Efficient production of glutathione in Saccharomyces cerevisiae via a synthetic isozyme system.

Glutathione, a tripeptide consisting of cysteine, glutamic acid, and glycine, has multiple beneficial effects on human health. Previous studies have focused on producing glutathione in Saccharomyces cerevisiae by overexpressing γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), which are the rate-limiting enzymes involved in the glutathione biosynthetic pathway. However, the production yield and titer of glutathione remain low due to the feedback inhibition on GSH1. To overcome this limitation, a synthetic isozyme system consisting of a novel bifunctional enzyme (GshF) from Gram-positive bacteria possessing both GSH1 and GSH2 activities, in addition to GSH1/GSH2, was introduced into S. cerevisiae, as GshF is insensitive to feedback inhibition. Given the HSP60 chaperonin system mismatch between bacteria and S. cerevisiae, co-expression of Group-I HSP60 chaperonins (GroEL and GroES) from Escherichia coli was required for functional expression of GshF. Among various strains constructed in this study, the SKSC222 strain capable of synthesizing glutathione with the synthetic isozyme system produced 240 mg L-1 glutathione with glutathione content and yield of 4.3% and 25.6 mgglutathione /gglucose , respectively. These values were 6.6-, 4.9-, and 4.3-fold higher than the corresponding values of the wild-type strain. In a glucose-limited fed-batch fermentation, the SKSC222 strain produced 2.0 g L-1 glutathione in 67 h. Therefore, this study highlights the benefits of the synthetic isozyme system in enhancing the production titer and yield of value-added chemicals by engineered strains of S. cerevisiae.

Perturbation of the peptidoglycan network and utilization of the signal recognition particle-dependent pathway enhances the extracellular production of a truncational mutant of CelA in Escherichia coli.

Caldicellulosiruptor bescii is the most thermophilic, cellulolytic bacterium known and has the native ability to utilize unpretreated plant biomass. Cellulase A (CelA) is the most abundant enzyme in the exoproteome of C. bescii and is primarily responsible for its cellulolytic ability. CelA contains a family 9 glycoside hydrolase and a family 48 glycoside hydrolase connected by linker regions and three carbohydrate-binding domains. A truncated version of the enzyme (TM1) containing only the endoglucanase domain is thermostable and actively degrades crystalline cellulose. A catalytically active TM1 was successfully produced via the attachment of the PelB signal peptide (P-TM1), which mediates post-translational secretion via the SecB-dependent translocation pathway. We sought to enhance the extracellular secretion of TM1 using an alternative pathway, the signal recognition particle (SRP)-dependent translocation pathway. The co-translational extracellular secretion of TM1 via the SRP pathway (D-TM1) resulted in a specific activity that was 4.9 times higher than that associated with P-TM1 overexpression. In batch fermentations, the recombinant Escherichia coli overexpressing D-TM1 produced 1.86 ± 0.06 U/ml of TM1 in the culture medium, showing a specific activity of 1.25 ± 0.05 U/mg cell, 2.7- and 3.7-fold higher than the corresponding values of the strain overexpressing P-TM1. We suggest that the TM1 secretion system developed in this study can be applied to enhance the capacity of E. coli as a microbial cell factory for the extracellular secretion of this as well as a variety proteins important for commercial production.

Evaluation of phage adsorption to Salmonella Typhimurium exposed to different levels of pH and antibiotic.

This study was designed to evaluate the physicochemical properties of phage P22 in different pH and antibiotic levels as measured by growth kinetics, phage adsorption, and lytic activity. P22 was susceptible to acidic pHs and stable above pH 4. The latent period of P22 was 45 min and burst size was 34 phages/cell. The adsorption ability of phage to Salmonella Typhimurium was varied depending on the multiplicity of infections (MOIs). The latent period was reduced to 6.84, 4.02, and 1.72 h, respectively, on the levels of the host at 104, 106, and 108 CFU/ml. No significant differences in adsorption were observed between pH 4 and pH 7, but the lytic activities were significantly enhanced at the presence of ceftriaxone (CEA) and ciprofloxacin (CIP) at pH 7. Therefore, the phages combined with antibiotics can be a promising therapeutic tool to control antibiotic-resistant bacteria. This results provide a better understanding of host-phages interactions in different environmental conditions.

