High cell density sequential batch fermentation for enhanced propionic acid production from glucose and glycerol/glucose mixture using Acidipropionibacterium acidipropionici.

BACKGROUND: Propionic acid fermentation from renewable feedstock suffers from low volumetric productivity and final product concentration, which limits the industrial feasibility of the microbial route. High cell density fermentation techniques overcome these limitations. Here, propionic acid (PA) production from glucose and a crude glycerol/glucose mixture was evaluated using Acidipropionibacterium acidipropionici, in high cell density (HCD) batch fermentations with cell recycle. The agro-industrial by-product, heat-treated potato juice, was used as N-source. RESULTS: Using 40 g/L glucose for nine consecutive batches yielded an average of 18.76 ± 1.34 g/L of PA per batch (0.59 gPA/gGlu) at a maximum rate of 1.15 gPA/L.h, and a maximum biomass of 39.89 gCDW/L. Succinic acid (SA) and acetic acid (AA) were obtained as major by-products and the mass ratio of PA:SA:AA was 100:23:25. When a crude glycerol/glucose mixture (60 g/L:30 g/L) was used for 6 consecutive batches with cell recycle, an average of 35.36 ± 2.17 g/L of PA was obtained per batch (0.51 gPA/gC-source) at a maximum rate of 0.35 g/L.h, and reaching a maximum biomass concentration of 12.66 gCDW/L. The PA:SA:AA mass ratio was 100:29:3. Further addition of 0.75 mg/L biotin as a supplement to the culture medium enhanced the cell growth reaching 21.89 gCDW/L, and PA productivity to 0.48 g/L.h, but also doubled AA concentration. CONCLUSION: This is the highest reported productivity from glycerol/glucose co-fermentation where majority of the culture medium components comprised industrial by-products (crude glycerol and HTPJ). HCD batch fermentations with cell recycling are promising approaches towards industrialization of the bioprocess.

A novel synergistic enzyme-antibiotic therapy with immobilization of mycobacteriophage Lysin B enzyme onto Rif@UiO-66 nanocomposite for enhanced inhaled anti-TB therapy; Nanoenzybiotics approach.

The emergence of antibiotic-resistant and phage-resistant strains of Mycobacterium tuberculosis (M. tuberculosis) necessitates improving new therapeutic plans. The objective of the current work was to ensure the effectiveness of rifampicin and the mycobacteriophage LysB D29 (LysB)enzyme in the treatment of multi-drug resistant tuberculosis (MDR-TB) infection, where new and safe metal-organic framework (MOF) nanoparticles were used in combination. UiO-66 nanoparticles were synthesized under mild conditions in which the antimycobacterial agent (rifampicin) was loaded (Rif@UiO-66) and LysB D29 enzyme immobilized onto Rif@UiO-66, which were further characterized. Subsequently, the antibacterial activity of different ratios of Rif@UiO-66 and LysB/Rif@uio-66 against the nonpathogenic tuberculosis model Mycobacterium smegmatis (M. smegmatis) was evaluated by minimum inhibitory concentration (MIC) tests. Impressively, the MIC of LysB/Rif@uio-66 was 16-fold lower than that of pure rifampicin. In vitro and in vivo toxicity studies proved that LysB/Rif@UiO-66 is a highly biocompatible therapy for pulmonary infection. A biodistribution assay showed that LysB/Rif@UiO-66 showed a 5.31-fold higher drug concentration in the lungs than free rifampicin. A synergistic interaction between UiO-66, rifampicin and the mycobacteriophage lysB D29 enzyme was shown in the computational method (docking). Therefore, all results indicated that the LysB/Rif@UiO-66 nanocomposite exhibited promising innovative enzyme-antibiotic therapy for tuberculosis treatment.

Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays.

