Streptomyces clavuligerus is well-known for synthesizing several &beta / -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.

iv In recent years, outbreaks of infections associated with raw and minimally processed fruits and vegetables have been reported. Possible sources for contamination are irrigation water, manure, wash water, handling by workers and contact with contaminated surfaces. Pathogens can occur on raw and minimally processed produce at populations ranging from 103 to 109 CFU/g and able to survive and sometimes grow under various storage conditions. The objective of this study was to analyse the growth/survival of Salmonella Enteritidis at spot-inoculated or stem-injected cherry tomatoes during passive modified atmosphere packaging (MAP), controlled atmosphere (CA) and air storage at 7 and 22&deg / C. Low density polyethylene (LDPE) with a package size of 10x10 cm2 for 25&plusmn / 2 g tomatoes was used for MAP storage in which the gas composition equilibrated to 6% O2/ 4% CO2 and a carbon dioxide incubator was used for CA storage in which the CO2 level was monitored and maintained as 5% through the term of storage at 7 and 22&deg / C. During the research, the effect of ozone treatment (5-30 mg/L ozone gas for 0-20 min) was also considered for surface sanitation. The results demonstrate that S.Enteritidis can survive and/or grow during the storage of tomatoes depending on the location site of the pathogen on fruit, suspension cell density and storage temperature. During MAP, CA and air storage, S.Enteritidis with initial population of 7.0 log10 CFU/tomato survived on tomato surfaces with an approximate decrease of 4.0-5.0 log10 CFU/tomato in population within the storage period / however, in the case of initial population of 3.0 log10 CFU/tomato, cells died completely on day 4 during MAP storage and on day 6 during CA and air storage. The death rate of S.Enteritidis on the surfaces of tomatoes that were stored in MAP was faster than that of stored in air. Storage temperature was effective on the survival of S.Enteritidis for the samples stored at ambient atmosphere / cells died completely on day 6 at 7&deg / C and on day 8 at 22&deg / C. Stem scars provided protective environments for Salmonella / an approximate increase of 1.0 log10 CFU/tomato in stem-scar population was observed during MAP, CA and air storage at 22&deg / C within the period of 20 days. Cells survived with no significant change in number at 7&deg / C. The development of the microbial association in tomatoes was dominated by lactic acid bacteria (LAB). The pH values of the tomatoes changed approximately from 4.0 to 3.0 during the storage period. LAB grew well under all atmospheric conditions with or without the presence of S.Enteritidis. Gaseous ozone treatment has bactericidal effect on S.Enteritidis, inoculated on the surface of the tomatoes. 5 mg/L ozone gas treatment was not effective. 30 mg/L ozone gas treatment affected surface color.

The main objective of this study was to investigate the effects of high hydrostatic pressure on the stability of blood constituents for the purpose of an effective reduction of viral and bacterial count. The effect of HHP treatment on the several blood constituents were analyzed at different HHP levels at 25 0C for 5 minutes. The bovine blood as the model material was separated into two major parts / namely, serum and blood cells by centrifugation. Erythrocytes were found to be mostly stable up to 220 MPa pressure treatment displaying only surface modifications, but the cells lose their morphology at 350 MPa. White Blood Cells and platelets were found to be more sensitive, being degraded at around 110 MPa pressures putting an upper limit for the HHP treatment for the whole blood. But serum components and parameters studied showed much higher stability up to 220 MPa pressure level. The HHP treated blood cells were analyzed by FTIR spectroscopic technique and found to be stable in the macromolecular level. HHP treated proteins display only minimal changes in their secondary structures shown by the artificial neural network and curve fitting studies. Changes in the lipid bands indicated the changes in the membranes of the blood cells. In the microbiologic part of the study, Listeria innocua was found to be more stable than Bovine Herpes Virus type 1 as the model bacterium and virus respectively and their inactivation levels were compared with that of blood constituents.

