The 2nd International Symposium "VERA JOHANIDES"   Regulation of biosynthesis of immunosuppressant FK506 (tacrolimus) by Streptomyces tsukubaensis     dr. Dušan GORANOVIČ Zagreb, May 2013

FK506 (takrolimus – Tsukuba macrolide immunosupressant) medically important polyketide immunosupressant, produced by Streptomyces tsukubaensis FK506 is synthesized by a hybrid PKS/NRPS system FK506 (takrolimus) Streptomyces tsukubaensis

methoxymalonyl region modification-regulation region FK506 gene cluster 83 kb, 25 genes PKS region methoxymalonyl region modification-regulation region allylmalonyl region Goranovič et al. 2010. Journal of Biological Chemistry. Kosec et al. 2012. Metabolic Engineering.     DHCHC   Allylmalonyl-CoA Chorismate         Pipecolic acid   Lysine Methoxymalonyl-ACP

Regulation of secondary metabolite biosynthesis complex regulation of secondary metabolism global (pleiotropic) regulation pathway specific regulation Corre et al., 2008 www.bacteriainphotos.com Corre et al., 2008 Tenconi et al., 2013

identification of 17 new genes, among them 3 regulatory genes Whole genome sequencing of Streptomyces tsukubaensis NRRL 18488 and the annotation of the FK506 gene cluster identification of 17 new genes, among them 3 regulatory genes fkbC fkbB fkbP fkbA fkbO fkbD fkbM fkbL

Goranovič et al. 2012. BMC Microbiology. Whole genome sequencing of Streptomyces tsukubaensis NRRL 18488 and the annotation of the FK506 gene cluster identification of 17 new genes, among them 3 regulatory genes Goranovič et al. 2012. BMC Microbiology. fkbC fkbB fkbP fkbA fkbO fkbD fkbM fkbN fkbQ fkbR allM allN fkbG allA allK allD allP allO allS fkbH fkbI fkbJ fkbK fkbL allR

Study of the effect of regulatory genes on FK506 production fkbC fkbB fkbO fkbP fkbA PfkbB allA allK allD allN inactivation allM allN allP allO allS fkbG fkbI fkbK fkbD fkbN inactivation fkbN fkbQ fkbR inactivation fkbR PallA PfkbG PfkbN PfkbR allR fkbH fkbJ fkbL fkbM Goranovič et al., 2012

FK506 production of S. tsukubaensis regulatory gene mutants ↑55% ↑38% ↑30% ↓80% NP Goranovič et al., 2012

Analysis of potential target gene promoter activity using the reporter system based on chalcone synthase rppA gene from S. erythraea Analysis of promoter activity by measuring expression of rppA in liquid media after 6 days of cultivation in a complex production medium (light gray – WT S. tsukubaensis, dark gray – ΔfkbR, white – ΔfkbN) Goranovič et al., 2012

Analysis of potential target gene promoter activity using RT-PCR fkbG (methoxymalonyl-ACP biosynthetic genes a possible target of the FkbN regulator) expression of fkbN in sync with the onset of FK506 production Goranovič et al., 2012

References Corre C., Song L., O'Rourke S., Chater K.F., Challis G.L. 2008. 2-Alkyl-4-hydroxymethylfuran-3-carboxylic acids, antibiotic production inducers discovered by Streptomyces coelicolor genome mining. Proc Natl Acad Sci U S A, 105, 45: 17510-17515. Tenconi E., Guichard P., Motte P., Matagne A., Rigali S. 2013. Use of red autofluorescence for monitoring prodiginine biosynthesis. J Microbiol Methods, 93, 2: 138-143. Kuščer E., Coates N., Challis I., Gregory M., Wilkinson B., Sheridan R., Petković H. 2007. Roles of rapH and rapG in positive regulation of rapamycin biosynthesis in Streptomyces hygroscopicus. Journal of Bacteriology, 189, 13: 4756-4763. Mo S., Kim D.H., Lee J.H., Park J.W., Basnet D.B., Ban Y.H., Yoo Y.J., Chen S.W., Park S.R., Choi E.A., Kim E., Jin Y.Y., Lee S.K., Park J.Y., Liu Y., Lee M.O., Lee K.S., Kim S.J., Kim D., Park B.C., Lee S.G., Kwon H.J., Suh J.W., Moore B.S., Lim S.K., Yoon Y.J. 2011. Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues. Journal of the American Chemical Society, 133, 4: 976-985. Magdevska V., Gaber R., Goranovič D., Kuščer E., Boakes S., Duran Alonso M.B., Santamaria R.I., Raspor P., Leadlay P.F., Fujs Š., Petković H. 2010. Robust reporter system based on chalcone synthase rppA gene from Saccharopolyspora erythraea. Journal of Microbiological Methods, 83, 2: 111-119. Goranovič D., Kosec G., Mrak P., Fujs Š., Horvat J., Kuščer E., Kopitar G., Petković H. 2010. Origin of the allyl group in FK506 biosynthesis. Journal of Biological Chemistry, 285, 19: 14292-14300. Goranovič D., Blažič M., Magdevska V., Horvat J., Kuščer E., Polak T., Santos-Aberturas J., Martinez-Castro M., Barreiro C., Mrak P., Kopitar G., Kosec G., Fujs Š., Martin J.F., Petković H. 2012. FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis. BMC Microbiology, 12, 1: doi:10.1186/1471-2180-12-238: 31 str. Kosec G., Goranovič D., Mrak P., Fujs Š., Kuščer E., Horvat J., Kopitar G., Petković H. 2012. Novel chemobiosynthetic approach for exclusive production of FK506. Metabolic Engineering, 14, 1: 39-46.

The 2nd International Symposium "VERA JOHANIDES"   Regulation of biosynthesis of immunosuppressant FK506 (tacrolimus) by Streptomyces tsukubaensis     dr. Dušan GORANOVIČ Zagreb, May 2013