In a precarious world of rapidly growing pandemics, the field of vaccine production has witnessed considerable growth. Bacillus Calmette-Guérin (BCG) is a live-attenuated vaccine and a part of the immunization program in 157 countries. The quality control is based on a potency test through viable cell enumeration. The colony-forming unit (CFU) assay is the official method, however, it often yields fluctuating results, suffers from medium cracking, and requires lengthy analysis (~ 28 days). Flow cytometric analysis was proposed earlier, but it was coupled with a Coulter counter for measuring the entire bacterial population (live/dead). In the present study, thiazole orange/propidium iodide dyes supplemented with fluorogenic reference beads were employed for viable counting, eliminating the need for a Coulter counter. Both the flow cytometry and the colorimetric technique employing tetrazolium salt were validated and compared to the CFU assay. The colorimetric assay displayed high precision, accuracy, and a strong positive correlation with the CFU assay. The flow cytometry assay demonstrated high precision and a notable ability to distinguish different forms of BCG cells (live, injured, and dead). It also exhibited a perfect positive correlation with the CFU assay. Both methods reduced the analysis time by > 26 days and eliminated the need for human intervention by automating the test.

Membrane-based continuous fermentation with cell recycling for propionic acid production from glycerol by Acidipropionibacterium acidipropionici.

BACKGROUND: Microbial production of propionic acid (PA) from renewable resources is limited by the slow growth of the producer bacteria and product-mediated inhibition. The present study evaluates high cell density continuous PA fermentation from glycerol (Gly) using Acidipropionibacterium acidipropionici DSM 4900 in a membrane-based cell recycling system. A ceramic tubular membrane filter of 0.22 µm pore size was used as the filtering device for cell recycling. The continuous fermentations were run sequentially at dilution rates of 0.05 and 0.025 1/h using varying glycerol concentrations and two different yeast extract concentrations. RESULTS: PA volumetric productivity of 0.98 g/L.h with a product yield of 0.38 gPA/gGly was obtained with 51.40 g/L glycerol at a yeast extract concentration of 10 g/L. Increasing the glycerol and yeast extract concentrations to 64.50 g/L and 20 g/L, respectively, increased in PA productivity, product yield, and concentration to 1.82 g/L.h, 0.79 gPA/gGly, and 38.37 g/L, respectively. However, lowering the dilution rate to 0.025 1/h reduced the production efficiency. The cell density increased from 5.80 to 91.83 gCDW/L throughout the operation, which lasted for a period of 5 months. A tolerant variant of A. acidipropoinici exhibiting growth at a PA concentration of 20 g/L was isolated at the end of the experiment. CONCLUSIONS: Applying the current approach for PA fermentation can overcome several limitations for process industrialization.

Irinotecan-gut microbiota interactions and the capability of probiotics to mitigate Irinotecan-associated toxicity.

BACKGROUND: Irinotecan is a chemotherapeutic agent used to treat a variety of tumors, including colorectal cancer (CRC). In the intestine, it is transformed into SN-38 by gut microbial enzymes, which is responsible for its toxicity during excretion. OBJECTIVE: Our study highlights the impact of Irinotecan on gut microbiota composition and the role of probiotics in limiting Irinotecan-associated diarrhea and suppressing gut bacterial ß-glucuronidase enzymes. MATERIAL AND METHODS: To investigate the effect of Irinotecan on the gut microbiota composition, we applied 16S rRNA gene sequencing in three groups of stool samples from healthy individuals, colon cancer, and Irinotecan treated patients (n = 5/group). Furthermore, three Lactobacillus spp.; Lactiplantibacillus plantarum (L. plantarum), Lactobacillus acidophilus (L. acidophilus), Lacticaseibacillus rhamnosus (L. rhamnosus) were used in a single and mixed form to in-vitro explore the effect of probiotics on the expression of ß-glucuronidase gene from E. coli. Also, probiotics were introduced in single and mixed forms in groups of mice before the administration of Irinotecan, and their protective effects were explored by assessing the level of reactive oxidative species (ROS) as well as studying the concomitant intestinal inflammation and apoptosis. RESULTS: The gut microbiota was disturbed in individuals with colon cancer and after Irinotecan treatment. In the healthy group, Firmicutes were more abundant than Bacteriodetes, which was the opposite in the case of colon-cancer or Irinotecan treated groups. Actinobacteria and Verrucomicrobia were markedly present within the healthy group, while Cyanobacteria were noted in colon-cancer and the Irinotecan-treated groups. Enterobacteriaceae and genus Dialister were more abundant in the colon-cancer group than in other groups. The abundance of Veillonella, Clostridium, Butryicicoccus, and Prevotella were increased in Irinotecan-treated groups compared to other groups. Using Lactobacillus spp. mixture in mice models significantly relieved Irinotecan-induced diarrhea through the reduction of both ß-glucuronidase expression and ROS, in addition to guarding gut epithelium against microbial dysbiosis and proliferative crypt injury. CONCLUSIONS: Irinotecan-based chemotherapy altered intestinal microbiota. The gut microbiota participates greatly in determining both the efficacy and toxicity of chemotherapies, of which the toxicity of Irinotecan is caused by the bacterial ß-glucuronidase enzymes. The gut microbiota can now be aimed and modulated to promote efficacy and decrease the toxicity of chemotherapeutics. The used probiotic regimen in this study lowered mucositis, oxidative stress, cellular inflammation, and apoptotic cascade induction of Irinotecan.

