Applied Microbiology and Biotechnology最新文献

Protein-glutamine glutaminases (PGs; EC 3.5.1.44) have gained attention in the food industry due to their application in plant protein products. The recently discovered PG from Bacteroides helcogenes (PGB) has especially been shown to provide promising characteristics for improving the techno-functional properties of plant proteins. A prerequisite for food enzymes, such as the PG, is their production with an expression host that meets food safety and yield requirements. The antibiotic-free and secretory production of the PGB was targeted in this study using the undomesticated Bacillus subtilis 007. The CRISPR/Cas9-mediated approach enabled specific genomic PGB integrations, while simultaneously deleting unwanted B. subtilis traits. Firstly, the PGB expression cassette was integrated into the sigF gene, leading to an asporogenic strain and extracellular activity of 4.1 µkat/L_culture in bioreactor cultivations. However, excessive foaming hampered the production process tremendously. Consequently, a second PGB copy was integrated into the sfp locus, which is involved in the production of lipopeptides, such as surfactin. As a result, the PGB activity was increased to 5.4 µkat/L_culture, and foaming during cultivation was reduced significantly. The introduction of a third PGB copy for preventing cell motility did not increase production; however, the integration into the well-established amyE locus improved the PGB yield during reactor cultivations. A final extracellular activity of 9.5 µkat/L_culture was reached. The multiple genomic integrations of the PGB gene enabled the efficient PGB secretion in an optimized B. subtilis host without the need for antibiotics.

• Site-specific PGB integration enabled by genome sequencing of B. subtilis 007.

• Antibiotic-free and secretory PGB production with an optimized B. subtilis host.

• Increased PGB production reaching 9.5 µkat/L_culture.

Since plastics pose the greatest threat to humanity, it is essential to find an economic and sustainable solution to combat environmental pollution. In this study, the ability of polyhydroxyalkanoates (PHA) production by the halophilic bacterium Halovirbrio sp. HP20-59 in the presence of different carbon sources was examined. The strain showed a selective substrate preference, with the highest PHA production (reaching up to 73% of cell dry weight) in the presence of galactose, while fructose, arabinose, glycerol and xylose resulted in lower accumulation. Phylogenetic analysis based on the 16S rRNA gene sequence and whole-genome sequencing confirmed the HP20-59 strain as a novel species within the Oceanospirillales order. Draft genome showed a size of 4,165,370 bp with a GC content of 55.1% and a complete set of pha genes. The comparative analysis of the phaC gene identified a 638 amino acid-long class I poly(R)-hydroxyalkanoic acid synthase, showing 91% similarity to Halovibrio variabilis and 89% similarity to species within the Vreelandella genus, suggesting a possible horizontal gene transfer of the pha gene cluster. These findings highlight the unique genetic and metabolic characteristics of Halovibrio sp. HP20-59, making it a promising candidate for industrial PHA production and a valuable resource for research on sustainable biopolymers.

Multidrug-resistant (MDR) Pseudomonas aeruginosa poses a critical challenge in clinical settings because of its resistance to conventional antibiotics. This study investigated the antibacterial potential of silica-coated silver nanoparticles (SiO₂@AgNPs) against MDR P. aeruginosa and explored their synergistic interactions with selected antibiotics. A total of 450 pus samples were processed for bacterial isolation, and P. aeruginosa was identified using standard microbiological methods. MDR strains were confirmed using MIC-based VITEK antimicrobial susceptibility testing and RT-PCR for resistance genes. The antibacterial activity of the SiO₂@AgNPs was assessed using the microbroth dilution method. A checkerboard assay was conducted against MDR isolates to determine the synergy between SiO₂@AgNPs and ciprofloxacin, meropenem, and ceftazidime-avibactam. The synthesized nanoparticles were characterized using transmission electron microscopy (TEM), Fourier-transform infrared (FTIR) spectroscopy, and X-ray diffraction (XRD) analysis. Of the 450 pus samples, 100 P. aeruginosa isolates were identified, of which 13 were classified as MDR P. aeruginosa. SiO₂@AgNPs exhibited effective antibacterial activity, with an MIC of 500 µg/mL against MDR P. aeruginosa. Checkerboard assays demonstrated strong synergy with meropenem and ceftazidime-avibactam (FICI = 0.375) and partial synergy with ciprofloxacin (FICI = 0.625–1.0625). TEM revealed spherical particles with an average size of 10 nm, FTIR confirmed SiO₂ functional groups, and XRD revealed crystalline silver nanoparticles within an amorphous silica matrix. These findings indicate that SiO₂@AgNPs possess potent antibacterial activity against MDR P. aeruginosa and can enhance the efficacy of certain antibiotics, highlighting their potential in combination therapy against resistant strains.

● Silica-coated silver nanoparticles effectively inhibited MDR P. aeruginosa.

● SiO₂@AgNPs enhance the efficacy of meropenem and ceftazidime-avibactam.

