The 2 nd International Symposium " VERA JOHANIDES "

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

2. 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

3. 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

4. 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

5. 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 fkbO fkbA fkbL fkbD fkbM fkbP fkbD fkbM fkbL

6. 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 fkbO fkbA fkbL fkbD fkbM fkbP allA allK allD allM allN allP allO allS fkbG fkbI fkbK fkbD fkbN fkbQ fkbR allR fkbM fkbH fkbJ fkbL

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

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

9. Analysis of potential target gene promoter activity using the reporter system based on chalcone synthase rppA gene fromS. erythraea Analysis of promoter activity by measuring expression ofrppA 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

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

11. 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. 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. 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 Streptomycestsukubaensis. 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.

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