Bio-based production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with modulated monomeric fraction in Escherichia coli.

In this study, we applied metabolic engineering and bioprocessing strategies to enhance heterologous production of an important biodegradable copolymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), with a modulated 3-hydroxyvalerate (3-HV) monomeric fraction from structurally unrelated carbon of glycerol in engineered Escherichia coli under different oxygenic conditions. We used our previously derived propanologenic (i.e., 1-propanol-producing) E. coli strain with an activated genomic Sleeping beauty mutase (Sbm) operon as a host for heterologous expression of the phaCAB operon. The 3-HV monomeric fraction was modulated by regulating dissimilated carbon flux channeling from the tricarboxylic acid (TCA) cycle into the Sbm pathway for biosynthesis of propionyl-CoA, which is a key precursor to (R)-3-hydroxyvaleryl-CoA (3-HV-CoA) monomer. The carbon flux channeling was regulated either by manipulating a selection of genes involved in the TCA cycle or varying oxygenic condition of the bacterial culture. With these consolidated strategies being implemented, we successfully achieved high-level PHBV biosynthesis with a wide range of 3-HV monomeric fraction from ~ 4 to 50 mol%, potentially enabling the fine-tuning of PHBV mechanical properties at the biosynthesis stage. We envision that similar strategies can be applied to enhance bio-based production of chemicals derived from succinyl-CoA. KEY POINTS: • TCA cycle engineering was applied to enhance 3-HV monomeric fraction in E. coli. • Effects of oxygenic conditions on 3-HV incorporation into PHBV in E. coli were investigated. • Bacterial cultivation for high-level PHBV production in engineered E. coli was performed.

Strain engineering for high-level 5-aminolevulinic acid production in Escherichia coli.

Herein, we report the development of a microbial bioprocess for high-level production of 5-aminolevulinic acid (5-ALA), a valuable non-proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5-ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied clustered regularly interspersed short palindromic repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5-ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl-CoA for enhanced 5-ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high-level 5-ALA biosynthesis from glycerol (~30 g L-1 ) under both microaerobic and aerobic conditions, achieving up to 5.95 g L-1 (36.9% of the theoretical maximum yield) and 6.93 g L-1 (50.9% of the theoretical maximum yield) 5-ALA, respectively. This study represents one of the most effective bio-based production of 5-ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio-based production.

New insights into affinity proteins for HER2-targeted therapy: Beyond trastuzumab.

Human epidermal growth factor receptor 2 (HER2) is known as a potential target for both cancer treatment and diagnosis. One of the most interesting HER2-targeted therapeutics is an affinity protein which selectively recognizes and binds to a defined target. Trastuzumab is a monoclonal antibody which has been approved as the first affinity proteins for treatment of some HER2-positive cancers including breast cancer. Despite initial response to trastuzumab, the majority of patients with metastatic HER2-positive breast cancer still show resistance to the therapy. Recently, various anti-HER2 affinity proteins, including antibodies, antibody fragments (e.g., Fab and scFv) and other protein scaffolds (e.g., affibody and DARPin), alone or fused/conjugated with therapeutic agents (e.g., proteins, drugs and radioisotopes) have been developed to overcome the trastuzumab resistance. Here, we review these engineered affinity proteins which are either clinically approved or under evaluation. Modern technologies and future prospects for their clinical applications in cancer treatment are also discussed.

Integrated strain engineering and bioprocessing strategies for high-level bio-based production of 3-hydroxyvalerate in Escherichia coli.

