127NISR RESEARCH GRANT
2003 Young Investigator Research Grant

Characterization of the Biosynthetic Gene Cluster of Goadsporin which Promote Secondary Metabolism and Sporulation in Actinomycetes
Hiroyasu ONAKA
Biotechnology Research Center, Toyama Prefectural University
Research aims
Goadsporin which was isolated from Streptomyces sp.
TP-A0584 is a 19-amino-acid polypeptide containing four
oxazole and two thiazole rings within its backbone (Fig. 1).
Goadsporin promotes secondary metabolism and morphogenesis in actinomycetes. There are no related reports on
the substance which induces the secondary metabolism andmorphogenesis in a wide variety of different Streptomycesspecies. The research aims of this study are to identify andcharacterize the goadsporin biosynthetic gene cluster, andpropose the mode of action of it.
Methods and results
Cloning of the goadsporin biosynthetic gene cluster
Goadsporin is a peptide compound, therefore we proposed that goadsporin was biosynthesized ribosomally. It ispredicted that unusual amino acids contained in goadsporin,thiazole, oxazole, and methyloxazole are derived from cysteine, serine, and threonine. We synthesized oligonucleotide
probe corresponding to the precursor sequence of goadsporin and did colony hybridization with the oligoprobe.
Several clones were obtained, and one of positive clone,pGSBC1 was selected.
Heterologous expression of goadsporin in Streptomyces
lividans
pGSBC1 was transformed into S. lividans TK23, that is asurrogate host for heterologous expression. pGSBC1 wasconstructed on the basis of pTOYAMAcos cosmid vector
which is integrated into the specific chromosome attC siteFig. 1.

in actinomycetes. pGSBC1 integrated strain produced
goadsporin. Nucleotide sequencing of the pGSBC1 fragment revealed that godA is located on the end of the fragment and 14 ORFs are located on the downstream of godA
spanning 20-kb. Subcloning experiments revealed that essential region for goadsporin biosynthesis is between godA
and orf5 spanning 20-kb as shown in Fig. 2. In conclusion,
the ten genes, godA, godB, godC, godD, godE, godF, godG,godH, godI, and godR are responsible for goadsporin
biosynthesis.
Proposed overall biosynthetic pathway of goadsporin
A proposed biosynthetic pathway for the goadsporin isshown in Fig. 3. The 49-residue godA polypeptide is a substrate, and it processed by putative goadsporin synthetase,godD, E, F, and G. They introduce six heterocycles andconverts GodA to proGoadsporin. Proteolysis of proGoadsporin is performed by GodB which contains a conserved
peptidase domain. Finally GodH, which encodes the putative acetyltransferase, catalyzes the N-acetylation of N-terminal to end goadsporin biosynthesis. GodI may be responsible for the resistance of goadsporin and/or translocationof goadsporin and its biosynthesis apparatus.
Conclusion
Goadsporin which was isolated from Streptomyces sp.
TP-A0584 is a 19-amino-acid polypeptide containing fouroxazole and two thiazole rings within its backbone. Except
for goadsporin, linear polypeptides containing thiazole andoxazole rings have not been isolated from actinomycetes.
Goadsporin promotes secondary metabolism and morpho-

The chemical structure of goadsporin.

1-kb

godA godB

godC
Fig. 2.

godD

godE

orf2 orf3
godF godG godH godR orf1

Genetic organization of the goadsporin biosynthetic gene cluster.

godI

orf4
orf5

128

Fig. 3.

Proposed overall biosynthetic pathway of goadsporin.

genesis in actinomycetes. For example, it promotes the formation of red pigment and sporulation at a concentration of
1 m M and growth inhibition occurred at more than 1 m M in
Streptomyces lividans. Such an activity was observed
against to a wide variety of actinomycetes, however no activity to the other organisms. The biosynthesis of goadsporin would be started by the translation of godA structuralgene, and then the serine residue is modified to oxazole ordehydroalanine, threonine is to methyloxazole, and cysteinis to thiazole. godD, godE, godF, and godG products would
involve in such a post translational modification. Finally,
the peptide sequence of N-terminal region would be digested and acetylated to afford mature goadsporin by godC
and godH products. The genetic information of goadsporin

biosynthesis was applied to the goadsporin derivative
biosynthesis. The site directed mutagenesis of godA has
generated five molecules of the derivative.
References
H. Onaka, H. Tabata, Y. Igarashi, Y. Sato and T. Furumai, Goadsporin, achemical substance which promotes secondary metabolism and morphogenesis in streptomycetes. I. Purification and Characterization, The
Journal of Antibiotics, 54, 1036–1044, (2001)
Y. Igarashi, Y. Kan, K. Fujii, T. Fujita, K. Harada, H. Naoki, H. Tabata, H.
Onaka and T. Furumai, Goadsporin, a chemical substance which promotes secondary metabolism and morphogenesis in streoptomycetes. II.
Structure determination, The Journal of Antibiotics, 54, 1045–1053(2001)