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Noda Institute for Scientific Research GRANT
2012 Young Investigator Research Grant

Analysis of the regulatory genes encoded on a Streptomyces
linear plasmid and its application for antibiotic production
Kenji Arakawa
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University

Research aims
The filamentous soil bacteria Streptomyces are characterized by the ability to produce a wide variety of secondary
metabolites including antibiotics. In many Streptomyces
species, small diffusible signaling molecules, such as Afactor and virginia butanolide, control antibiotic production
and morphological differentiation.
Streptomyces rochei strain 7434AN4 carries three linear
plasmids (pSLA2-L, -M, and -S) and produces two structurally unrelated polyketide antibiotics: lankacidin (LC)and lankamycin (LM) (Fig. 1). The biosynthetic gene clusters of these antibiotics are located on the giant linear plasmid pSLA2-L.1) In addition, many regulatory genes havebeen identified on pSLA2-L, including a biosynthetic gene(srrX), six tetR-type repressor genes (srrA-F), and threeSARP (Streptomyces antibiotic regulatory protein) genes(srrY, srrZ, and srrW). We revealed that srrX and srrA constitute the signaling molecule/receptor system in S.
rochei.2–4) In this study, we analyzed the following topics:(1) structural diversity of the signaling molecules termed“SRBs (Streptomyces rochei butenolides)” that induce LCand LM production at nanomolar concentrations; (2)genome mining of cryptic biosynthetic gene clusters bygenetic manipulation of the regulatory genes.
Methods
Antibiotic production was analyzed by high performanceliquid chromatography (HPLC) and thin layer chromatography (TLC). Metabolites were purified by Sephadex LH-20
and/or silica gel chromatography. NMR spectra were re-corded on a JEOL LA-500 spectrometer equipped with afield gradient accessory. Electrospray ionization (ESI) andatmospheric pressure chemical ionization (APCI) mass
spectra were measured using a LTQ Orbitrap XL mass
spectrometer (Thermo Fisher Scientific, USA).
Results
Structural diversity of the signaling molecules SRBs
Our group has determined the structure of two novel signaling molecules, SRB1 and SRB2, which induce LC andLM production in Streptomyces rochei. Based on ESI-MSand extensive NMR analyses, SRB1 and SRB2 were determined to be a 2-(1′-hydroxyl-6′-oxo-8′-methylnonyl)-3methyl-4-hydroxybut-2-en-1,4-olide and 2-(1′-hydroxyl-6′oxo-8′-methyldecyl)-3-methyl-4-hydroxybut-2-en-1,4olide, respectively (Fig. 2).5) In this study, we synthesized
the structural variants of these SRBs (2,3-dihydro-SRBs
and 6′-deoxo-SRBs) to analyze their biological activity. Wefound that 2,3-dihydro-SRBs were unable to induce LC andLM production, suggesting the importance of the C2-C3
double bond for activity. On the other hand, 6′-deoxo-SRBs
exhibited induced LC and LM production, suggesting that
the C-6′ ketone group in SRBs is not directly related to activity. Several potential genes for SRB biosynthesis werefound clustered with srrX (orf 85) on pSLA2-L (Fig. 2): an
NAD-dependent dehydrogenase gene, srrG (orf 81); a
phosphatase gene, srrP (orf 83); a P450 hydroxylase gene,srrO (orf 84); and a thioesterase gene, srrH (orf 86). Comprehensive mutational analysis to confirm the identity andfunction of the SRB biosynthetic genes is in progress in ourlaboratory.
1.

Fig. 1. (A) Chemical structure of lankacidin C (LC) and lankamycin (LM) isolated from Streptomyces rochei 7434AN4. (B) Gene organization of a linear
plasmid pSLA2-L.

		

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Fig. 2. (A) The biosyntheic gene cluster for the signaling molecules SRB1 and SRB2 on pSLA2-L. (B) Structure of SRB1 and SRB2 isolated fromS. rochei.

2. Genome mining of cryptic biosynthetic gene clustersby genetic manipulation of the regulatory genes
Mutation of srrB, a tetR-type transcriptional repressor
gene, resulted in the overproduction of both LC and LM,
suggesting that srrB has a negative role in antibiotic biosynthesis. This result led us to develop a genome mining
approach in S. rochei to discover new secondary metabolites by activation of silent cryptic gene clusters. A triple
mutant with an srrB mutation and blocked in the biosynthetic pathway of both LC and LM accumulated moderateamounts of a UV-active compound, which was not present
in the parent strain or in any of the single/double mutants.
This compound contains a unique azoxyalkene chromophore, whose biosynthetic pathway is under investigation.

might be a powerful tool to identify bioactive secondary
metabolites of cryptic biosynthetic pathways.

Conclusion

4)

In general, the Streptomyces genome contains over 20genes or gene clusters associated with the biosynthesis ofsecondary metabolites. Under normal laboratory conditions, nearly 70–80% of biosynthetic genes are transcriptionally silent. Genetic manipulation of regulatory genes

References
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Arakawa, K., Mochizuki, S., Yamada, K., Noma, T., Kinashi, H.
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