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


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2012-12

AUTHORS

Dušan Goranovič, Marko Blažič, Vasilka Magdevska, Jaka Horvat, Enej Kuščer, Tomaž Polak, Javier Santos-Aberturas, Miriam Martínez-Castro, Carlos Barreiro, Peter Mrak, Gregor Kopitar, Gregor Kosec, Štefan Fujs, Juan F Martín, Hrvoje Petković

ABSTRACT

BACKGROUND: FK506 (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. RESULTS: Three 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. CONCLUSIONS: Our 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. More... »

PAGES

238

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/1471-2180-12-238

DOI

http://dx.doi.org/10.1186/1471-2180-12-238

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1010979935

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/23083511


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