As petro-based production generates numerous environmental impacts and their associated technological concerns, bio-based production has been well recognized these days as a modern alternative to manufacture chemical products in a more renewable, environmentally friendly, and sustainable manner. Herein, we report the development of a microbial bioprocess for high-level and potentially economical production of 3-hydroxyvalerate (3-HV), a valuable special chemical with multiple applications in chemical, biopolymer, and pharmaceutical industries, from glycerol, which can be cheaply and renewably refined as a byproduct from biodiesel production. We used our recently derived 3-HV-producing Escherichia coli strains for bioreactor characterization under various culture conditions. In the parental strain, 3-HV biosynthesis was limited by the intracellular availability of propionyl-CoA, whose formation was favored by anaerobic conditions, which often compromised cell growth. With appropriate strain engineering, we demonstrated that 3-HV can be effectively produced under both microaerobic (close to anaerobic) and aerobic conditions, which determine the direction of dissimilated carbon flux toward the succinate node in the tricarboxylic acid (TCA) cycle. We first used the ∆sdhA single mutant strain, in which the dissimilated carbon flux was primarily directed to the Sleeping beauty mutase (Sbm) pathway (via the reductive TCA branch, with enhanced cell growth under microaerobic conditions, achieving 3.08 g L-1 3-HV in a fed-batch culture. In addition, we used the ∆sdhA-∆iclR double mutant strain, in which the dissimilated carbon flux was directed from the TCA cycle to the Sbm pathway via the deregulated glyoxylate shunt, for cultivation under rather aerobic conditions. In addition to demonstrating effective cell growth, this strain has shown impressive 3-HV biosynthesis (up to 10.6 g L-1), equivalent to an overall yield of 18.8% based on consumed glycerol, in aerobic fed-batch culture. This study not only represents one of the most effective bio-based production of 3-HV from structurally unrelated carbons to date, but also highlights the importance of integrated strain engineering and bioprocessing strategies to enhance bio-based production.Key points• TCA cycle engineering was applied to enhance 3-HV biosynthesis in E. coli. • Effects of oxygenic conditions on 3-HV in E. coli biosynthesis were investigated. • Bioreactor characterization of 3-HV biosynthesis in E. coli was performed.

Production of cellulosic butyrate and 3-hydroxybutyrate in engineered Escherichia coli.

Being the most abundant renewable organic substance on Earth, lignocellulosic biomass has acted as an attractive and cost-effective feedstock for biobased production of value-added products. However, lignocellulosic biomass should be properly treated for its effective utilization during biotransformation. The current work aimed to demonstrate biobased production of butyrate and 3-hydroxybutyrate (3-HB) in engineered Escherichia coli using pretreated and detoxified aspen tree (Populus tremuloides) wood chips as the feedstock. Various bioprocessing and genetic/metabolic factors limiting the production of cellulosic butyrate and 3-HB were identified. With these developed bioprocessing strategies and strain engineering approaches, major carbons in the hydrolysate, including glucose, xylose, and even acetate, could be completely dissimilated during shake-flask cultivation with up to 1.68 g L-1 butyrate, 8.95 g L-1 3-HB, and minimal side metabolites (i.e., acetate and ethanol) being obtained. Our results highlight the importance of consolidating bioprocess and genetic engineering strategies for effective biobased production from lignocellulosic biomass.

Effect of Twine-arginine Translocation-signaling Fusion System and Chaperones Co-expression on Secretory Expression of Somatropin.

BACKGROUND: Twine-arginine translocation (TAT) system is one of the exporting systems in Escherichia coli which could transport fully/semi-correctly folded proteins outside the reductive cytoplasmic space. In combination with co-expression with a chaperone system, the correctly folded proteins could be transported to oxidative periplasmic space and culture media to pass the main limitations in E. coli expression system such as misfolding and inclusion body formation. MATERIALS AND METHODS: To study the effectiveness of signaling sequences and chaperone co-expression on the translocation of expressed protein, somatropin was selected as the target. Two common signal sequences in TAT system (TorA and SufI) were added at the N-terminal of somatropin and the cassettes were co-expressed in E. coli BL21 (DE3) by a chaperone team including DnaK/J-GrpeE. RESULTS: The expression pattern studies including Western blotting and sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that somatropin is expressed in two cassettes. However, the pattern was different for two signaling sequences. CONCLUSION: The results confirmed that the approach of using TAT-signaling sequences and co-expression with the chaperone team could enhance translocation of protein to periplasmic space and culture media compared to control groups. Western blotting results showed that the signal sequence TorA could transport more expressed proteins to the periplasmic space and culture media in comparison with SufI. However, there was a considerable amount of human growth hormone in the cytoplasm which could not be transported outside the cytoplasmic space.

Improvement of solubility and refolding of an anti-human epidermal growth factor receptor 2 single-chain antibody fragment inclusion bodies.

