FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis

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.

Phage P1-Derived Artificial Chromosomes Facilitate Heterologous Expression of the FK506 Gene Cluster

“…KCTC 11604BP, over-expression of the fkbR homologue tcs7 decreased FK506 production, while tcs7 deletion elevated FK506 yields [32]. In contrast, in S. tsukubaensis NRRL 18488 over-expression of fkbR increased FK506 production levels, while deletion had the opposite effect [33]. We introduced fkbR into S. coelicolor M1146/PAC20N#3 in the same manner as fkbN , but observed a decrease of 44% (0.69 mg L −1 , Figure 4B).…”

“…Over-expression of fkbN increased FK506 yields in S. tsukubaensis NRRL 18488 [33] and Streptomyces sp. KCTC 11604BP [32] and fkbN is present in all other identified FK506 gene clusters [30].…”

“…The two molecules differ only in one position with the presence of either an allyl (FK506) or ethyl (FK520) side chain (Figure 1A). Several recent studies have greatly expanded our understanding of FK506 and FK520 biosynthesis, including the identification of the enzyme FkbO as a chorismate hydrolase that generates the first FK506/FK520 precursor [28], the enzymes responsible for constructing allylmalonyl CoA or ethylmalonyl CoA for the two different side chains of FK506 and FK520 [29], [30], [31] and genes whose products positively or negatively regulate FK506 expression [32], [33]. Introduction of precursors into culture media or over-expression of certain components of the FK506 gene cluster have also led to improvements in FK506 yields [34], [35].…”

Two Master Switch Regulators Trigger A40926 Biosynthesis in Nonomuraea sp. Strain ATCC 39727

“…5). Several members of the LAL family are pathway-specific transcriptional activators of secondary-metabolite gene clusters in a range of actinomycetes (13,14,(26)(27)(28)(29)(30)(31)(32). Recently, two LAL proteins were identified as pleiotropic regulators affecting various cellular processes in Streptomyces coelicolor (15).…”

Positive Feedback Regulation of stgR Expression for Secondary Metabolism in Streptomyces coelicolor

“…They have been structurally well characterized to have a conserved helix-turn-helix (HTH) motif at the N terminus for DNA binding and a regulatory domain for substrate or inducer binding at the C terminus (2). Consistent with their wide distributions and great quantities, they have diverse and conserved regulatory functions in bacteria for primary metabolism (3,4), secondary metabolism (5,6), stress responses (7), cell division (8), virulence (9,10), protection (11), etc. Typically, most LTTRs function as the global transcriptional regulators by directly binding to the promoters of their regulons.…”