Streptomyces clavuligerus is a Gram-positive, filamentous bacterium which produces several important secondary metabolites, including isopenicillin N, cephamycin C and the &beta / -lactamase inhibitor clavulanic acid. Among these compounds, clavulanic acid is being used in combination with commonly used &beta / -lactam antibiotics in order to fight against bacterial infections that are resistant to such antibiotics. Among these combinations, Augmentin, composed of amoxicillin and clavulanic acid, is the most widely prescribed drug and has a market value of more than one billion dollars per year. There are two genes that act in regulation of clavulanic acid biosynthesis: ccaR located in cephamycin C gene cluster and claR located in clavulanic acid gene cluster. The goal of this study is to improve clavulanic acid production capacities of both wild type and industrial S. clavuligerus strains by integrating extra copies of claR gene into S.clavuligerus genome and its overexpression via a multicopy plasmid. Although previously has shown to be quite effective on wild type S. clavuligerus strains, claR overexpression in the industrial strain used in this study yielded only 1.4-fold increase in volumetric and 1.7-fold increase in specific CA production by the recombinant strains MA28 and MA16, respectively.

The members of the genus Streptomyces are well-known for their capacity to synthesize a vast repertoire of secondary metabolites, including many useful antibiotics and proteins. Streptomyces clavuligerus is the producer of the medically important &amp / #946 / -lactam antibiotics such as cephamycin C and the potent &amp / #946 / -lactamase inhibitor clavulanic acid. The aspartate pathway of S. clavuligerus is an important primary metabolic pathway providing substrates for &amp / #946 / -lactam synthesis. This pathway uses L-aspartic acid as the precursor for the biosynthesis of the amino acids L-lysine, L-methionine, L-isoleucine, L-threonine and several important metabolic intermediates. L-&amp / #945 / -aminoadipic acid (&amp / #945 / -AAA) required for &amp / #946 / -lactam synthesis is a catabolic product of L-lysine produced from the lysine branch of the aspartate pathway. The carbon flow through the L-lysine-specific branch of aspartate pathway is limiting for the formation of cephamycin C. Formation of L-homoserine from aspartate semialdehyde (ASA) is the first step of the other branch of the aspartate pathway leading to L-threonine, L-isoleucine and L-methionine synthesis and is catalyzed by homoserine dehydrogenase (HSD, EC 1.1.1.3). Regulation of the activity or biosynthesis of the HSD of S. clavuligerus determines the availability of ASA for the biosynthesis of L-lysine and &amp / #945 / -AAA. The gene encoding for homoserine dehydrogenase (hom) was previously cloned from S. clavuligerus NRRL 3585 and characterized in our laboratory. In this study, the hom gene was disrupted via insertion of a kanamycin resistance cassette into this gene which was subsequently transferred to S. clavuligerus cells using the Streptomyces plasmid vector pIJ486. A hom mutant of S. clavuligerus (AK39) was formed through integration into the chromosome by double crossing over and the effects of hom disruption on cephamycin C yields were investigated. Disruption of hom gene resulted in a 1.7 to 2.0 fold increase in specific cephamycin C production in chemically defined medium (CDM).

