Dataset Information

Origin of the allyl group in FK506 biosynthesis.

ABSTRACT: FK506 (tacrolimus) is a secondary metabolite with a potent immunosuppressive activity, currently registered for use as immunosuppressant after organ transplantation. FK506 and FK520 are biogenetically related natural products that are synthesized by combined polyketide synthase/nonribosomal peptide synthetase systems. The entire gene cluster for biosynthesis of FK520 from Streptomyces hygroscopicus var. ascomyceticus has been cloned and sequenced. On the other hand, the FK506 gene cluster from Streptomyces sp. MA6548 (ATCC55098) was sequenced only partially, and it was reasonable to expect that additional genes would be required for the provision of substrate supply. Here we report the identification of a previously unknown region of the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 containing genes encoding the provision of unusual building blocks for FK506 biosynthesis as well as a regulatory gene. Among others, we identified a group of genes encoding biosynthesis of the extender unit that forms the allyl group at carbon 21 of FK506. Interestingly, we have identified a small independent diketide synthase system involved in the biosynthesis of the allyl group. Inactivation of one of these genes, encoding an unusual ketosynthase domain, resulted in an FK506 nonproducing strain, and the production was restored when a synthetic analog of the allylmalonyl-CoA extender unit was added to the cultivation medium. Based on our results, we propose a biosynthetic pathway for the provision of an unusual five-carbon extender unit, which is carried out by a novel diketide synthase complex.

SUBMITTER: Goranovic D

PROVIDER: S-EPMC2863216 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Origin of the allyl group in FK506 biosynthesis.

Goranovic Dusan D Kosec Gregor G Mrak Peter P Fujs Stefan S Horvat Jaka J Kuscer Enej E Kopitar Gregor G Petkovic Hrvoje H

The Journal of biological chemistry 20100301 19

FK506 (tacrolimus) is a secondary metabolite with a potent immunosuppressive activity, currently registered for use as immunosuppressant after organ transplantation. FK506 and FK520 are biogenetically related natural products that are synthesized by combined polyketide synthase/nonribosomal peptide synthetase systems. The entire gene cluster for biosynthesis of FK520 from Streptomyces hygroscopicus var. ascomyceticus has been cloned and sequenced. On the other hand, the FK506 gene cluster from S ...[more]

PMID: 20194504

Similar Datasets

Project description: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.

Dataset Information

Origin of the allyl group in FK506 biosynthesis.

Publications

Origin of the allyl group in FK506 biosynthesis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets