Medium optimization for cephamycin c overproduction and comparison of antibiotic production by ask, hom, and ask+hom recombinants of streptomyces clavuligerus

Ünsaldı, Eser
Streptomyces clavuligerus is well-known for synthesizing several β-lactam antibiotics like cephamycin C which is produced through aspartic acid pathway initiated by aspartokinase (Ask) enzyme encoded by ask. Four different strains were constructed in our laboratory to increase cephamycin C production by S. clavuligerus. TB3585 and BA39 contained extra copies of ask gene on a multicopy plasmid, control strains TBV and BAV contained vector only in wild type strain NRRL3585 and hom-minus background, AK39, respectively. In this study, the effects of carbon and nitrogen sources incorporated into chemically defined medium were investigated for optimum growth and cephamycin C production by AK39. A modified-chemically defined medium (mCDM) was obtained by increasing the asparagine concentration two-fold and replacing glycerol with sucrose. Subsequently, growth and cephamycin C production by recombinant S. clavuligerus strains (TB3585, AK39, BA39, BAV, TBV) in Tryptic Soy Broth (TSB) and mCDM were compared. The specific antibiotic production in mCDM by TB3585 was 3.3- and 3.2-fold higher than TBV at 72h and 96h, respectively. Aspartokinase activity of S. clavuligerus recombinants was measured to verify the ask overexpression. TB3585 showed the highest activity at 48h. Finally, intracellular amino acid pools of the strains were measured to relate the Ask activity and antibiotic production to the amino acid content within the cells. AK39 was shown to have the highest intracellular levels of lysine, leading to cephamycin C precursor synthesis; lysine plus threonine, exerting concerted feedback inhibition on Ask enzyme; methionine, which cannot be produced by AK39 like threonine due to hom disruption.


Optimization of regeneration and agrobacterium mediated transformation of sugar beet (Beta vulgaris L.)
Baloğlu, Mehmet Cengiz; Yücel, Ayşe Meral; Department of Biology (2005)
In this study, optimization of a transformation and regeneration system via indirect and direct organogenesis in cotyledon, hypocotyl, petiole, leaf and shoot base tissues of sugar beet (Beta vulgaris L. cv. ELK 345 and 1195) was investigated. Two different germination, three different callus induction and shoot induction medium was used for indirect organogenesis of sugar beet cultivar ELK 345. Except cotyledon, other explants (hypocotyl, petiole and leaf) produced callus. However no shoot development was ...
Integration of clavaminate synthase 2 gene into the chromosome of an industrial strain of Streptomyces Clavuligerus for enhanced clavulanic acid production
Vanlı, Güliz; Özcengiz, Gülay; Özkan, Melek; Department of Biotechnology (2010)
Streptomyces clavuligerus is a gram-positive, filamentous bacterium which has a great ability to produce secondary metabolites including isopenicillin N, cephamycin C and a beta-lactamase inhibitor clavulanic acid. Clavulanic acid (CA) which is a bicyclic beta-lactam, inhibits most of class A beta-lactamases by binding irreversibly to the serine hydroxyl group at the active center of beta-lactamases and resulting in the stable acyl-enzyme complexes. Clavaminate synthase (CAS) is one of the best characterize...
New Steroidal 4-Aminoquinolines Antagonize Botulinum Neurotoxin Serotype A in Mouse Embryonic Stem Cell Derived Motor Neurons in Postintoxication Model.
Konstantinović, J; Kiriş, Erkan; Kota, KP; Kugelman-Tonos, J; Videnović, M; Cazares, LH; Terzić, Jovanović; Verbić, TŽ; Andjelković, B; Duplantier, AJ; Bavari, S; Šolaja, BA (American Chemical Society (ACS), 2018-02-22)
The synthesis and inhibitory potencies against botulinum neurotoxin serotype A light chain (BoNT/A LC) using in vitro HPLC based enzymatic assay for various steroidal, benzothiophene, thiophene, and adamantane 4-aminoquinoline derivatives are described. In addition, the compounds were evaluated for the activity against BoNT/A holotoxin in mouse embryonic stem cell derived motor neurons. Steroidal derivative 16 showed remarkable protection (up to 89% of uncleaved SNAP-25) even when administered 30 min postin...
In vivo half life of nanoencapsulated L-asparaginase
Baran, ET; Ozer, N; Hasırcı, Vasıf Nejat (2002-12-01)
In the present study, antileukemic enzyme L-asparaginase (ASNase) was encapsulated into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules in order to decrease the immunogenicity and toxicity of the enzyme and to increase its in vivo half life in mice.
Quantitative expression analysis of the genes potentially involved in clavulanic acid overproduction in streptomyces clavuligerus
Aktaş, Caner; Özcengiz, Gülay; Department of Molecular Biology and Genetics (2018)
Streptomyces clavuligerus is the producer of the medically important β-lactam antibiotics, including cephamycin C (CC) and the potent β-lactamase inhibitor clavulanic acid. (CA). We have already undertaken an extensive comparative proteomic analysis by using both 2-DE-MALDI-MS and GeLC-MS approaches between an industrial CA overproducer, namely DEPA and the reference strain NRRL3585. In this context, we documented several differentially expressed (over- and under-represented) proteins accounting for high CA...
Citation Formats
E. Ünsaldı, “Medium optimization for cephamycin c overproduction and comparison of antibiotic production by ask, hom, and ask+hom recombinants of streptomyces clavuligerus,” M.S. - Master of Science, Middle East Technical University, 2010.