Mechanistic study of copper oxide, zinc oxide, cadmium oxide, and silver nanoparticles-mediated toxicity on the probiotic Lactobacillus reuteri.

The use of metal/metal oxide nanoparticles (NPs) in consumer products has increased dramatically. Accordingly, human exposure to these NPs has increased. Lactobacillus reuteri, a member of the beneficial gut microbiota, is essential for human health. In the present study, the toxic effect of three metal oxides (CuO, ZnO, and CdO) and one metal (Ag) NPs on L. reuteri were investigated in vitro. L. reuteri was susceptible to all the prepared NPs in a dose-dependent manner, visualized as an increase in the zones of inhibition and a significant reduction in the maximum specific growth rates (µmax). The minimal inhibitory concentrations were 5.8, 26, 560, and 560 µg/mL for CdO-, Ag-, ZnO-, and CuO-NPs, respectively, and the respective minimal bactericidal concentrations were 60, 70, 1500, and 1500 µg/mL. Electron microscopic examinations revealed the adsorption of the prepared NPs on L. reuteri cell surface, causing cell wall disruption and morphological changes. These changes were accompanied by significant leakage of cellular protein content by 214%, 191%, 112%, and 101% versus the untreated control when L. reuteri was treated with CdO-, Ag-, CuO-, and ZnO-NPs, respectively. NPs also induced oxidative damage, where the malondialdehyde level was significantly increased, and glutathione content was significantly decreased. Quantifying the DNA damage using comet assay showed that CuONPs had the maximum DNA tail length (8.2 px vs. 2.1 px for the control). While CdONPs showed the maximum percentage of DNA in tail (15.5% vs. 3.1%). This study provides a mechanistic evaluation of the NPs-mediated toxicity to a beneficial microorganism.

The Specific Capsule Depolymerase of Phage PMK34 Sensitizes Acinetobacter baumannii to Serum Killing.

