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