Assessment of phage-mediated inhibition of Salmonella Typhimurium treated with sublethal concentrations of ceftriaxone and ciprofloxacin.

This study was designed to evaluate the synergistic effect of phage (P22) and antibiotic on the inhibition of Salmonella Typhimurium exposed to ceftriaxone (CEF) and ciprofloxacin (CIP). The effect of phage and antibiotic treatments was evaluated by plaque size, disk diffusion, antibiotic susceptibility and phage multiplication assays. The sequential treatment effect of phage and antibiotic was carried out in different treatment order and time for 12 h at 37°C. P22 plaque sizes were increased by 28 and 71%, respectively, in the presence of CEF and CIP. The clear zone sizes in disk diffusion assay were significantly increased to >37 mm in the presence of CEF and CIP compared to the control (28-31 mm). Pre-treatment with P22 enhanced the antimicrobial effect of CIP, showing >2 log reduction after a 12 h incubation. Phage P22 combined with antibiotics (CEF and CIP) effectively inhibited the growth of S. Typhimurium depending on the treatment order and time. These results provide useful information for understanding the synergistic effect of phage and antibiotic treatment which can be an effective option to control antibiotic resistant pathogens.

The Role of Bacterial Membrane Vesicles in the Dissemination of Antibiotic Resistance and as Promising Carriers for Therapeutic Agent Delivery.

The rapid emergence and spread of antibiotic-resistant bacteria continues to be an issue difficult to deal with, especially in the clinical, animal husbandry, and food fields. The occurrence of multidrug-resistant bacteria renders treatment with antibiotics ineffective. Therefore, the development of new therapeutic methods is a worthwhile research endeavor in treating infections caused by antibiotic-resistant bacteria. Recently, bacterial membrane vesicles (BMVs) have been investigated as a possible approach to drug delivery and vaccine development. The BMVs are released by both pathogenic and non-pathogenic Gram-positive and Gram-negative bacteria, containing various components originating from the cytoplasm and the cell envelope. The BMVs are able to transform bacteria with genes that encode enzymes such as proteases, glycosidases, and peptidases, resulting in the enhanced antibiotic resistance in bacteria. The BMVs can increase the resistance of bacteria to antibiotics. However, the biogenesis and functions of BMVs are not fully understood in association with the bacterial pathogenesis. Therefore, this review aims to discuss BMV-associated antibiotic resistance and BMV-based therapeutic interventions.

Variability in the Adaptive Response of Antibiotic-Resistant Salmonella Typhimurium to Environmental Stresses.

This study was designed to evaluate the resistance phenotype and genotype of wild type (WT)-, cefotaxime (CET)-, and ciprofloxacin (CIP)-induced Salmonella Typhimurium ATCC 19585, CIP-resistant Salmonella Typhimurium ATCC 19585, Salmonella Typhimurium CCARM 8009, and Salmonella Typhimurium KCCM 40253 before and after exposure to pH 4.5, 4% NaCl, and heat at 42°C. The susceptibilities of WT Salmonella Typhimurium ATCC 19585 and WT Salmonella Typhimurium KCCM 40253 to all antibiotics tested in this study were decreased after CET and CIP induction with the exception with kanamycin, meropenem, and polymyxin B. The highest ß-lactamase activities were 2.8 and 3.3 nmol/(min·mL), respectively, at the WT- and CET-induced Salmonella Typhimurium CCARM 8009. FT-IR spectra were found to be dominant at the region from 1,700 to 1,500 cm-1 corresponding to proteins such as amides I, II, and III. The relative expression levels of efflux pump-related genes (acrA, acrB, and TolC), porin-related gene (ompC), virulence-related gene (stn), adhesion-related gene (fimA), and stress-induced alternative sigma factor (rpoS) varied in the antibiotic resistance and stress exposure. This study provides useful information for understanding the antibiotic resistance profile, physicochemical property, and gene expression pattern in Salmonella Typhimurium in association with the induction of antibiotic resistance and exposure to environmental stresses.