The rising antimicrobial resistance is particularly alarming for Acinetobacter baumannii, calling for the discovery and evaluation of alternatives to treat A. baumannii infections. Some bacteriophages produce a structural protein that depolymerizes capsular exopolysaccharide. Such purified depolymerases are considered as novel antivirulence compounds. We identified and characterized a depolymerase (DpoMK34) from Acinetobacter phage vB_AbaP_PMK34 active against the clinical isolate A. baumannii MK34. In silico analysis reveals a modular protein displaying a conserved N-terminal domain for anchoring to the phage tail, and variable central and C-terminal domains for enzymatic activity and specificity. AlphaFold-Multimer predicts a trimeric protein adopting an elongated structure due to a long α-helix, an enzymatic ß-helix domain and a hypervariable 4 amino acid hotspot in the most ultimate loop of the C-terminal domain. In contrast to the tail fiber of phage T3, this hypervariable hotspot appears unrelated with the primary receptor. The functional characterization of DpoMK34 revealed a mesophilic enzyme active up to 50 °C across a wide pH range (4 to 11) and specific for the capsule of A. baumannii MK34. Enzymatic degradation of the A. baumannii MK34 capsule causes a significant drop in phage adsorption from 95% to 9% after 5 min. Although lacking intrinsic antibacterial activity, DpoMK34 renders A. baumannii MK34 fully susceptible to serum killing in a serum concentration dependent manner. Unlike phage PMK34, DpoMK34 does not easily select for resistant mutants either against PMK34 or itself. In sum, DpoMK34 is a potential antivirulence compound that can be included in a depolymerase cocktail to control difficult to treat A. baumannii infections.

Engineering a Lysin with Intrinsic Antibacterial Activity (LysMK34) by Cecropin A Fusion Enhances Its Antibacterial Properties against Acinetobacter baumannii.

Bacteriophage-encoded lysins are increasingly reported as alternatives to combat Acinetobacter baumannii infections, for which limited therapeutic options are available. Some lysins, such as LysMK34, have a C-terminal amphipathic helix allowing them to penetrate the otherwise-impermeable outer membrane barrier. Another approach to kill Gram-negative pathogens with lysins relies on fusion of a peptide with outer membrane-permeabilizing properties to the lysin. In this work, we aimed to leverage the intrinsic antibacterial activity of LysMK34 by fusing the peptide cecropin A to its N terminus via a linker of three Ala-Gly repeats, resulting in engineered LysMK34 (eLysMK34). The engineered lysin has an improved antibacterial activity compared to that of the parental lysin, LysMK34, in terms of MICs (0.45 to 1.2 µM), killing rate, and killing extent. eLysMK34 has a ≥2-fold-increased activity against stationary-phase cells, and the bactericidal effect becomes less dependent on the intracellular osmotic pressure. In particular, colistin-resistant strains become highly susceptible to eLysMK34, and enhanced antibacterial activity is observed in complement-deactivated human serum. These observations demonstrate that fusion of a lysin with intrinsic antibacterial activity with a selected outer membrane-permeabilizing peptide is a useful strategy to further improve the in vitro antibacterial properties of such lysins. IMPORTANCE Phage lysins are a new class of enzyme-based antibiotics that increasingly gain interest. Lysins kill cells through rapid degradation of the peptidoglycan layer, resulting in sudden osmotic lysis. Whereas Gram-positive bacteria are readily susceptible to the actions of lysins, Gram-negative bacteria are naturally resistant, as the outer membrane protects their peptidoglycan layer. This work reveals that fusing an outer membrane-permeabilizing peptide to a lysin with intrinsic antibacterial activity results in a superior lysin that shows improved robustness in its antibacterial activity, including against the most worrisome colistin-resistant A. baumannii strains.

The inhibitory effect of dextranases from Bacillus velezensis and Pseudomonas stutzeri on Streptococcus mutans biofilm.

Background and Objectives: Dental caries is a breakdown of the teeth enamel due to harmful bacteria, lack of oral hygiene, and sugar consumption. The acid-producing bacterium Streptococcus mutans is the leading cause of dental caries. Dextranase is an enzyme that can degrade dextran to low molecular weight fractions, which have many therapeutic and industrial applications. The purpose of the present study was to isolate a novel dextranase-producing bacteria from a source (molasses). The cell-free extracts containing dextranases were tested as antibiofilm agents. Materials and Methods: Dextranase-producing bacteria were identified using phenotypic and genotypic methods such as 16S rRNA gene sequencing and enzymatic characterization. Results: The highest six dextranase-producing bacterial isolates were Bacillus species. The best conditions for dextranase productivity were obtained after 72 hours of culture time at pH 7. The addition of glucose to the medium enhanced the production of the enzymes. The cell-free extract of the six most active isolates showed remarkable activity against biofilm formation by Streptococcus mutans ATCC 25175. The highest inhibition activities reached 60% and 80% for Bacillus velezensis and Pseudomonas stutzeri, respectively. Conclusion: Therefore, our study added to the current dextranase-producing bacteria with potential as a source of dextranases.