Single chain variable fragment antibodies (scFvs) have attracted many attentions due to their small size, faster bio-distribution and better penetration in to the target tissues, and ease of expression in Escherichia coli. Although, scFv expression in E. coli usually leads to formation of inclusion bodies (IBs). The aim of this research was to improve solubilizing and refolding conditions for IBs of scFv version of pertuzumab (anti-human epidermal growth factor receptor 2 (HER2) antibody). After protein overexpression in E. coli BL21 (DE3), bacterial cells were lysed and IBs were extracted via repeated washing and centrifugation. The effect of different types, concentrations, pHs, and additive of denaturing agents on IBs solubility were evaluated. More than 40 refolding additives were screened and combinations of 10 of the best additives were check out using Plackett-Burman design to choose three refolding additives with the most positive effect on refolding of the scFv. Response surface methodology (RSM) was used to optimize the concentration of adopted additives. The most efficient buffer to solubilize IBs was a buffer containing 6 M urea with 6 mM beta mercaptoethanol, pH 11. The optimum concentration of three buffer additives for refolding of the scFv was 23 mM tricine, 0.55 mM arginine, and 14.3 mM imidazole. The bioactivity of the refolded scFv was confirmed by immunohistochemical staining of breast cancer tissue, a specific binding based method. The systematic optimization of refolding buffer developed in the present work will contribute to improve the refolding of other scFv fragments.

Twin arginine translocation system in secretory expression of recombinant human growth hormone.

Recombinant protein production in E. coli has several advantages over other expression systems. Misfolding, inclusion body formation, and lack of eukaryotic post translational modification are the most disadvantages of this system. Exporting of correctly folded proteins to the outside of reductive cytoplasmic environment through twin-arginine system could help to pass these limiting steps. Two signal sequences, TorA and SufI are used at N-terminal of human growth hormone (hGH) bearing DsbA gene sequence at C-terminal to enhance folding. The synthetic cassettes including the signal sequence, hGH and DsbA were transformed into E. coli BL21 (DE3) to study the effect of signal sequence and DsbA chaperone on translocation and folding of the protein. The results confirmed using signal sequence at N-terminal of targeted protein and coexpression with DsbA could transport proteins to the periplasmic space and culture media compared to control groups. Although there is no protein band of somatropin in SDS-Page of culture media samples when using SufI as signaling sequence, the study demonstrated TorA signal sequence could transport the target protein to the culture media. However, there was a considerable amount of hGH in periplasmic space when using SufI compared to control.

Optimization of the expression of phaC2 encoding poly (3-hydroxyalkanoate) synthase from Pseudomonas aeruginosa PTCC1310 in Fad B deleted Escherichia coli.

BACKGROUND: Poly3-hydroxyalkanoates (PHAs) are potential candidates for the industrial production of biodegradable plastics. Therefore, in the present study, expression and activity of one of the enzymes involved in the PHA synthesis, phaC2 (isolated from Pseudomonas aeruginosa PTCC1310), were investigated in Fad B deleted Escherichia coli. MATERIALS AND METHODS: The inserts obtained from recombinant pTZ57R plasmids were ligated into the pGEX-5x-1 expression vector and then transformed into Fad B deleted E. coli cells using the heat shock method. This protein was then expressed using isopropyl beta-d-thiogalactoside (IPTG) as an inducer. By changing expression conditions such as IPTG and glucose concentration, time and temperature of incubation with IPTG, the expression conditions were optimized. RESULTS: The optimum condition for the expression of this enzyme was: 1.5 mM IPTG, 1 mM glucose, incubated at 37°C for 2 hours. CONCLUSION: We obtained functional expression of the phaC2 gene and investigated various conditions that could influence the expression of protein to optimize production of PHA synthase enzymes. This would allow us to study PHA production in large quantities.

Release Studies on Ciprofloxacin Loaded Non-ionic Surfactant Vesicles.

BACKGROUND: Development of new drug carriers would be an interesting approach if it allowed increased efficacy of antibiotics and a reduction in doses, thus reducing the risk of developing resistance. As with most drug carriers, niosomes have been used to improve the selective delivery and the therapeutic index of antimicrobial agents. METHODS: In this study, different formulation of niosomes containing ciprofloxacin (CPFX), Span (20, 60 or 80), Tween (20, 60 or 80) and cholesterol were prepared by film hydration method. The release of the drug from different formulations was studied by using Franz diffusion cell. The niosomes were further characterized by optical microscopy and particle size analysis, and their antimicrobial activity was evaluated. RESULTS: Size of the niosomes was significantly dependent on the amount of cholesterol and surfactant type and varied from 8.56 to 61.3 µm. The entrapment efficiency of CPFX niosomes prepared by remote loading was more than 74%. Niosomes composed of Span/Tween 60 provided a higher CPFX release rate than other formulations. The obtained results indicated a diffusion-based mechanism for drug leakage through bilayers. All formulations presented more antibacterial activity as compared to free CPFX solution. CONCLUSION: Niosomal CPFX appears to be a promising approach in the management of bacterial infections, especially ophthalmic ones, and should be further evaluated by in vivo experiments.

