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. methoxymalonyl region modification-regulation region
FK506 gene cluster
83 kb, 25 genes
PKS region
methoxymalonyl region
modification-regulation region
allylmalonyl region
Goranovič et al Journal of Biological Chemistry.
Kosec et al 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
Corre et al., 2008
Tenconi et al., 2013

5. identification of 17 new genes, among them 3 regulatory genes
Whole genome sequencing of Streptomyces tsukubaensis NRRL 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

6. Goranovič et al. 2012. BMC Microbiology.
Whole genome sequencing of Streptomyces tsukubaensis NRRL and the annotation of the FK506 gene cluster
identification of 17 new genes, among them 3 regulatory genes
Goranovič et al 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

7. 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

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 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

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 fkbN in 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 Alkyl-4-hydroxymethylfuran-3-carboxylic acids, antibiotic production inducers discovered by Streptomyces coelicolor genome mining. Proc Natl Acad Sci U S A, 105, 45:
Tenconi E., Guichard P., Motte P., Matagne A., Rigali S Use of red autofluorescence for monitoring prodiginine biosynthesis. J Microbiol Methods, 93, 2:
Kuščer E., Coates N., Challis I., Gregory M., Wilkinson B., Sheridan R., Petković H Roles of rapH and rapG in positive regulation of rapamycin biosynthesis in Streptomyces hygroscopicus. Journal of Bacteriology, 189, 13:
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 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:
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 Robust reporter system based on chalcone synthase rppA gene from Saccharopolyspora erythraea. Journal of Microbiological Methods, 83, 2:
Goranovič D., Kosec G., Mrak P., Fujs Š., Horvat J., Kuščer E., Kopitar G., Petković H Origin of the allyl group in FK506 biosynthesis. Journal of Biological Chemistry, 285, 19:
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 FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis. BMC Microbiology, 12, 1: doi: / : 31 str.
Kosec G., Goranovič D., Mrak P., Fujs Š., Kuščer E., Horvat J., Kopitar G., Petković H Novel chemobiosynthetic approach for exclusive production of FK506. Metabolic Engineering, 14, 1:

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