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Expression of the endogenous and heterologous clavulanic acid cluster in Streptomyces flavogriseus: why a silent cluster is sleeping

  • Applied genetics and molecular biotechnology
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Abstract

Clusters for clavulanic acid (CA) biosynthesis are present in the actinomycetes Streptomyces flavogriseus ATCC 33331 and Saccharomonospora viridis DSM 43017. These clusters, which are silent, contain blocks of conserved genes in the same order as those of the Streptomyces clavuligerus CA cluster but assembled in a different organization. S. flavogriseus was grown in nine different media, but clavulanic acid production was undetectable using bioassays or by high-performance liquid chromatography analyses. Reverse-transcriptase polymerase chain reaction (RT-PCR) of S. flavogriseus CA biosynthesis genes showed that the regulatory genes ccaR and claR and some biosynthetic genes were expressed whereas expression of cyp, orf12, orf13, and oppA2 was undetectable. The ccaR gene of S. clavuligerus was unable to switch on CA production in S. flavogriseus::[Pfur-ccaR C], but insertion of a cosmid carrying the S. clavuligerus CA cluster (not including the ccaR gene) conferred clavulanic acid production on S. flavogriseus::[SCos-CA] particularly in TBO and YEME media; these results suggests that some of the S. flavogriseus CA genes are inactive. The known heptameric sequences recognized by CcaR in S. clavuligerus are poorly or not conserved in S. flavogriseus. Quantitative RT-PCR analysis of the CA gene clusters of S. clavuligerus and S. flavogriseus showed that the average expression value of the expressed genes in the former strain was in the order of 1.68-fold higher than in the later. The absence of CA production by S. flavogriseus can be traced to the lack of expression of the essential genes cyp, orf12, orf13, orf14, and oppA2. Heterologous expression of S. clavuligerus CA gene cluster in S. flavogriseus::[SCos-CA] was 11- to 14-fold lower than in the parental strain, suggesting that the genetic background of the host strain is important for optimal production of CA in Streptomyces.

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Acknowledgments

This work was supported by grants BIO2009-09820 and LE046A11-2 from the Spanish Ministry of Economy and Competitivity and the Junta de Castilla y León, respectively. R. Álvarez-Álvarez and Y. Martínez-Burgo received PFU fellowships from the Spanish Ministry of Education, Culture and Sports. We appreciate the collaboration of Dr. T. López-García in the design of oligonucleotides and RT-qPCR experiments and the reception of plasmid pFL1272 from Dr. F. Lombo.

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  1. Microbiology Section, Department of Molecular Biology, Faculty of Biology and Environmental Sciences, Vegazana Campus, University of León, León, 24071, Spain

    R. Álvarez-Álvarez, Y. Martínez-Burgo, J.F. Martín & P. Liras

  2. Institute of Biotechnology INBIOTEC, Av. Real 1, León, 24006, Spain

    R. Álvarez-Álvarez, R. Pérez-Redondo & P. Liras

  3. Fundamental Biology Department, Faculty of Medicine and University Institute of Oncology from the Principado de Asturias (I.U.O.P.A), University of Oviedo, Oviedo, 33006, Spain

    A.F. Braña

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  1. R. Álvarez-Álvarez
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Álvarez-Álvarez, R., Martínez-Burgo, Y., Pérez-Redondo, R. et al. Expression of the endogenous and heterologous clavulanic acid cluster in Streptomyces flavogriseus: why a silent cluster is sleeping. Appl Microbiol Biotechnol 97, 9451–9463 (2013). https://doi.org/10.1007/s00253-013-5148-7

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