Microbial etiologies of ventilator-associated pneumonia (VAP) in intensive care unit of Beni-Suef University's Hospital.

BACKGROUND: Ventilator-associated pneumonia (VAP) is a major health problem for people intubated in intensive care units (ICUs), leading to increased mortality rates, hospital stay, and treatment costs. In the present study, the core pathogens causing VAP in Beni-Suef University's Hospital, Egypt, was investigated over a study period of 2 years (2017-2019). RESULTS: Of a total of 213 patients subjected to mechanical ventilation, 60 have developed VAP during their stay in the ICU. The mortality rate reached 41.7% among VAP patients. Sixty bacteria were isolated from an endotracheal aspirate of hospitalized patients. The different isolates were cultured followed by running biochemical tests, sensitivity assays, and automated VITEK®2 System analysis. Unexpectedly, all the isolates were Gram-negative bacteria. Klebsiella pneumoniae were the main pathogen encountered (27/60 isolates) followed by Acientobacter baumannnii (7/60) and other microorganisms belonging to the genera Moraxella, Escherichia, and Pseudomonas (11/60). Antibiotic sensitivity testing was performed via the VITEK®2 System using up to 16 different antibiotics representing 8 different antibiotic classes and subclasses (aminoglycosides, carbapenems, fluoroquinolones, penicillin/ß-lactamase inhibitor, extended-spectrum cephalosporins, aminopenicillins, aminopenicillins/ß-lactamase inhibitor, folic acid synthesis inhibitor). Majority of the isolates (28/60) showed a remarkable extensive drug resistance (XDR) pattern, while 15 isolates were multi-drug resistant (MDR) and only 6 were pan-drug resistant (PDR) with regard to antibiotics under evaluation. CONCLUSION: The association of VAP with multi-drug-resistant bacteria is alarming, and rapid management is crucial. Identification of core pathogens is essential for identifying the most appropriate technique for infection control.

Metabolic potential of the moderate halophile Yangia sp. ND199 for co-production of polyhydroxyalkanoates and exopolysaccharides.

Yangia sp. ND199 is a moderately halophilic bacterium isolated from mangrove samples in Northern Vietnam, which was earlier reported to grow on several sugars and glycerol to accumulate poly(hydroxyalkanoates) (PHA). In this study, the potential of the bacterium for co-production of exopolysaccharides (EPS) and PHA was investigated. Genome sequence analysis of the closely related Yangia sp. CCB-M3 isolated from mangroves in Malaysia revealed genes encoding enzymes participating in different EPS biosynthetic pathways. The effects of various cultivation parameters on the production of EPS and PHA were studied. The highest level of EPS (288 mg/L) was achieved using sucrose and yeast extract with 5% NaCl and 120 mM phosphate salts but with modest PHA accumulation (32% of cell dry weight, 1.3 g/L). Growth on fructose yielded the highest PHA concentration (85% of CDW, 3.3 g/L) at 90 mM phosphate and higher molecular weight EPS at 251 mg/L yield at 120 mM phosphate concentration. Analysis of EPS showed a predominance of glucose, followed by fructose and galactose, and minor amounts of acidic sugars.

Heterologous expression of lytic polysaccharide monooxygenases (LPMOs).

