Influence of environmental and genetic factors on 3-hydoxypropionaldehyde production by Lactobacillus reuteri.

The influence of environmental factors such as glycerol concentration, time of production, presence of Escherichia coli, and two different strains of Lactobacillus reuteri (ATCC 55730 and ATCC 53608) on 3-hydroxypropionaldehyde (3-HPA) production was analyzed. Additionally, the influence of those factors on gene expression in the 3-HPA production pathway was evaluated. The genes studied were GldC, cbiP, and Lreu_1734. The results of this study showed that the principal environmental factors that influence 3-HPA production are glycerol concentration and Lactobacillus reuteri strain. As glycerol concentration increased, 3-HPA content increased. The greatest 3-HPA concentration (56.6 mM ±5.99) was achieved by L. reuteri ATCC 55730. Gene expression was also affected by environmental factors. Factor that showed the greatest influence were also strain and glycerol concentration. The genes cbiP, GldC, and Lreu_1734 had basal gene expression in glycerol absence; however, glycerol regulated its expression. Glycerol induced overexpression of cbiP and GldC genes (Strain ATCC 53608), probably to ensure its efficient utilization. On the contrary, glycerol concentration suppressed Lre_1734 expression in both analyzed strains, as a mechanism for 3-HPA accumulation. Down-regulation was observed in all the genes tested in strain ATCC 55730, probably due to feedback inhibition by 3-HPA.

Oral Biofilm Composition, Dissemination to Keratinocytes, and Inflammatory Attenuation Depend on Probiotic and Synbiotic Strain Specificity.

Several inflammatory diseases are characterized by a disruption in the equilibrium between the host and its microbiome. Due to the increase in resistance, the use of antibiotics for the widespread, nonspecific killing of microorganisms is at risk. Pro-microbial approaches focused on stimulating or introducing beneficial species antagonistic toward pathobionts may be a viable alternative for restoring the host-microbiome equilibrium. Unfortunately, not all potential probiotic or synbiotic species and even subspecies (to strain level) are equally effective for the designated pathology, leading to conflicting accounts of their efficacy. To assess the extent of these species- and strain-specific effects, 13 probiotic candidates were evaluated for their probiotic and synbiotic potential with glycerol on in vitro oral biofilms, dissemination from biofilms to keratinocytes, and anti-inflammatory activity. Species- and strain-specific effects and efficacies were observed in how they functioned as probiotics or synbiotics by influencing oral pathobionts and commensals within biofilms and affected the dissemination of pathobionts to keratinocytes, ranging from ineffective strains to strains that reduced pathobionts by 3 + log. In addition, a minority of the candidates exhibited the ability to mitigate the inflammatory response of LPS-stimulated monocytes. For a comprehensive assessment of probiotic therapy for oral health, a judicious selection of fully characterized probiotic strains that are specifically tailored to the designated pathology is required. This approach aims to challenge the prevailing perception of probiotics, shifting the focus away from "form over function." Rather than using unproven, hypothetical probiotic strains from known genera or species, one should choose strains that are actually functional in resolving the desired pathology before labelling them probiotics.

Insights into the Mechanisms of Reuterin against Staphylococcus aureus Based on Membrane Damage and Untargeted Metabolomics.

Reuterin is a dynamic small-molecule complex produced through glycerol fermentation by Limosilactobacillus reuteri and has potential as a food biopreservative. Despite its broad-spectrum antimicrobial activity, the underlying mechanism of action of reuterin is still elusive. The present paper aimed to explore the antibacterial mechanism of reuterin and its effects on membrane damage and the intracellular metabolome of S. aureus. Our results showed that reuterin has a minimum inhibitory concentration of 18.25 mM against S. aureus, based on the 3-hydroxypropionaldehyde level. Key indicators such as extracellular electrical conductivity, membrane potential and permeability were significantly increased, while intracellular pH, ATP and DNA were markedly decreased, implying that reuterin causes a disruption to the structure of the cell membrane. The morphological damage to the cells was confirmed by scanning electron microscopy. Subsequent metabolomic analysis identified significant alterations in metabolites primarily involved in lipid, amino acid, carbohydrate metabolism and phosphotransferase system, which is crucial for cell membrane regulation and energy supply. Consequently, these findings indicated that the antibacterial mechanism of reuterin initially targets lipid and amino acid metabolism, leading to cell membrane damage, which subsequently results in energy metabolism disorder and, ultimately, cell death. This paper offers innovative perspectives on the antibacterial mechanism of reuterin, contributing to its potential application as a food preservative.

