Role of the cmcH-ccaR intergenic region and ccaR overexpression in cephamycin C biosynthesis in Streptomyces clavuligerus

Date

2013-07-01

Author

Kurt, Aslihan
Alvarez-Alvarez, Ruben
Liras, Paloma
Özcengiz, Gülay

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

287
views

0
downloads

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 Delta RI, 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 PglpF 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.

Subject Keywords

Biotechnology, Applied Microbiology and Biotechnology, General Medicine

URI

https://hdl.handle.net/11511/43252

Journal

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

DOI

https://doi.org/10.1007/s00253-013-4721-4

Collections

Department of Biology, Article

Suggestions

OpenMETU
Core

Overexpression of a serine alkaline protease gene in Bacillus licheniformis and its impact on the metabolic reaction network Çalık, Pınar; Oliver, SG; Ozdamar, TH (Elsevier BV, 2003-05-20) This work reports on cloning of serine alkaline protease (SAP) encoding gene subC to a multi-copy plasmid and its expression in Bacillus licheniformis with the quantitative impact of overexpression of the subC gene on metabolic flux distributions. Bioprocess characteristics of the wild-type and the recombinant B. licheniformis were investigated in a defined simple synthetic medium with glucose as the sole carbon source under well-defined bioreactor-operation conditions. Significant physiological changes wer...
Determination and metabolic engineering of rate limiting reactions in aromatic amino acid pathway in Bacillus subtilis for L-phenylaianine production Guzide, Calik; Yasemin, Demirci; Pinar, Calik; Ozdamar, Tuncer H. (2007-09-01) Rate limiting reactions in the aromatic-group amino acid pathway (AAAP) in Bacillus subtilis for l-phenylalanine (Phe) production were determined as reported elsewhere (Özçelik-Şenver et al., 2004). The biochemical reactions in the AAAP start with the reaction using phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) synthesised in the glycolysis pathway and the PPP, respectively, leading to the formation of Phe via chorismate by branching at prephenate. The branch-point metabolites E4P supplied in vi...
Expression of chitinase A (chiA) gene from a local isolate of Serratia marcescens in Coleoptera-specific Bacillus thuringiensis Okay, S.; Tefon, B. E.; ÖZKAN, MELEK; Özcengiz, Gülay (Wiley, 2008-01-01) Aims: The present study focused on cloning and expression of chiA gene from a highly chitinolytic local isolate of Serratia marcescens in an anti-Coleopteran Bacillus thuringiensis and comparison of the characteristics of the native and recombinant ChiAs.
Influence of controlled-pH and uncontrolled-pH operations on recombinant benzaldehyde lyase production by Escherichia coli Çalık, Pınar; Demir, AS (Elsevier BV, 2006-03-02) To select the host microorganism having the highest benzaldehyde lyase (BAL) production capacity, pUC 18::bal gene was transferred into four Escherichia coli strains. As the highest enzyme activity was obtained with E. coli K12 (ATCC 10798) carrying pUC18::bal gene, BAL production medium was designed for K 12. Using the designed medium containing 8.0 kg m(-3) glucose, 5.0 kg m(-3) (NH4)(2)HPO4 and the salt solution, the effects of uncontrolled-pH and controlled-pH operations were investigated at uncontrolle...
Influence of oxygen transfer on benzaldehyde lyase production by recombinant Escherichia coli BL21(DE3) pLySs Angardi, Vahideh; Çalık, Pınar; Department of Chemical Engineering (2007) In this study, the effects of oxygen transfer conditions on the synthesis of the enzyme benzaldehyde lyase as intracellular in recombinant E. coli BL21 (DE3) pLysS was investigated sistematically and a comprehensive model was developed to determine benzaldehyde lyase activity. For this purpose, the research program was carried out in mainly two parts. In the first part of study, the effects of oxygen transfer together with the mass transfer coefficient (KLa), enhancement factor E (=KLa/KLao), volumetric oxy...

Citation Formats

A. Kurt, R. Alvarez-Alvarez, P. Liras, and G. Özcengiz, “Role of the cmcH-ccaR intergenic region and ccaR overexpression in cephamycin C biosynthesis in Streptomyces clavuligerus,” APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, pp. 5869–5880, 2013, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/43252.