Improved biological activity of a single chain antibody fragment against human epidermal growth factor receptor 2 (HER2) expressed in the periplasm of Escherichia coli.

A novel monoclonal antibody against human epidermal growth factor receptor 2 (HER2), i.e., pertuzumab (Perjeta®) developed by Genentech, has been verified to be effective in treating metastatic HER2-overexpressing breast cancer. The fact that the presence of the Fc region of the anti-HER2 is uncritical for growth inhibition of tumor cells suggests the potential biological activity of the associated antibody fragments. In the present study, we report functional expression of anti-HER2his-scFv, a single-chain variable fragment (scFv) derived from pertuzumab, in the periplasm of Escherichia coli and its purification. Biological activity of the soluble scFv produced in this manner was characterized using immunofluorescent staining, immunocytochemistry, flow cytometry and cytotoxicity assay. The effect of anti-HER2his-scFv on HER2 dimerization was also assessed by tyrosine kinase assay. It was observed that the purified scFv had a high specificity and affinity to HER2 receptors expressed on the surface of tumor cells with a selective cytotoxic effect on HER2-overexpressing SK-OV-3 cells. In addition, anti-HER2his-scFv was able to suppress phosphorylation of HER2 in the presence of heregulin. The results suggest that anti-HER2his-scFv can be a potential candidate for various therapeutic and diagnosis applications.

Microbial quality survey of sunscreen products in Iranian market.

BACKGROUND: Microbial contamination of cosmetic products is very crucial because of their daily use and direct contact with the skin. These products are at high risk for microbial contamination from various sources such as environment, consumer's hands, body sweat and during the time of manufacturing. Therefore, this study aimed to investigate the microbial quality of sunscreens products, manufactured in or imported to or formulated in local pharmacies in Iran. MATERIALS AND METHODS: The microbial quality were determined in three different levels; the intact product (at the time of purchase) and after three and after six months of opening it. Total Aerobic Viable Count (TAVC) and the presence of coliforms, Pseudomonas aeruginosa, Staphylococcus aureus, molds, and yeasts were studied. RESULTS: At the time of purchase, 40, 73.3 and 43.3 percentage of Iranian made, imported and pharmacy formulated sunscreens were contaminated with at least one of the objectionable microorganisms, respectively. After three months of opening it, 36.6, 70 and 46.6 percentage of Iranian made, imported and pharmacy formulated sunscreens were contaminated with at least one of the objectionable microorganisms, respectively. The percentages of contaminated samples were 36.6, 70 and 50 after six months of opening for Iranian made, imported and pharmacy formulated sunscreens, respectively. CONCLUSION: Microbial contamination of these sunscreens products is a potential health risk for consumers. It seems that it is necessary to inspect and monitor the products during the manufacturing and shelf life period. It is highly recommended to control and regulate cosmetic products by health organizations to ensure the quality and safety of this kind of products.

Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli.

The human epidermal growth factor receptor (HER) family plays an important role in cell growth and signaling and alteration of its function has been demonstrated in many different kinds of cancer. Receptor dimerization is necessary for the HER signal transduction pathway and tyrosine kinase activity. Recently, several monoclonal antibodies have been developed to directly interfere with ligand-HER receptor binding and receptor dimerization. A single chain variable fragment (ScFv) is a valuable alternative to an intact antibody. This report describes the production and purification of an ScFv specific for domain II of the HER2 receptor in Escherichia coli BL21 (DE3) cytoplasm. The majority of expressed of anti-her2his-ScFv protein was produced as inclusion bodies. A Ni-NTA affinity column was used to purify the anti-her2his-ScFv protein. The molecular weight of anti-her2his-ScFv protein was estimated to be approximately 27 kDa, as confirmed by SDS-PAGE and Western blotting assay. The anti-her2his-ScFv showed near 95 % purity and reached a yield of approximately 29 mg/l in flask fermentation. The purified anti-her2his-ScFv showed its biological activity by binding to HER2 receptor on the surface of BT-474 cells. This ScFv may be a potential pharmaceutical candidate for targeting tumour cells overexpressing HER2 receptor.