Effective encapsulation of reuterin-producing Limosilactobacillus reuteri in alginate beads prepared with different mucilages/gums.

The mainly aim of this study was to use mucilaginous solutions obtained from tamarind, mutamba, cassia tora, psyllium and konjac powdered to encapsulate reuterin-producing Limosilactobacillus reuteri in alginate beads by extrusion technique. In the particles were determined the bacterial encapsulation efficiency, cell viability during storage and survival under simulated gastric and intestinal conditions. Moreover, the reuterin production, its entrapment into the beads and the influence on viability of encapsulated microorganism were evaluated. Scanning electron microscopy and Fourier Transform Infrared spectroscopy were employed to characterize the produced particles. The beads showed a relatively spherical shape with homogenous distribution of L. reuteri. The use of gums and mucilages combined with alginate improved the encapsulation efficiency (from 93.2 to 97.4%), the viability of encapsulated bacteria during refrigerated storage (especially in prolonged storage of 20, 30 and 60 days) and the survival after exposure to gastric and enteric environments (from 67.7 to 76.6%). The L. reuteri was able to produce reuterin via bioconversion of glycerol in the film-forming solutions, and the entrapment of the metabolite was improved using konjac, mutamba and tamarind mucilaginous solutions in the encapsulation process (45, 44.57 and 41.25%, respectively). Thus, our findings confirm the great potential of these hydrocolloids to different further purposes, enabling its application as support material for delivery of chemical or biological compounds.

Production, Storage Stability, and Susceptibility Testing of Reuterin and Its Impact on the Murine Fecal Microbiome and Volatile Organic Compound Profile.

Background: Probiotics are generally considered as safe, but infections may rarely occur in vulnerable patients. Alternatives to live microorganisms to manage dysbiosis may be of interest in these patients. Reuterin is a complex component system exhibiting broad spectrum antimicrobial activity and a possible candidate substance in these cases. Methods: Reuterin supernatant was cultured from Lentilactobacillus diolivorans in a bioreactor in a two-step process. Storage stability at -20°C and effect of repeated freeze-thaw cycles were assessed by high performance liquid chromatography (HPLC). Antimicrobial activity was tested against Clostridium difficile, Listeria monocytogenes, Escherichia coli, Enterococcus faecium, Staphylococcus (S.) aureus, Staphylococcus epidermidis, Streptococcus (S.) agalactiae, Propionibacterium acnes, and Pseudomonas aeruginosae. Male BALBc mice were gavage fed with reuterin supernatant (n = 10) or culture medium (n = 10). Fecal volatile organic compounds (VOC) were assessed by gas chromatography mass spectroscopy; the microbiome was examined by 16S rRNA gene sequencing. Results: The supernatant contained 13.4 g/L reuterin (3-hydroxypropionaldehyde; 3-HPA). 3-HPA content remained stable at -20°C for 35 days followed by a slow decrease of its concentration. Repeated freezing/thawing caused a slow 3-HPA decrease. Antimicrobial activity was encountered against S. aureus, S. epidermidis, and S. agalactiae. Microbiome analysis showed no differences in alpha and beta diversity markers. Linear discriminant effect size (LEfSe) analysis identified Lachnospiraceae_bacterium_COE1 and Ruminoclostridium_5_uncultured_Clostridiales_ bacterium (in the reuterin medium group) and Desulfovibrio_uncultured_ bacterium, Candidatus Arthromitus, Ruminococcae_NK4A214_group, and Eubacterium_xylanophilum_group (in the reuterin group) as markers for group differentiation. VOC analysis showed a significant decrease of heptane and increase of 3-methylbutanal in the reuterin group. Conclusion: The supernatant produced in this study contained acceptable amounts of 3-HPA remaining stable for 35 days at -20°C and exhibiting an antimicrobial effect against S. aureus, S. agalactiae, and S. epidermidis. Under in vivo conditions, the reuterin supernatant caused alterations of the fecal microbiome. In the fecal, VOC analysis decreased heptane and increased 3-methylbutanal were encountered. These findings suggest the high potential of the reuterin system to influence the intestinal microbiome in health and disease, which needs to be examined in detail in future projects.

Reuterin-producing Limosilactobacillus reuteri: Optimization of in situ reuterin production in alginate-based filmogenic solutions.