● Nanoparticle-antibiotic combinations may offer new strategies for treating resistant infections.

Nutritional supplements such as trace element-enriched yeasts are becoming increasingly popular to overcome the worldwide problem of zinc (Zn) deficiency. Unlike selenium-enriched yeast, which is already authorized in the European Union, Zn-enriched yeasts (ZnY) have not yet been approved for food purposes in the European Union, as their evaluation is still ongoing, demanding more comprehensive data regarding the Zn species present in ZnY. This study screens ten different industrial yeast strains regarding their Zn-enrichment quota, with further characterization of selected strains using spectroscopic and proteomic approaches. Microfermentation experiments on the industrial yeasts showed Zn levels spanning 0.06–51 pg/cell. Large-scale fermentation in bioreactors was carried out with two strains excelling in either biomass or Zn accumulation. A combination of inductively coupled plasma mass spectrometry (ICP-MS) and various spectroscopic methods confirmed the Zn enrichment, while suggesting that fractions of the Zn accumulated on the cell surface, with simultaneously high values of phosphorus being present. Speciation via X-ray absorption spectroscopy (XAS) analyses revealed that Zn species are transformed and Zn is coordinated to P-O-ligands and to amino acid ligands in both strains. Proteomic analysis showed that ZnY cells moved from a Zap1-governed Zn balance to an intracellular excess response, implying cellular Zn uptake. This study demonstrates that, in a Zn-excess medium, industrial yeast strains exhibit variability in Zn-accumulation capacity, cellular Zn-localization, and regulatory responses involving the expression of Zn-binding proteins. The presented findings contribute to optimizing industrial fermentation processes for producing Zn-rich yeast biomass and enhance the understanding of Zn regulation in yeast, aiding in the approval of Zn-enriched yeasts for supplements and novel food applications.

• Zn enrichment in yeasts is strongly time and strain dependent

• Zn proteome changes under Zn excess suggest that Zn is partly internalized in the yeast cells

• Beside proteins, phosphorous compounds seem to be Zn-binding ligands in Zn-enriched yeast

The permeabilized cells of two Pantoea anthophila strains (BI 55.2 and BI 69.1) were evaluated for galactosylation of phenolic compounds with different molecular structure (gallic acid, caffeic acid, catechin, phlorizin, puerarin and mangiferin), the highest conversion was observed on puerarin (36.5 ± 8.4 and 53.3 ± 7.1), followed by phlorizin (33.5 ± 4.6 and 41.8 ± 0.4) and caffeic acid (21.4 ± 0.05 and 32.5 ± 0.5), respectively, no reaction was observed on mangiferin. They also exhibited a high catalytic promiscuity for most of the phenolic compounds (3 to 7 different galactosides). BI 69.1 was selected for a further kinetic characterization on phlorizin and puerarin as acceptors, elongation of the galactosyl change was observed, the mass spectrometry determined by UPLC-ESI-Qtof-MS showed the synthesis of digalactosides (759.1 and 739.08 m/z) from monogalactosides as starters (597.13 and 577.12 m/z) for phlorizin and puerarin, respectively. The major phenolic galactoside was purified and the molecular structure was elucidated by NMR, corresponding to a β-D-(1 → 6) puerarin monogalctoside.

• Permeabilized cells efficiently galactosylate diverse phenolic substrates

• High catalytic promiscuity observed with formation of multiple galactosides

• NMR confirmed a β-D-(1→6) monogalactoside structure derived from puerarin

The persistence of commensal bacteria and administered probiotics in the human gut depends to some extent on their capacity to metabolize diet and host-derived glycans. N,N′-Diacetylchitobiose (N-acetylglucosamine-β-1,4-N-acetylglucosamine; ChbNAc) is a component of N-glycosylated proteins and also the major degradation product of chitin. We have identified in Lacticaseibacillus paracasei BL23 a gene cluster, named chb, involved in the catabolism of ChbNAc. The cluster encodes a transcriptional regulator (ChbR), a cellobiose-type phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) IIC (ChbC), IIA (ChbA) and IIB (ChbB) components, a DUF3284-containing protein (ChbD), and a glycoside hydrolase of the newly identified GH170 family (ChbE). Inactivation of chbC or chbE prevents the growth of L. paracasei in ChbNAc, suggesting that the PTS is involved in its transport and phosphorylation, and that the putative hydrolase ChbE may be acting on the resulting phosphorylated ChbNAc. An L. paracasei mutant with inactivated nagA, encoding an N-acetylglucosamine-6P deacetylase, was also defective in ChbNAc utilization, indicating that the transformation of N-acetylglucosamine-6P into glucosamine-6P by NagA is necessary for ChbNAc metabolism. Transcriptional analysis showed that the chb genes and the nagA gene are regulated by substrate-specific induction mediated by the transcriptional repressors ChbR and NagR, respectively. In addition, both transcriptional regulators repressed the nagB gene, which encodes a glucosamine-6P deaminase that catalyzes the conversion of glucosamine-6P into the glycolytic intermediate fructose-6P. We characterized for the first time the genes responsible for ChbNAc metabolism in a member of the Lactobacillales. The chb and nag clusters may constitute a strategy that allows L. paracasei to adapt to the gastrointestinal environment.

