Sefamisin C üretiminin artıtılması için besiyeri optimizasyonu ve streptomyces clavuligerus ask, hom ve ask+hom rekombinantlarının antibiyotik üretimlerinin karşılaştırılması.

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.M.S. - Master of Scienc

Streptomyces clavulıgerus’un iki ayrı sefamisin c aşırı üretici suşu ve bir endüstriyel klavulanik asit aşırı üreticisinin standard suş NRRL 3585 ile karşılaştırmalı proteomik analizi.

In this study, two cephamycin C (CC) overproducers, Streptomyces clavuligerus AK39 and TB3585 strains, and a clavulanic acid (CA) overproducer S. clavuligerus DEPA were subjected to proteomic analysis to elucidate their differential protein expression profiles when compared to that of the standard strain S. clavuligerus NRRL 3585. Two proteomics approaches were employed: 2DE technique coupled with MALDI-TOF/MS and LC-MS/MS. By using two techniques, a total of 40 proteins were identified as upregulated while 70 proteins were downregulated in AK39. For TB3585 strain, a total of 44 proteins were overrepresented while 50 proteins were underrepresented. For both AK39 and TB3585, “Hypothetical/Unknown Proteins” category harbored higher numbers of upregulated proteins which was followed by “Secondary Metabolism”. Both CC overproducers specifically overexpressed enzymes related with CC biosynthesis and underexpressed proteins involved in the biosynthesis of diverse secondary metabolites like clavams and polyketides. As for the DEPA strain, a total of 80 proteins were upregulated, and 174 proteins were downregulated. Upregulated protein categories in DEPA were ranked as “Hypothetical/Unknown”, “Others/General Function”, “DNA Replication, Recombination, Repair, Transcription” and “Secondary Metabolism”, respectively. Three CA biosynthetic enzymes as well as CcaR were overrepresented whereas enzymes of two other secondary metabolites were underrepresented along with two important global regulators. A decrease in amino acid metabolism, particularly in methionine biosynthesis, appeared as one of the prominent mechanisms of success of DEPA strain as a prolific producer of CA. Results described herein provide useful information for further improvement of CC and CA production in S. clavuligerus with rational approaches.  Ph.D. - Doctoral Progra

FEN BİLİMLERİ ENSTİTÜSÜ/LİSANSÜSTÜ TEZ PROJESİ

S. CLAVULİGERUS NRRL3585'DE ASK OVEREKPRESYONU, HOM DELESYONU VE HOM GENİ TAHRİP EDİLMİŞ MUTANTTA ASK GENİ AMPLİFİKASYONUNUN ETKİLERİNİN PROTEOMİK ÖLÇEKTE ARAŞTIRILMAS

Homologous expression of aspartokinase (ask) gene in Streptomyces clavuligerus and its hom-deleted mutant: Effects on cephamycin C production

In this study, the effect of homologous multiple copies of the ask gene, which encodes aspartokinase catalyzing the first step of the aspartate pathway, on cephamycin C biosynthesis in S. clavuligerus NRRL 3585 and its hom mutant was investigated. The intracellular pool levels of aspartate pathway amino acids accorded well with the Ask activity levels in TB3585 and AK39. When compared with the control strain carrying vector alone without any gene insert, amplification of the ask gene in the wild strain resulted in a maximum of 3.1- and 3.3-fold increase in specific, 1.7- and 1.9-fold increase in volumetric cephamycin C production when grown in trypticase soy broth (TSB) and a modified chemically defined medium (mCDM), respectively. However, expression of multicopy ask gene in a hom-deleted background significantly decreased cephamycin C yields when the cells were grown in either TSB or mCDM, most probably due to physiological disturbance resulting from enzyme overexpression and high copy number plasmid burden in an auxotrophic host, respectively

Proteome-wide alterations in an industrial clavulanic acid producing strain of Streptomyces clavuligerus

AbstractThe usefulness of genetic/metabolic engineering for further improvement of industrial strains is subject of discussion because of the general lack of knowledge on genetic alterations introduced by iterative cycles of random mutagenesis in such strains. An industrial clavulanic acid (CA)-overproducer Streptomyces clavuligerus DEPA was assessed to understand proteome-wide changes that have occurred in a local industrial CA overproducer developed through succesive mutagenesis programs. The proteins that could be identified corresponded to 33 distinct ORFs for underrepresented ones and 60 ORFs for overrepresented ones. Three CA biosynthetic enzymes were overrepresented in S. clavuligerus DEPA; carboxyethylarginine synthase (Ceas2), clavaldehyde dehydrogenase (Car) and carboxyethyl-arginine beta-lactam-synthase (Bls2) whereas the enzymes of two other secondary metabolites were underrepresented along with two important global regulators [two-component system (TCS) response regulator (SCLAV_2102) and TetR-family transcriptional regulator (SCLAV_3146)] that might be related with CA production and/or differentiation. γ-butyrolactone biosynthetic protein AvaA2 was 2.6 fold underrepresented in S. clavuligerus DEPA. The levels of two glycolytic enzymes, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase and phosophoglycerate kinase were found decreased while those of dihydrolipoyl dehydrogenase (E3) and isocitrate dehydrogenase, with two isoforms were found as significantly increased. A decrease of amino acid metabolism, methionine biosynthesis in particular, as well as S-adenosylmethionine synthetase appeared as one of the prominent mechanisms of success of S. clavuligerus DEPA strain as a prolific producer of CA. The levels of two enzymes of shikimate pathway that leads to the production of aromatic amino acids and aromatic secondary metabolites were also underrepresented. Some of the overrepresented stress proteins in S. clavuligerus DEPA included polynucleotide phosphorylase/polyadenylase (PNPase), ATP-dependent DNA helicase, two isoforms of an anti-sigma factor and thioredoxin reductase. Downregulation of important proteins of cell wall synthesis and division was recorded and a protein with β-lactamase domain (SCLAV_p1007) appeared in 12 isoforms, 5 of which were drastically overrepresented in DEPA strain. These results described herein provide useful information for rational engineering to improve CA production in Streptomyces clavuligerus