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.

'Streptomyces tsukubaensis' was the first tacrolimus producer strain identified. Although it has been included in the Streptomyces genus, its taxonomic position has not been rigorously determined. By using a polyphasic approach, we have established that the tacrolimus producer strain 'S. tsukubaensis' NRRL 18488 represents a unique species in the Streptomyces genus, which is phylogenetically distant from other subsequently described producers. This fact means a horizontal transference of the tacrolimus-producing gene cluster. Physiology, nutrient requirement, and molecular genetics analyses of tacrolimus biosynthesis in 'S. tsukubaensis' necessitate chemically defined or semi-defined media, which work as a jigsaw puzzle and allow for pieces (nutrients) exchange. To date, studies related to 'S. tsukubaensis' have been mainly focused in the improvement of tacrolimus production using complex industrial fermentation media, which difficulty allows testing of tacrolimus overproduction enhancers or inhibitors because of the presence of non-defined substances. In the present work, two semi-defined media were developed in order to study the main factors involved in tacrolimus production in 'S. tsukubaensis'.

The RNA polymerase (RNAP) omega factor () forms a complex with the ␣ 2 ␤␤ core of this enzyme in bacteria. We have characterized the rpoZ gene of Streptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of the rpoZ gene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the ⌬rpoZ strain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ⌬rpoZ mutant with the wild-type rpoZ allele restored both phenotype and antibiotic production. Interestingly, the rpoZ gene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed that rpoZ promoter activity was increased in a ⌬phoP background, it can be concluded that rpoZ is controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation in Streptomyces.In bacteria, the RNA polymerase (RNAP) complex plays a central role in transcription and is a target for regulation of primary metabolism (6,7,44). The rpoZ gene encodes the RNAP omega () subunit, which forms a complex with the ␣ 2 ␤␤Ј core of this enzyme. The subunit has been identified in the RNAPs of most free-living bacteria. This protein is functionally homologous to the RpoK subunit of the archaeal RNA polymerase complex and the RPB6 subunit of the eukaryotic RNA polymerases I, II, and III (32). In Escherichia coli the subunit interacts with the ␤Ј subunit and promotes assembly of the RNA polymerase complex (14, 32), although it is not essential for survival in this bacterium (13).Streptomyces spp. are soil-dwelling bacteria that are notorious for their ability to produce thousands of antibiotics, pigments, antitumor agents, immunomodulators, and a variety of other bioactive secondary metabolites (1, 2, 8). Differential expression of secondary metabolism genes occurs following nutrient depletion (34), but the transcriptional control mechanisms that govern the onset of secondary metabolites are still obscure (29). The rpoZ gene of Streptomyces kasugaensis has been shown to be required for antibiotic production and morphological differentiation but is not essential for growth (21). A DNA fragment containing the rpoZ gene was shown to complement an S. kasugaensis pleiotropic mutant deficient in aerial mycelium formation and kasugamycin biosynthesis. Although sigma factors in Streptomyces have received considerable attention in relation to the expression of antibiotic biosynthetic genes (9,19,20), the role of the RNAP subunit is still obscure.The expression of many genes involved in antibiotic biosynthesis is negatively controlled by the phosphate concentration in the medium (reviewed in references 28 and 30). Limitati...

The current off-patent state of tacrolimus (FK506) has opened the hunting season for new generic pharmaceutical formulations of this immunosuppressant. This fact has boosted the scientific and industrial research on tacrolimus for the last 5 years in order to improve its production. The fast discovery of tacrolimus producer strains has generated a huge number of producers, which presents the biosynthetic cluster of FK506 as a high promiscuous genetic region. For the first time, the current state-of-the-art on the tacrolimus biosynthesis, production improvements and drug purification is reviewed. On one hand, all the genes involved in the tacrolimus biosynthesis, in addition to the traditional PKS/NRPS, as well as their regulation are analysed. On the other hand, tacrolimus direct and indirect precursors are reviewed as a straight manner to improve the final yield, which is a current trend in the field. Twenty years of industrial and scientific improvements on tacrolimus production are summarised, whereas future trends are also drafted.

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