Lytic polysaccharide monooxygenases (LPMOs) are relatively new enzymes that have been discovered 10 years ago. LPMOs comprise a diverse group of enzymes which play a pivotal role in the depolymerization of sugar-based biopolymers including cellulose, hemicellulose, chitin, and starch. Their mechanism of action relies on the correct coordination of a copper ion in the active site, which is partly composed of the N-terminal histidine. Therefore, correct secretion and folding of these copper-enzymes is fundamental for obtaining a catalytic activity. LPMOs occur in all kingdoms of life; they have been found in viruses, bacteria and eukaryotes, including fungi, plants and animals. In many cases, using homologous expression of these proteins is not feasible and an alternative organism, which can be cultured and is able to heterologously express the protein of interest, is required for studying enzyme properties. Therefore, we made an extensive compilation of expression techniques used for LPMOs the expression and characterization of which have been reported to date. In the current review, we provide a summary of the different techniques, including expression hosts and vectors, secretion methods, and culturing conditions, that have been used for the overexpression and production of this important class of enzymes at laboratory scale. Herein, we compare these techniques and assess their advantages and disadvantages.

Lysin LysMK34 of Acinetobacter baumannii Bacteriophage PMK34 Has a Turgor Pressure-Dependent Intrinsic Antibacterial Activity and Reverts Colistin Resistance.

The prevalence of extensively and pandrug-resistant strains of Acinetobacter baumannii leaves little or no therapeutic options for treatment for this bacterial pathogen. Bacteriophages and their lysins represent attractive alternative antibacterial strategies in this regard. We used the extensively drug-resistant A. baumannii strain MK34 to isolate the bacteriophage PMK34 (vB_AbaP_PMK34). This phage shows fast adsorption and lacks virulence genes; nonetheless, its narrow host spectrum based on capsule recognition limits broad application. PMK34 is a Fri1virus member of the Autographiviridae and has a 41.8-kb genome (50 open reading frames), encoding an endolysin (LysMK34) with potent muralytic activity (1,499.9 ± 131 U/µM), a typical mesophilic thermal stability up to 55°C, and a broad pH activity range (4 to 10). LysMK34 has an intrinsic antibacterial activity up to 4.8 and 2.4 log units for A. baumannii and Pseudomonas aeruginosa strains, respectively, but only when a high turgor pressure is present. The addition of 0.5 mM EDTA or application of an osmotic shock after treatment can compensate for the lack of a high turgor pressure. The combination of LysMK34 and colistin results in up to 32-fold reduction of the MIC of colistin, and colistin-resistant strains are resensitized in both Mueller-Hinton broth and 50% human serum. As such, LysMK34 may be used to safeguard the applicability of colistin as a last-resort antibiotic.IMPORTANCEA. baumannii is one of the most challenging pathogens for which development of new and effective antimicrobials is urgently needed. Colistin is a last-resort antibiotic, and even colistin-resistant A. baumannii strains exist. Here, we present a lysin that sensitizes A. baumannii for colistin and can revert colistin resistance to colistin susceptibility. The lysin also shows a strong, turgor pressure-dependent intrinsic antibacterial activity, providing new insights in the mode of action of lysins with intrinsic activity against Gram-negative bacteria.

Exploring the Enzymatic and Antibacterial Activities of Novel Mycobacteriophage Lysin B Enzymes.

Mycobacteriophages possess different sets of lytic enzymes for disruption of the complex cell envelope of the mycobacteria host cells and release of the viral progeny. Lysin B (LysB) enzymes are mycolylarabinogalactan esterases that cleave the ester bond between the arabinogalactan and mycolic acids in the mycolylarabinogalactan-peptidoglycan (mAGP) complex in the cell envelope of mycobacteria. In the present study, four LysB enzymes were produced recombinantly and characterized with respect to their enzymatic and antibacterial activities. Examination of the kinetic parameters for the hydrolysis of para-nitrophenyl ester substrates, shows LysB-His6 enzymes to be active against a range of substrates (C4-C16), with a catalytic preference towards p-nitrophenyl laurate (C12). With p-nitrophenyl butyrate as substrate, LysB-His6 enzymes showed highest activity at 37 °C. LysB-His6 enzymes also hydrolyzed different Tween substrates with highest activity against Tween 20 and 80. Metal ions like Ca2+ and Mn2+ enhanced the enzymatic activity of LysB-His6 enzymes, while transition metal ions like Zn2+ and Cu2+ inhibited the enzymatic activity. The mycolylarabinogalactan esterase activity of LysB-His6 enzymes against mAGP complex was confirmed by LC-MS. LysB-His6 enzymes showed marginal antibacterial activity when tested alone against Mycobacterium smegmatis, however a synergetic activity was noticed when combined with outer membrane permealizers. These results confirm that LysB enzymes are lipolytic enzymes with potential application as antimycobacterials.