In this study, the effects of bioprocess operation parameters on recombinant human growth hormone (rhGH) production by P. pastoris were systematically investigated. In this frame, first, for the extracellular expression and purification of human growth hormone by recombinant P. pastoris the cDNA of hGH, fused with a polyhistidine tag and also fused with a target site for the Factor Xa protease in which cleavage produces a mature N- and C- termini of rhGH, was cloned into pPICZ&amp / #945 / A plasmid and the constructed system within the plasmid, pPICZ&amp / #945 / A::hGH, was integrated to AOX1 locus of P. pastoris and expressed under alcohol oxidase promoter which is induced by methanol. With dot-blot analysis, the appropriate two strains producing human growth hormone at high levels and having different methanol utilization phenotype (Mut+ and Muts) were chosen among the other transformants. Then, the effects of methanol concentrations on the expression of rhGH and cell growth were analyzed and both of the phenotypes were compared in defined and complex media in laboratory scale air filtered shake bioreactors. The highest rhGH concentration for Mut+ and MutS, was found as 0.052 kg m-3 and 0.16 kg m-3, respectively, at 2 %(v/v) methanol concentration in complex medium. When methanol was used as the sole carbon source in defined medium, Muts phenotype had very low specific growth rate on methanol due to the intrinsic characteristics of it, therefore detectable rhGH was not observed, on the other hand, optimum rhGH concentration produced by Mut+ strain was found as 0.032 kg m-3 at 3% (v/v) methanol concentration in defined medium. In mixed system (glycerol/methanol) which is also defined, when the optimum glycerol concentration, 30 kg m-3, was used, Muts produced the highest rhGH, 0.110 kg m-3, at 1% (v/v) methanol concentration and any increase in methanol concentration resulted in lower rhGH production, on the other hand, Mut+ strain produced 0.060 kg m-3 rhGH at 4% (v/v) methanol concentration, which indicated that higher rhGH production capacity of Mut+ strain was obtained at high methanol concentrations. Using the designed defined medium for Mut+ phenotype where methanol was used as the sole carbon source with an optimum concentration of 3% (v/v), the effects of oxygen transfer on rhGH production, by-product formation, and cell growth, oxygen transfer and fermentation characteristics were investigated by using pilot scale bioreactor. Oxygen transfer effects on rhGH production were investigated at QO/VR=0.5 vvm / N=250, 500, 625, 750 min-1 conditions. The variations in rhGH , cell, amino acid and organic acid concentrations with the cell cultivation time, specific cell growth rate, the oxygen uptake rate, the liquid phase coefficient by using the dynamic method, maintenance coefficient for oxygen and yield coefficients were determined. The highest rhGH concentration was obtained at 0.5 vvm, 500 min-1 condition as 0.023 kg m-3 with 5.37 kg m-3 cell density.

In this study, novel polyketide biosynthesis gene cluster of Aspergillus fumigatus was characterized and functionally analyzed. Analysis of the newly sequenced A. fumigatus genome for laccases, which are involved in melanin biosynthesis and detoxification in fungi, resulted in several putative laccase and multicopper oxidase gene sequences, one of which, Afu4g14490 (tpnJ), was selected for further characterization. The predicted amino acid sequence TpnJp showed 63% identity with the dihydrogeodin oxidase of Aspergillus terreus, which is involved in the biosynthesis of the antifungal geodin. When the genome region of tpnJ was investigated, the presence of a polyketide biosynthesis gene cluster containing 13 genes, hypothesized to be responsible for the production of trypacidin and monomethylsulochrin, was realized. By a comparative genomics approach, a putative geodin biosynthesis gene cluster containing 13 genes, including dihydrogeodin oxidase, in A. terreus and a putative trypacidin biosynthesis gene cluster containing 13 genes in N. fischeri were established. Targeted deletions of the polyketide synthase (tpnC) and multicopper oxidase (tpnJ) genes confirmed the hypothesis that TpnCp, a three-domain minimal polyketide synthase, is involved in trypacidin and monomethylsulochrin biosynthesis in A. fumigatus. TpnCp is the first fungal minimal polyketide synthase whose functional role was experimentally identified. Moreover, the fact that LC-MS analysis of DtpnJ strain showed the absence of trypacidin and the presence of a higher amount of monomethylsulochrin in DtpnJ strain, confirmed the hypothesis that TpnJp is involved in the oxidation of monomethylsulochrin into trypacidin. This novel multicopper oxidase having high substrate specificity is given the name monomethylsulochrin oxidase.

