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Title of Journal: Appl Microbiol Biotechnol

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Abbravation: Applied Microbiology and Biotechnology

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Springer Berlin Heidelberg

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DOI

10.1007/bf00254209

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1432-0614

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Expression of the endogenous and heterologous clav

Authors: R ÁlvarezÁlvarez Y MartínezBurgo R PérezRedondo AF Braña JF Martín P Liras
Publish Date: 2013/08/22
Volume: 97, Issue: 21, Pages: 9451-9463
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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 highperformance liquid chromatography analyses Reversetranscriptase polymerase chain reaction RTPCR 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 flavogriseusPfurccaR C but insertion of a cosmid carrying the S clavuligerus CA cluster not including the ccaR gene conferred clavulanic acid production on S flavogriseusSCosCA 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 RTPCR 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 168fold 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 flavogriseusSCosCA was 11 to 14fold lower than in the parental strain suggesting that the genetic background of the host strain is important for optimal production of CA in StreptomycesThis work was supported by grants BIO200909820 and LE046A112 from the Spanish Ministry of Economy and Competitivity and the Junta de Castilla y León respectively R ÁlvarezÁlvarez and Y MartínezBurgo received PFU fellowships from the Spanish Ministry of Education Culture and Sports We appreciate the collaboration of Dr T LópezGarcía in the design of oligonucleotides and RTqPCR experiments and the reception of plasmid pFL1272 from Dr F Lombo

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