Applied Microbiology and Biotechnology最新文献

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

Environmental stresses due to climate changes, such as high temperatures and land degradation, significantly impact crop yield, making innovative strategies necessary to increase plant stress tolerance. This study investigates the potential of plant growth-promoting rhizobacteria (PGPR) to enhance wheat resilience under multiple environmental stresses, such as high salinity and temperature. For this, 15 bacterial strains were isolated from the rhizosphere and roots of Pancratium maritimum and screened for their ability to withstand high salinity (50–600 mM NaCl) and elevated temperatures (up to 42 °C). The isolates were identified by 16S rRNA sequencing and tested for their PGP traits under combined abiotic stresses. Most of the strains exhibited PGP features, such as biofilm formation, phosphate solubilization, and phytohormone production. To enhance the growth of wheat plants, used as a model crop of commercial interest, three different consortia were designed and tested in vitro. The consortium (CONSIII), composed of Serratia marcescens ERA6, Enterobacter cloacae ERA9, and Bacillus proteolyticus ESOB2, provided synergistic effects that led to an enhancement in plant growth and stress resilience in vitro. This positive effect was confirmed in pot trials under double abiotic stress (37 °C, 132 mM NaCl), where CONSIII was able to boost the root and shoot growth, increase chlorophyll and carotenoid content, and enhance antioxidant activity, mitigating reactive oxygen species accumulation. These findings underscore the potential of PGPR consortia as bioinoculants for sustainable agriculture, demonstrating their effectiveness in the simultaneous presence of salinity and heat stresses—a challenging and under-investigated environmental scenario.

• PGPR strains isolated from Pancratium maritimum rhizosphere are able to grow and exhibit PGP traits under combined salinity and heat conditions

• The formulated consortium of PGPR strains (CONSIII) significantly enhances wheat growth and stress resilience under a multi-stress environment

• CONSIII increases plant biomass, pigment content, and antioxidant activity, proving its value as a sustainable bioinoculant

Thioredoxins (TRXs) are small, conserved redox-active proteins that play central roles in oxidative stress responses. Here, we identified and functionally characterized a novel thioredoxin, MpTRX1, from the newly isolated yeast Metschnikowia persimmonesis. The full-length MpTRX1 gene was cloned and expressed in Escherichia coli and Saccharomyces cerevisiae to analyze its biochemical and physiological functions. MpTRX1 encodes a 103-amino-acid protein containing a canonical CXXC redox motif, and structural modeling confirmed a conserved thioredoxin fold. Recombinant MpTRX1 exhibited clear disulfide reductase activity in both DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)) and insulin reduction assays. Mutation of either catalytic cysteine residue abolished activity, confirming their essential roles. Moreover, heterologous expression of MpTRX1 in S. cerevisiae enhanced tolerance to hydrogen-peroxide-induced oxidative stress. Although the functional assays were conducted in a heterologous system, these findings demonstrate that MpTRX1 is a bona fide thioredoxin that may contribute to oxidative stress protection in M. persimmonesis. This work provides the first molecular characterization of a protein from M. persimmonesis and establishes a foundation for future studies on its potential ecological and biotechnological applications.

• Identification of MpTRX1, a novel thioredoxin from M. persimmonesis.

• Recombinant MpTRX1 reduces both chemical and protein substrates.

• Overexpression of MpTRX1 enhances oxidative stress tolerance in S. cerevisiae.

In nature, remarkable geological structures, such as stromatolites, thrombolites, and beachrocks, are formed through biocementation, a process involving the successive dissolution and reprecipitation of CaCO₃. This research demonstrates mimicking natural cement via bio-sintering of limestone, a process that involves successive dissolution and reprecipitation of limestone facilitated by bacteria under ambient environmental conditions. When the bacterium Acetobacter aceti (ATCC 15973) was introduced into a mixture of ethanol and limestone powder, the pH dropped rapidly, leading to the dissolution of limestone into calcium acetate. After ethanol was fully consumed, the pH gradually increased due to acetate oxidation, causing biocement crystals to precipitate. All reaction rates were measured, and the products characterized through Fourier transform infrared spectroscopy, scanning electron microscopy, quantitative X-ray diffraction, and a particle size analyzer. The detailed characterization indicates that the precipitate is mainly calcite and that the dissolved calcium carbonate sinters microbially. This pathway offers new opportunities in biocementation research by using limestone as a direct calcium source, reducing the overall carbon footprint, and eliminating the need for urea or other chemical additives. The biocement produced shows potential for practical applications in soil stabilization, eco-concrete, and other sustainable construction solutions, providing a foundation for environmentally conscious alternatives in next-generation construction. 

• Novel bio-sintering emulates natural CaCO₃ cementation via microbial action.

• Acetobacter aceti mediates limestone dissolution and calcite precipitation.

• The process forms calcite without urea or synthetic additives.

Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus causing high mortality in neonatal piglets and posing a significant threat to the swine industry. Evidence indicates that PDCoV has cross-species transmission potential and may pose a zoonotic risk, emphasizing the need for reliable serological tools for epidemiological surveillance and vaccine evaluation. Here, we developed a double-antigen sandwich enzyme-linked immunosorbent assay (DAgS-ELISA) based on the PDCoV S1 protein, expressed in CHO cells and used as both coating and HRP-conjugated antigen. The assay reliably detected PDCoV-specific antibodies in sera from pigs, chickens, rabbits, and mice, showing high sensitivity (92.86%) and specificity (99.11%) as determined by receiver operating characteristic curve analysis, with excellent reproducibility. No cross-reactivity was observed with antibodies against other common swine pathogens. Concordance with indirect immunofluorescence assay was 96.18% (kappa = 0.923), and assay results correlated strongly with neutralizing antibody titers (Pearson r = 0.865). Overall, this S1-based DAgS-ELISA provides a sensitive, specific, and cross-species applicable method for PDCoV serological detection, supporting its use for epidemiological surveillance and evaluation of vaccine-induced neutralizing antibodies.

• A novel S1-based double-antigen sandwich ELISA was established for PDCoV detection.

• The assay shows high sensitivity, strong specificity, and broad cross-species applicability.

• ELISA results strongly correlate with neutralizing antibody titers, aiding vaccine evaluation.