Does the tissue engineering architecture of poly(3-hydroxybutyrate) scaffold affects cell-material interactions?

A critical element in tissue engineering involves the fabrication of a three-dimensional scaffold. The scaffold provides a space for new tissue formation, supports cellular ingrowth, and proliferation and mimics many roles of the extracellular matrix. Poly(3-hydroxybutyrate) (PHB) is the most thoroughly investigated member of the polyhydroxyalkanoates (PHAs) family that has various degrees of biocompatibility and biodegradability for tissue engineering applications. In this study, we fabricated PHB scaffolds by utilizing electrospinning and salt-leaching procedures. The behavior of monkey epithelial kidney cells (Vero) and mouse mesenchymal stem cells (mMSCs) on these scaffolds was compared by the MTS assay and scanning electron microscopy. Additionally, this study investigated the mechanical and physical properties of these scaffolds by measuring tensile strength and modulus, dynamic contact angle and porosity. According to our results, the salt-leached scaffolds showed more wettability and permeability, but inferior mechanical properties when compared with nanofibrous scaffolds. In terms of cell response, salt-leached scaffolds showed enhanced Vero cell proliferation, whereas both scaffolds responded similarly in the case of mMSCs proliferation. In brief, nanofibrous scaffolds can be a better substrate for cell attachment and morphology.

Optimization of the Expression of Genes Encoding Poly (3-hydroxyalkanoate) Synthase from Pseudomonas aeruginosa PTCC 1310 in Escherichia coli.

Over the years, the use of plastics has complicated the problem of disposal of solid wastes. One strategy to reduce plastic waste is the use of biodegradable plastics. A group of these plastics are polyhydroxyalkanoates (PHAs). To date more than 250 different microorganisms are known to synthesize and accumulate PHA. Most Pseudomonas strains are able to accumulate mcl-PHA. In previous studies, the phaC1 and phaC2 genes were identified in Pseudomonas aeruginosa (P.aeruginosa) PTCC 1310 and were cloned. The aim of this study was to express these genes and optimize the conditions for their expression. The inserts obtained from vectors pTZPHAC1 and pTZPHAC2 were subcloned into pET15b expression vector. After transformation of competent Escherichia coli (E.coli) BL21 (DE3) cells with recombinant plasmids, expression was induced using IPTG. By changing expression conditions such as IPTG concentration, time and temperature of incubation with IPTG, the expression conditions for these enzymes were optimized, and the obtained results were compared using proper statistical analysis. The PHA synthase genes were induced with IPTG and the expressed 62 kDa protein was observed and purified. By changing expression conditions, 1 mM IPTG, 37 °C and a 2 hr incubation provided the highest level of protein production in E.coli cells. These results suggest that induction condition of PhaC genes can influence expression of PHA synthase enzymes.

Developing an extended genomic engineering approach based on recombineering to knock-in heterologous genes to Escherichia coli genome.

Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage λ (λ Red) and flippase-flippase recognition targets (FLP-FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P( trc ) and P( araB )), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol) under various genetic backgrounds (i.e., HB101 and DH5α). The expression of the inserted foreign genes was subjected to regulation using appropriate inducers [isopropyl ß-D: -1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive genomic engineering of E. coli, but also paves an effective way to "tailor" plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic engineering.

Antimicrobial resistance pattern of bacterial isolates from burn wounds in an Iranian University Hospital.

