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The 2 nd International Symposium " VERA JOHANIDES " Regulation of biosynthesis of immunosuppressant FK506 (tacrolimus) by Streptomyces tsukubaensis dr. Dušan GORANOVIČ Zagreb, May 2013. FK506 (takrolimus – T sukuba m acroli de im m u no s upressant ) - PowerPoint PPT Presentation
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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
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.
fkbC fkbB fkbP fkbAfkbO
allN fkbGallA allK allP allO
fkbH
fkbI
fkbJ
fkbK
fkbL allR
fkbD fkbN fkbQ fkbR allD allSallM
fkbM
DHCHC
H3CO
O
N O
O
OO
HO
OCH3
HO
OCH3
O
OH
Pipecolic acid
Lysine
NH
COOH
HO
HO COOH
Chorismate
S
O
CoA
COOH
Allylmalonyl-CoA
HOOC ACP
O
Methoxymalonyl-ACP
Regulation of secondary metabolite biosynthesis
• complex regulation of secondary metabolism
• global (pleiotropic) regulation
• pathway specific regulation
Corre et al., 2008
Tenconi et al., 2013
www.bacteriainphotos.com
Corre et al., 2008
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 fkbAfkbO
fkbD
fkbMfkbL
fkbL fkbMfkbD
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 fkbAfkbO
fkbD
fkbM
fkbN fkbQ fkbR allM allN fkbGallA allK allD allP allO allS
fkbH
fkbI
fkbJ
fkbK
fkbL allR
fkbL fkbD fkbM
Study of the effect of regulatory genes on FK506 production
fkbC fkbB fkbP fkbAfkbO
fkbD
fkbM
fkbN fkbQ fkbR allM allN fkbGallA allK allD allP allO allS
fkbH
fkbI
fkbJ
fkbK
fkbL allR
allNinactivation
fkbNinactivation
fkbRinactivation
PallA PfkbG PfkbN PfkbR
PfkbB
Goranovič et al., 2012
FK506 production of S. tsukubaensis regulatory gene mutants
Goranovič et al., 2012
↑38%
↑55%
↑30%
↓80%
NP
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 possibletarget of the FkbN regulator)
• expression of fkbN in sync withthe onset of FK506 production
Goranovič et al., 2012
ReferencesCorre 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 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