Enantioselective sulfoxidation using Streptomyces glaucescens GLA.0.

Asymmetric oxidation of prochiral sulfides is a direct means for production of enantiopure sulfoxides which are important in organic synthesis and the pharmaceutical industry. In the present study, Streptomyces glaucescens GLA.0 was employed for stereoselective oxidation of prochiral sulfides. Growing cells selectively catalyzed the oxidation of phenyl methyl sulfide to the corresponding sulfoxide. Only very little overoxidation was observed, resulting in minor amounts of the unwanted sulfone. Addition of isopropyl alcohol as a co-solvent, time of substrate addition and composition of the reaction media resulted in enhanced phenyl methyl sulfide biotransformation. The concentration of the undesired by-product (sulfone) was as low as 4% through the reaction course under optimal reaction conditions. The results show that S. glaucescens GLA.0 is a promising whole-cell biocatalyst for preparing highly enantiopure (R)-phenyl methyl sulfoxide in high yield (90%) with an enantiomeric excess (ee) exceeding 99%.

The Preclinical and Clinical Progress of Bacteriophages and Their Lytic Enzymes: The Parts are Easier than the Whole.

The therapeutic potential of phages has been considered since their first identification more than a century ago. The evident concept of using a natural predator to treat bacterial infections has, however, since then been challenged considerably. Initially, the vast success of antibiotics almost eliminated the study of phages for therapy. Upon the renaissance of phage therapy research, the most provocative and unique properties of phages such as high specificity, self-replication and co-evolution prohibited a rapid preclinical and clinical development. On the one hand, the typical trajectory followed by small molecule antibiotics could not be simply translated into the preclinical analysis of phages, exemplified by the need for complex broad spectrum or personalized phage cocktails of high purity and the more complex pharmacokinetics. On the other hand, there was no fitting regulatory framework to deal with flexible and sustainable phage therapy approaches, including the setup and approval of adequate clinical trials. While significant advances are incrementally made to eliminate these hurdles, phage-inspired antibacterials have progressed in the slipstream of phage therapy, benefiting from the lack of hurdles that are typically associated with phage therapy. Most advanced are phage lytic enzymes that kill bacteria through peptidoglycan degradation and osmotic lysis. Both phages and their lytic enzymes are now widely considered as safe and have now progressed to clinical phase II to show clinical efficacy as pharmaceutical. Yet, more initiatives are needed to fill the clinical pipeline to beat the typical attrition rates of clinical evaluation and to come to a true evaluation of phages and phage lytic enzymes in the clinic.

Lactobacillus reuteri NAD(P)H oxidase: Properties and coexpression with propanediol-utilization enzymes for enhancing 3-hydroxypropionic acid production from 3-hydroxypropionaldehyde.

Lactobacillus reuteri metabolizes glycerol through propanediol-utilization (Pdu) pathway to 1,3-propanediol (1,3-PD) via 3-hydroxypropionaldehyde (3-HPA) as intermediate. In the resting cells, the oxidized co-factor obtained in the reaction is regenerated by simultaneous oxidation of 3-HPA to 3-hydroxypropionic acid (3-HP) using propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL) and propionate kinase (PduW). We have earlier shown that the use of resting cells of recombinant Escherichia coli expressing the oxidative pathway gives the highest theoretical yield of 1 mol 3-HP per mol 3-HPA but is limited by cofactor depletion. In the present study, the gene encoding the enzyme NAD(P)H oxidase (LreuNox) that utilizes molecular oxygen as substrate, was isolated from L. reuteri and heterologously overexpressed in E. coli. LreuNox has a pH optimum of 6 and exhibits Vmax of 101.1 ± 2.2 U/mg with NADH, which is 30% higher than that for NADPH. Co-expression of LreuNox with PduP, PduL and PduW in E. coli enhances the biocatalytic lifetime as well as productivity at least two-fold compared to that achieved without co-factor regeneration.