OBJECTIVE: About 73% of death cases in the first 5 days after burning are due to infection complications. The aim of this study was to identify the causing agents of infections in burn patients and the sensitivity pattern of them to the commonly used antimicrobials in an Iranian Burn center University Hospital. METHODS: In this cross-sectional study, patients who were admitted to one of the Iranian Burn center University hospitals in 2009 and had nosocomial infection due to burn wound, whom received antimicrobial agents for therapeutic reasons, with a hospitalization period of more than 48 hours were enrolled. Gram stain analyses were performed to help identifying growing colonies. Differential tests for identification of pathogenic bacteria species were performed following primary tests. E-test strips of each antimicrobial were placed on the culture medium plate in order to determine the minimum inhibitory concentration Studied antimicrobials for isolated Gram-negative bacteria were meropenem, piperacillin/tazobactam, ceftriaxone, cotrimoxazole, and for Staphylococcus aureus, vancomycin, piperacillin/tazobactam, cotrimoxazole, and cephalothin. FINDINGS: Only 16% of Pseudomonas aeruginosa species were sensitive to meropenem, and 13% were sensitive to piperacillin/tazobactam. Ten out of 29 Klebsiella species (34%) were sensitive to meropenem and piperacillin/tazobactam. All isolated strains of Staphylococcus aureus were sensitive to vancomycine while they were all resistant to cotrimoxazole. CONCLUSION: Pseudomona, Klebsiella and Staphylococcoci are the most common species causing burn infection in this medical center. Results showed the importance of limiting irrational use of wide-spectrum antimicrobials and recommends strict management of infections in burn injury centers.

Synthesis of some new quinazolinone derivatives and evaluation of their antimicrobial activities.

WIDE RANGE OF QUINAZOLINONE BIOLOGICAL PROPERTIES INCLUDING: antibacterial, anticancer, and anti-inflammatory activities encouraged us to synthesis some fused quinazolinone derivatives. Anthranilic acid was condensed with chloro acylchloride followed by dehydration to form the benzoxazinone intermediate; subsequent addition of an amine provided the fused quinazolinones. Deoxyvasicinone which was previously synthesized by a multi step complex reactions was prepared in three steps using the following procedure: Log P values of the compounds were measured using the shake flask method in octanol/water solvent system. The synthesized compounds were evaluated against six strains of bacteria (three Gram-positive and three Gram-negative) and three strains of fungi. Overall results of antimicrobial tests showed that the compounds had better bacteriostatic activity against Gram-negative bacteria. The obtained results of MBC revealed that these compounds had more significant bacteriostatic than bactericidal activities. Almost all of the screened compounds showed good activity against C. albicans and A. niger. The obtained results of MFC indicated that these compounds had more significant fungistatic than fungicidal activities.

Overproduction of Clavulanic Acid by UV Mutagenesis of Streptomyces clavuligerus.

Clavulanic acid is produced industrially by fermentation of Streptomyces clavuligerus and researches have increased its production by strain improvement, recombinant DNA technology, and media composition and growth condition optimization. The main objective of this study was to increase the level of clavulanic acid production from Streptomyces clavuligerus (DSM 738), using UV irradiation. After incubation, the spores and aerial mycelia were scraped off the agar plate by a sterile loop. After passing through a cotton wool, the serially diluted spore suspension was spread on GYM- agar containing caffeine. The plates were irradiated with UV light, wrapped in aluminum foil and incubated. The colonies were sub-cultured again to express the mutations. An aliquot of the spore suspension prepared from the resulted culture was poured in GYM agar plates and incubated. The plates were overlaid with nutrient-agar containing penicillin G and Klebsiela pneumoniae, and incubated. The inhibition zone diameter was measured and compared with the wild type colony. Repeating this procedure, the overproducer mutants were selected. Concentration of clavulanic acid was determined by HPLC analysis. It was concluded that secondary metabolites, mainly antibiotics containing clavulanic acid, were produced about 6-7 days after the growth, and concentration of clavulanic acid was increased up to two-folds after UV mutagenesis.

Synthesis, antimicrobial evaluation and QSAR study of some 3-hydroxypyridine-4-one and 3-hydroxypyran-4-one derivatives.

A series of Mannich bases of 2-alkyl-3-hydroxy-pyridine-4-ones, namely 2-alkyl-3-hydroxy-5-N-piperidylmethyl or N,N-dialkylaminomethyl pyridine-4-ones 9, 10 and 15-18, two derivatives of N-aryl-2-methyl-3-hydroxy-pyridine-4-ones 19, 20 and two N-alkyl derivatives of maltol, 21 and 22 were prepared. They were screened for their antibacterial and antifungal activities against a variety of microorganisms using micro plate Alamar Blue((R)) assay (MABA) method. Multiple linear regressions (MLR) analysis was performed for the synthesized compounds as well as a series of pyridinone and pyranone derivatives 23-43 which have been synthesized and evaluated for antimicrobial activity by other researchers previously. Studied compounds showed a better quantitative structure-activity relationship (QSAR) model for the antimicrobial activity against Candida albicans and Staphylococcus aureus in comparison with other tested microorganisms.