NDLI: Role of the cmcH–ccaR intergenic region and ccaR overexpression in cephamycin C biosynthesis in Streptomyces clavuligerus

Content Provider	Springer Nature Link
Author	Kurt, Aslıhan Álvarez Álvarez, Rubén Liras, Paloma Özcengiz, Gülay
Copyright Year	2013
Abstract	The effect of the CcaR regulatory protein on expression of the cephamycin C gene cluster is studied. Quantitative reverse transcription PCR (qRT-PCR) expression analysis of the cephamycin biosynthesis genes in the ccaR-disrupted strain, S. clavuligerus ccaR::aph, revealed that in the absence of CcaR, the lat and cmcI genes expression was reduced 2,200- and 1,087-fold compared with the wild type. Expression of pcbAB–pcbC–cefD–cefE–cmcJ–cmcH and blp was 225- to 359-fold lower, while expression of pcbR–pbpA–bla and orf10 was only slightly affected if at all, indicating that resistance and regulatory genes are not under CcaR control as opposed to pathway biosynthetic genes. In the intergenic cmcH–ccaR region, a small messenger RNA (mRNA) overlaps with the cmcH transcription terminator. Deletion of 688 bp of the intergenic region results in a strain, S. clavuligerus ΔRI, still able to produce cephamycin C and clavulanic acid but at levels 30–40 % lower than the parental strain. Therefore, specific sequences in the intergenic region upstream of ccaR enhance the expression of ccaR but are not essential for its expression. Strains containing an additional ccaR gene integrated in the chromosome, S. clavuligerus pSET-PC, or multiple copies of ccaR expressed from the P glpF promoter, S. clavuligerus pAK23, were constructed. Fermentations of the pAK23 strain resulted in a 6.1-fold increase in specific cephamycin C production relative to the wild type. In the same experiments, qRT-PCR analysis of the cephamycin biosynthesis genes showed a 5.1-fold increase in ccaR expression and similar increases in expression of lat and cmcI, while expression of other cluster genes were increased in the order of 2- to 3-fold.
Starting Page	5869
Ending Page	5880
Page Count	12
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	97
Issue Number	13
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2013-01-30
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Cephamycin C Streptomyces clavuligerus ccaR Cephamycin C overproducer strains Biotechnology Microbiology Microbial Genetics and Genomics
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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