Biocementation of soil by calcite/aragonite precipitation using Pseudomonas azotoformans and Citrobacter freundii derived enzymes.

Microbial geotechnology is the use of microorganisms and/or their derivatives to alter engineering properties of soil for improving its stability, strength and stiffness. Ureases hydrolyze urea in the soil leading to CaCO3 precipitation, which binds soil particles together (biocementation). In the present study, nine Egyptian soils were screened for urease-producing bacteria, 15 isolates were obtained, and optimum urease producers were identified. Growth kinetics were measured at different pH values and in the presence of molasses as the sole carbon source. Citrobacter freundii and Pseudomonas azotoformans showed the highest extracellular urease activities of 45.5 ± 3.4 and 54.9 ± 3.5 U ml-1, respectively. Cell-free supernatants of these isolates mediated the precipitation of CaCO3 from the cementation solution (urea and CaCl2). The X-ray diffraction (XRD) of the precipitates revealed the formation of calcite and aragonite crystal forms. Sandy soil treated with the supernatants and evaluated by modified proctor and California bearing ratio (CBR) tests had significantly higher (P < 0.05) soil strength (CBR = ∼40% versus 30% for untreated soil). Scanning electron microscopy showed the CaCO3 precipitation resulting in reduction of the gaps between soil particles, hence confirming the biocementation phenomenon which is responsible for soil stabilization and the desired repairing effect on cracks. The use of urease-containing cell-free supernatant rather than the whole microorganism in biocementation lowers the risks of spreading pathogens to the environment and altering the microbial diversity at the application area.

Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).

Mycobacteriophage endolysins have emerged as a potential alternative to the current antimycobacterial agents. This study focuses on mycolylarabinogalactan hydrolase (LysB) enzymes of the α/ß-hydrolase family, which disrupt the unique mycolic acid layer of mycobacterium cell wall. Multiple sequence alignment and structural analysis studies showed LysB-D29, the only enzyme with a solved three-dimensional structure, to share several common features with esterases (lacking lid domain) and lipases (acting on long chain lipids). Sequence and structural comparisons of 30 LysB homology models showed great variation in domain organizations and total protein length with major differences in the loop-5 motif harboring the catalytic histidine residue. Docking of different p-nitrophenyl ligands (C4-C18) to LysB-3D models revealed that the differences in length and residues of loop-5 contributed towards wide diversity of active site conformations (long tunnels, deep and superficial funnels, shallow bowls, and a narrow buried cave) resembling that of lipases, cutinases, and esterases. A set of seven LysB enzymes were recombinantly produced; their activity against p-nitrophenyl esters could be related to their active site conformation and acyl binding site. LysB-D29 (long tunnel) showed the highest activity with long chain p-nitrophenyl palmitate followed by LysB-Omega (shallow bowl) and LysB-Saal (deep funnel).

Lactic acid bacteria: from starter cultures to producers of chemicals.

Lactic acid bacteria constitute a diverse group of industrially significant, safe microorganisms that are primarily used as starter cultures and probiotics, and are also being developed as production systems in industrial biotechnology for biocatalysis and transformation of renewable feedstocks to commodity- and high-value chemicals, and health products. Development of strains, which was initially based mainly on natural approaches, is also achieved by metabolic engineering that has been facilitated by the availability of genome sequences and genetic tools for transformation of some of the bacterial strains. The aim of this paper is to provide a brief overview of the potential of lactic acid bacteria as biological catalysts for production of different organic compounds for food and non-food sectors based on their diversity, metabolic- and stress tolerance features, as well as the use of genetic/metabolic engineering tools for enhancing their capabilities.