Applied Microbiology and Biotechnology最新文献

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

Yeasts and yeast-based products are nutrient-rich bioresources with broad applications in technologies for the production of food, feed, medicine, and cosmetics. However, traditional processing often results in non-specific lysis and suboptimal product quality. Yeast extract can be used as a flavor enhancer, nutritional supplement, or fermentation substrate, and the other components of the yeast cell wall and nucleic acids can be processed into bioactive materials, including glucans and nucleotides. These materials offer both nutritional and therapeutic benefits. Precision hydrolysis, leveraging the high specificity of tailored enzymes, has emerged as a superior strategy for maximizing the yield and functional quality of high-value yeast-based products. It provides superior outcomes by improving the quality of yeast-based products. Tailored enzymatic strategies, leveraging mechanistically focused core enzymes, including proteases, β-glucanases, and coupled nucleases-deaminases, have demonstrated superior efficiency, nutritional enhancement, and sensory refinement. This review focuses on the mechanistic properties of yeast processing enzymes, emphasizing their functional classification and applications in precision hydrolysis. It details how such enzymes are optimized for the targeted release and modification of high-value components. Additionally, the review highlights recent strategies for tailored biosynthesis of yeast processing enzymes, including enzyme discovery, heterologous expression systems, and machine-learning-guided optimization. This review aims to support future innovations that will promote the development of sustainable, high-value, and diversified yeast-based bioproducts by optimizing the biosynthesis of processing enzymes, thus lowering the overall cost of precision hydrolysis.

• Precision hydrolysis enables the controlled release of yeast components in a specific pattern, yielding high-quality, specific yeast-based products.

• By leveraging the highly specific effects of enzymes, targeted product refinement and superior characteristics under mild processing conditions can be achieved.

• To avoid the high cost of precision hydrolysis, continuous advances in enzyme discovery, protein engineering, and metabolic engineering technologies are vital.

The overexpression of proteins in Escherichia coli often results in the formation of inclusion bodies, which are biologically inactive, especially for proteins with exposed hydrophobic surfaces. Solubilization of inclusion bodies (IBs) and subsequent refolding is essential for obtaining correctly folded and active protein. However, protein refolding involves multiple steps—namely isolation, solubilization, and refolding—which is a labor-intensive process. In this study, we developed a strategy for soluble production and protein refolding. A fusion tag was applied to Burkholderia ambifaria lipase YCJ01, enabling abundant soluble expression in E. coli. Despite this, the soluble protein exhibited only partial enzymatic activity, suggesting an unfolded state of soluble lipase YCJ01. Lipase activity increased significantly after incubation with cosolvents, reaching 1003 U/mL, 754 U/mL, and 501 U/mL in 25% (v/w) glycerol, 15% (v/w) DMSO, and 4M trimethylamine N-oxide (TMAO) solutions, respectively. Correctly folded and highly active lipase YCJ01 with a natural N-terminus was obtained. Moreover, the cosolvent-induced refolding mechanism was elucidated through molecular dynamics simulations. Glycerol and DMSO were found to aggregate around hydrophobic regions of lipase, directly stabilizing structure by displacing water molecules and weakening water–protein hydrogen (H) bonds within the hydration shell. Conversely, TMAO molecules indirectly influenced the lipase structure by strengthening water–water H bonds.

• Cosolvents enhance lipase activity, with glycerol showing the highest improvement.

• MD simulations show glycerol and DMSO directly interact with hydrophobic regions.

• Glycerol and DMSO stabilize lipase directly, while TMAO enhances stability indirectly.

Flavonoid glycosides exhibit compromised bioavailability due to low membrane permeability. To address this limitation, we acetylated flavonoids through enzymatic reactions to increase bioavailability. This study first reported that Hesperetin-7-O-glucoside (Hes-7-G) was acetylated by galactoside acetyltransferase (GAT), and the apparent permeability (P_app) of the Caco-2 monolayer was increased by 69%, indicating the acetylated Hes-7-G application potential to improve bioavailability. Subsequently, we designed GAT mutants through comprehensive computational and experimental methods to improve the acetylation efficiency and elucidate the catalytic mechanism. Molecular Dynamics (MD) simulations found that Tyr483 and Met127 are key residues that control flavonoid binding through dynamic van der Waals interactions, while His115 and Thr113 mediated proton transfer accounts for 85–90% of the catalytic activity. Rational substitution of Pro148 with alanine (P148A) increased the flexibility of the cofactor binding ring and increased the catalytic efficiency (K_cat/K_M) by 21%. Average non-covalent interaction (aNCI) analysis revealed that regional selectivity in the glucose portion was controlled by hydrophobic interactions with Tyr483 and hydrogen bonding with Gly125, and rhamnose substitution caused spatial conflict. This work deciphered the structure-activity relationship of GAT, established a framework for protein engineering, and highlighted enzyme-driven acetylation as a sustainable strategy for optimizing flavonoid pharmacokinetics.

• Engineered acetyltransferase enhances flavonoid glycoside absorption.

• P148A mutation improves catalytic efficiency.

• Insight into the catalytic mechanism of GAT by flavonoid glycoside substrates.

The recombinant production of extracellular matrix proteins is a promising approach for replacing animal-derived materials in biomedical applications. K. phaffii represents a favorable expression host because it combines the ability of higher eukaryotes for secreted protein production with the ability to grow to high cell densities on simple, low-cost media. Additionally, this well-studied host allows for tight control of recombinant protein expression using the methanol-inducible AOX1 promoter. In this study, different methanol feeding strategies were evaluated to optimize the expression of a collagen-mimetic protein (ColMP-His). A methanol feed approach with carbon as a limiting nutrient resulted in the highest target protein production, whereas exponential feeding resulted in fast biomass accumulation with reduced protein expression. Moreover, the limited feeding strategy resulted in 25% lower oxygen consumption, despite the longer fermentation time, which has a positive impact on process cost efficiency. The application of a three-phases fermentation strategy with the addition of a preceding glycerol-fed batch phase to increase biomass did not improve product titers and was associated with reduced expression efficiency. A variation in the methanol feeding rate was also investigated for induction. A gradient-based methanol feed, which increased incrementally over time, achieved the highest final product concentration and sustained expression over extended fermentation periods. Compared with the initial process, the yield was increased by a factor of 11. Despite statistical limitations due to high variability, the results highlight the importance of adaptive process control in balancing cell growth and recombinant protein production. The presented gradient-based strategy provides a foundation for animal-free, scalable production of recombinant collagen materials.

• Methanol-limiting feed enhances collagen expression in Komagataella phaffii bioprocesses.

• Exponential feeding favors biomass but lowers protein yield and process efficiency

• Gradient feeding results in the highest collagen titers and sustained expression