• Lacticaseibacillus paracasei BL23 metabolizes N,N’-diacetylchitobiose

• The chb and nag gene clusters are involved in N,N’-diacetylchitobiose metabolism

• ChbR and NagR transcriptionally repressed the chb and nagAR clusters, respectively

Staphylococcus aureus is a major cause of hospital-acquired pneumonia, with methicillin-resistant strains contributing significantly to prolonged illness and mortality. Methicillin-resistant strains can be responsible for up to 75% of infections in certain countries. Therefore, the problem is described as severe, and the search for alternative methods of treatment of such infections is currently one of the priorities in healthcare. Bacteriophages, although historically underutilized, are re-gaining interest for their potential in treating bacterial infections. However, they do have their limitations such as specific ranges of activity and resistance development. Combining phages with antimicrobial agents such as lactoferrin—a natural protein with antimicrobial and anti-biofilm properties—may improve treatment outcomes. In this study, we evaluated the efficacy of three Kayviruses paired with lactoferrin against MRSA in infected pulmonary epithelial cell cultures. The combination significantly reduced bacterial viability, protected human cells from cytotoxic effects of bacterial infection, and decreased inflammasome activation. These findings suggest that phage-lactoferrin combinations may offer a promising, safer alternative for managing MRSA-related pneumonia and reducing dependence on traditional antibiotics.

•Phage lactoferrin mixture had no influence on A549 cells

•Lactoferrin increased phage efficacy and reduced influence of bacteria on cells

•Phage + Lf mixture limited inflammatory response similarly to phages and Lf alone

Pig slurry management has emerged as a pressing environmental challenge in the context of rapid population growth and intensified livestock production, highlighting the need for sustainable recovery technologies. While microalgae–bacteria (MB) systems offer promising opportunities for nutrient recycling, the high turbidity of raw pig slurry (PS) typically limits their direct application. This study proposes an innovative two-step treatment that combines microbial fuel cells (MFCs) with MB consortia to enhance both pollutant removal and resource recovery from raw PS with COD levels exceeding 18,000 mg·L⁻¹. Unlike conventional designs relying on perfluorinated membranes, the MFCs employed an ionic liquid [N_{8-10,8–10,8–10,1}⁺][Cl⁻] as a proton exchange medium, achieving 50% of COD removal and generating 57.27 ± 10.99 mW·m⁻². The effluent was subsequently treated with MB consortia, yielding biomass productivities of 0.1 to 0.2 g·L⁻¹·day⁻¹, comparable to chemical fertilizer-based controls. Cell density with pre-treated and untreated pig slurry also matched control levels. In pollutant recovery, the combined microbial fuel cell and microalgae-bacteria treatment achieved up to 67% recovery of COD, over 99% of N-NH₄⁺, and between 65 and 85% of P-PO₄³⁻. These findings highlight the potential of integrating MFCs with MB consortia as a strategy for raw pig slurry management, t-ransforming waste into renewable energy and bioresources.

• Pig slurry is transformed into biomass and bioenergy using sustainable technologies

• Microalgae-bacteria consortia enhance nutrient recovery and water treatment

• Ionic liquid microbial fuel cells support energy generation and COD reduction

Melanins are pigments widely distributed in microbial, plant, and animal kingdoms. Their UV–visible light shielding capacity, metal chelation ability, antioxidant, and antimicrobial properties make these pigments suitable for different industrial applications like in cosmetic and bioremediation fields. The actual manufacturing process relies on the extraction from animal tissues like the ink of Sepia officinalis and/or on synthetic chemical procedures. Streptomycetes might be the ideal candidates for the development of biotechnological processes of melanin production due to their ability to produce pigments as secondary metabolites, extracellularly released. Here, a new strain of Streptomyces nigra, capable of efficiently producing eumelanin, was isolated from soil samples in Messina, Sicily, Italy, and characterized first by 16S rRNA analysis and then by whole genome sequencing, with a complete gene clusters analysis. The strain ability of growing and producing melanin was tested on four media, including newly formulated ones, and by also optimizing temperature and pH conditions of growth, a melanin production of 2.45 ± 0.01 g/L was reached. The pigment, once produced under the optimal conditions, was purified and characterized by UV–visible, FT-IR, NMR, and EPR spectroscopy, revealing an eumelanin-like structure.

• A new Streptomyces nigra strain, MT6, was isolated and identified

• A new formulated medium boosted melanin production up to 2.45 g/L

• The extracellular pigment was characterized as eumelanin