The present study aimed detection of a human pathogen B. bugdorferi sensu lato species in suspected Lyme borreliosis (LB) patients in Turkey by PCR analysis and supportive serologic tests. The 152 clinical samples (140 serum and blood, 10 cerebrospinal fluid (CSF), 1 synovial fluid, 1 skin biopsy specimens) from 140 patients sent from 22 different cities of Turkey to The Spirochetal Diseases Diagnosis Laboratory of Central Veterinary Control and Research Institute were analysed. Serum samples were subjected to ELISA with a commercial kit and all of the blood, CSF, synovial fluid and skin biopsy samples were examined by PCR. In PCR analysis two primer sets targeting the ospA gene located on the plasmid and ribosomal 23S rRNA gene of B. burgdorferi sensu lato were used. The results indicated that 32,1% (45 of 140) seropositivity was detectable by ELISA. Our results support that there is a risk of acquiring LB in different regions of Turkey. Although considerable positive detections were recorded using serologic tests,none of the specimens were positive in PCR analysis. Further studies on PCR based methods for detection of B. burgdorferi sensu lato in patients with a high clinical probability of LB apparently may require that the specimen should be taken in the early phases and before the administration of any therapeutic agent.

The aim of this study was to analyze the effect of hydrogen peroxide and selected phenolic compounds on growth and catalase production of Aspergillus fumigatus. As a result of growing A. fumigatus at different temperatures it was observed that, growth and catalase production of this species were highest at 37 &deg / C. Catalase production was highest in the presence of 1 mM H2O2, yielding a significant 3 fold increase with respect to the control. Biomass was also increased by 1,44 fold with respect to the control sample. H2O2 increased catalase production possibly by inducing oxidative stress as biomass production significantly increased after the depletion of H2O2. Both gallic acid and trans-resveratrol significantly enhanced biomass generation of A. fumigatus (1,17 fold increase at 10 mM gallic acid and 1,45 fold increase at 3 mM resveratrol with respect to controls) and decreased extracellular catalase production (4,33 fold at 25 mM gallic acid and 16,7 fold decrease at 3 mM resveratrol with respect to controls) especially in the first 5 or 6 days of the cultivation where the anti-oxidant activity of the compounds were possibly at their maximum. A sudden and significant rise was observed in extracellular catalase activity between 5th and 7th days of the cultivation in phenolic compound applied samples, possibly owing to the depletion of the antioxidant activity of gallic acid and resveratrol followed by fungal cells&rsquo / response to a sudden increase of oxidative stress by boosting catalase production.

In Bacillus subtilis, genetic competence, sporulation and antibiotic production are controlled by quorum-sensing global regulatory mechanism. Bacilysin, being produced and excreted by certain strains of Bacillus subtilis, is a dipeptide antibiotic composed of L-alanine and L-anticapsin. We showed that the biosynthesis of bacilysin is under the control of quorum sensing global regulatory pathway through the action of ComQ/ComX, PhrC (CSF), ComP/ComA in a Spo0K (Opp)-dependent manner. Recently, the ywfBCDEF genes of B. subtilis 168 were shown to carry biosynthetic core function and renamed as bacABCDE operon. The objective of the present study is to elucidate the effects of previously-identified genes srfA, oppA, comA, phrC, phrF, phrK, comQ (comX), comP, spo0H, spo0A, abrB and codY on the expression of bacilysin biosynthetic operon bacABCDE. In order to monitor the expression of bac operon a B. subtilis strain, namely OGU1, containing a transcriptional bacA-lacZ fusion at bacA locus was constructed. Subsequently, each of the above-mentioned genes of cell density signaling was insertionally inactivated by transforming the competent cells of OGU1 with chromosomal DNA of the corresponding blocked mutant strains. The resulting strains and OGU1 as the control were cultured in PA medium and bacA-directed &amp / #946 / -galactosidase activities were monitored. bacA-lacZ expression was severely impaired in the srfA, oppA, comA, phrC, phrF, phrK, comQ (comX), comP, spo0H and spo0A disrupted mutants. On the other hand, in the abrB single mutant bacA expression level increased nearly 2-fold during exponential growth and in the codY mutant it severely decreased during the stationary phase.