Limosilactobacillus reuteri produces reuterin via glycerol anaerobic fermentation. This compound has antimicrobial properties and is used for food preservation purposes. Filmogenic solutions constituted of polysaccharides and glycerol are also employed, however, reuterin synthesis in filmogenic solutions has not yet been reported. Thus, the aim of this study was to optimize the in situ reuterin production by L. reuteri in alginate- and glycerol based-filmogenic solution, evaluating the survival of reuterin-producing bacteria during fermentation. The study consisted of a completely randomized design employing two L. reuteri strains (DSM 20016 and DSM 17938). The filmogenic solutions were obtained using sodium alginate (20 g/L) and two independent variables were studied: glycerol (0-300 mmol/L) and initial biomass of L. reuteri (≅6, 7, and 8 log CFU/mL). The samples were analyzed every 24 h for 72 h of anaerobic fermentation (37 °C). Both L. reuteri strains confirmed the potential for reuterin production and were susceptible to the metabolite produced. The highest reuterin production was achieved using L. reuteri DSM 20016. The initial microbial biomass of 8 log CFU/mL and 100 mmol/L of glycerol increased the reuterin production. However, higher conversion yields from glycerol to reuterin were obtained using 50 mmol/L of substrate.

The presence of 3-hydroxypropionate and 1,3-propanediol suggests an alternative path for conversion of glycerol to Acetyl-CoA.

BACKGROUND: In our recent study using [U-13C3]glycerol, a small subset of hamsters showed an unusual profile of glycerol metabolism: negligible gluconeogenesis from glycerol plus conversion of glycerol to 1,3-propanediol (1,3PDO) and 3-hydroxypropionate (3HP) which were detected in the liver and blood. The purpose of the current study is to evaluate the association of these unusual glycerol products with other biochemical processes in the liver. METHODS: Fasted hamsters received acetaminophen (400 mg/kg; n = 16) or saline (n = 10) intraperitoneally. After waiting 2 h, all the animals received [U-13C3]glycerol intraperitoneally. Liver and blood were harvested 1 h after the glycerol injection for NMR analysis and gene expression assays. RESULTS: 1,3PDO and 3HP derived from [U-13C3]glycerol were detected in the liver and plasma of eight hamsters (two controls and six hamsters with acetaminophen treatment). Glycerol metabolism in the liver of these animals differed substantially from conventional metabolic pathways. [U-13C3]glycerol was metabolized to acetyl-CoA as evidenced with downstream products detected in glutamate and ß-hydroxybutyrate, yet 13C labeling in pyruvate and glucose was minimal (p < 0.001, 13C labeling difference in each metabolite). Expression of aldehyde dehydrogenases was enhanced in hamster livers with 1,3PDO and 3HP (p < 0.05). CONCLUSION: Detection of 1,3PDO and 3HP in the hamster liver was associated with unorthodox metabolism of glycerol characterized by conversion of 3HP to acetyl-CoA followed by ketogenesis and oxidative metabolism through the TCA cycle. Additional mechanistic studies are needed to determine the causes of unusual glycerol metabolism in a subset of these hamsters.

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.

Coenzyme A-acylating propionaldehyde dehydrogenase (PduP) from Lactobacillus reuteri: kinetic characterization and molecular modeling.

3-Hydroxypropionic acid (3-HP), an important C3 chemical for a bio-based industry, is natively produced by Lactobacillus reuteri from glycerol. Conversion of glycerol occurs via the intermediate 3-hydroxypropionaldehyde (3-HPA), followed by an ATP-producing pathway initiated by the CoA-acylating propionaldehyde dehydrogenase (PduP). The pduP gene of L. reuteri was cloned and expressed in Escherichia coli and the recombinant enzyme was purified to homogeneity for characterization of its activity and properties. Kinetic studies with propionaldehyde as substrate showed a maximum specific activity of 28.9 U/mg, which is 80-fold higher than that reported previously. Maximum activity of 18 U/mg was obtained at 3-HPA concentration of 7 mM, above which substrate inhibition was observed. Substrate inhibition was also seen with coenzyme A at a concentration above 0.5mM and with NADP(+) above 9 mM. A structure of PduP is proposed based on homology modeling. In silico docking of the co-factors coenzyme A and NAD(+), respectively, showed a common binding site consisting of amino acids Thr145, Ile275, Cys277 and Ser417, which through site-directed mutagenesis to alanine and kinetic studies, were confirmed as essential for the catalytic activity of PduP.