. 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. FK506 (Tacrolimus) is a widely used immunosuppressant, produced by industrial fermentation processes using various Streptomyces species. Since its first clinical appearance in 1989 [1] it has been well established in medicine as an important immunosuppressant drug. MA6548 (ATCC53770) encoding the biosynthesis of this important drug was partially sequenced by Merck Research Laboratories [7,8,9,10]. Two entire gene clusters from Streptomyces sp. KCTC 11604BP and Streptomyces kanamyceticus KCTC 9225 [11], and a partial sequence of the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 [12] have been published, allowing for the first time a comparative analysis of gene clusters involved in the formation of FK506 by different Streptomyces strains
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