Applied Microbiology and Biotechnology最新文献

RNA viruses have high genetic diversity, allowing rapid adaptation to environmental pressures, such as disinfection. This diversity increases the likelihood of mutations influencing the viral sensitivity to disinfectants. Ethanol is widely used to control viral transmission; however, insufficient disinfection facilitates the survival of less-sensitive viruses. Further, the underlying mechanisms of ethanol-induced changes in viral sensitivity remain unclear. Here, we assessed the genetic characteristics of ethanol-adapted murine norovirus (MNV) and associated changes in viral sensitivity. Experimental ethanol-facilitated MNV adaptation and subsequent genetic characteristic evaluation of the whole genome sequence was performed. MNV was exposed to 70% ethanol for 5 s to achieve ± 3-log₁₀ inactivation. Twelve MNV populations were identified as “less sensitive,” consisting of nine treated and three control populations. Less-sensitive MNV populations exhibited significantly higher synonymous nucleotide diversity (πS) in ORF1 (p = 0.001), which encodes the non-structural protein, than sensitive populations. Ethanol sensitivity and πS were negatively correlated in ORF1 (R = − 0.49, p = 0.003), indicating that high genetic diversity in ORF1 could be linked to reduced ethanol sensitivity. This study demonstrates an association between nucleotide diversity in specific coding regions of the MNV genome and ethanol sensitivity. These findings are vital for improving disinfection methods and anticipating emerging viruses that are more resistant to disinfectants.

• Several MNV populations reduced sensitivity to ethanol.

• Higher synonymous diversity in ORF1 linked to reduced ethanol sensitivity.

• Synonymous mutations can influence viral adaptation to ethanol.

In recent years, there has been a surge in the production of kombucha—a functional beverage obtained via microbial fermentation of tea. However, fresh, unpasteurized kombucha is sensitive to quality deterioration as a result of, among other factors, oxidation. The addition of hops seems to be promising, due to their antioxidative properties, which may improve the stability of kombucha. However, aiming at retaining the highest antioxidative properties of kombucha, it remains unclear at which stage of the production process hops should be added. The study investigated the effect of hop supplementation during kombucha production on the basic physicochemical, antioxidative, and sensory properties of kombucha. Cascade hops in the concentrations 0.5 and 2 g/L were added at the onset of tea infusion and to the fresh, unpasteurized kombucha. The addition of hops (particularly at the pre-fermentation stage of production) led to a significant decrease in radical formation in the produced kombucha measured by electron spin resonance spectroscopy (ESR), which correlated with the higher DPPH antiradical activity and the elevated bitter α-acid content. From the sensory perspective, the post-fermentation addition of hops to kombucha resulted in a significantly higher rating of the overall quality. This enhancement was directly associated with heightened bitterness, increased presence of fruity and citrusy aromas, and a simultaneous reduction in the intensities of acetic and tea-related attributes. The data presented in this study are relevant for kombucha producers, who want to deliver a sensory-novel product in combination with an improved oxidative stability.

• Hop addition in kombucha production improves the antioxidative activity of the beverage.

• Hop α-acids display higher antioxidative properties in kombucha than polyphenols.

• Oxidative stability of kombucha fortified with hops depends on the timing of hops addition.

• Hop addition enriches the taste and aroma attributes of kombucha.

The enzyme D-sorbitol dehydrogenase (SLDH) facilitates the conversion of D-sorbitol to L-sorbose. While current knowledge of this enzyme class predominantly centers on Gluconobacter oxydans, the catalytic properties of enzymes from alternative sources, particularly their substrate specificity and coenzyme dependency, remain ambiguous. In this investigation, we conducted BLASTp analysis and screened out a novel SLDH (Fpsldh) from Faunimonas pinastri A52C2. The SLDH was then identified and characterized. Analysis of the purified enzyme revealed its dependence on NAD⁺/NADP⁺ and its specificity for L-sorbose production. Fpsldh demonstrated sustained catalytic activity over temperatures ranging from 27 to 37 ℃, with optimal performance observed at pH 8.0–10.0, and it exhibited no requirement for metal ions for activation. The K_m of Fpsldh is 7.51 mM. Furthermore, a Bacillus licheniformis host expressing Fpsldh was engineered. The resultant whole-cell catalyst yielded 13.19 g/L of L-sorbose after 33.6 h of transformation, obviating the need for exogenous cofactors. This study enhances our understanding of the catalytic properties of the SLDH family and introduces a novel method for L-sorbose production, a compound of considerable commercial value.

•New D-sorbitol dehydrogenase from Faunimonas pinastri A52C2 is characterized.

•Fpsldh is not PQQ but NAD⁺/NADP⁺-dependent.

•Bacillus licheniformis expressing Fpsldh can produce 13.19 g/L L-sorbose within 33.6 h.

Lignin, the most abundant renewable source of aromatic compounds on earth, remains underexploited in traditional biorefining. Fraxetin, a naturally occurring flavonoid, has garnered considerable attention in the scientific community due to its diverse and potent biological activities such as antimicrobial, anticancer, antioxidant, anti-inflammatory, and neurological protective actions. To enhance the green and value-added utilization of lignin, Saccharomyces cerevisiae was engineered as a cell factory to transform lignin derivatives to produce fraxetin. The expression of scopoletin 8-hydroxylase (S8H) and coumarin synthase (COSY) enabled S. cerevisiae to produce fraxetin from ferulic acid, one of the three principal monomers. The optimized fermentation strategies produced 19.1 mg/L fraxetin from ferulic acid by engineered S. cerevisiae. Additionally, the engineered cell factory achieved a fraxetin titer of 7.7 mg/L in lignin hydrolysate. This study successfully demonstrates the biotransformation of lignin monomers and lignin hydrolysate into fraxetin using a S. cerevisiae cell factory, thereby providing a viable strategy for the valorization of lignin.

• AtS8H showed substance specificity in the hydroxylation of scopoletin.

• AtCOSY and AtS8H were key enzymes for converting ferulic acid into fraxetin.

• Yeast was engineered to produce fraxetin from lignin hydrolysate.

Bacteriophage infections in bacterial cultures pose a significant challenge to industrial bioprocesses, necessitating the development of innovative antiphage solutions. This study explores the antiphage potential of indigo carmine (IC), a common FDA-approved food additive. IC demonstrated selective inactivation of DNA phages (P001, T4, T1, T7, λ) with the EC₅₀ values ranging from 0.105 to 0.006 mg/mL while showing no activity against the RNA phage MS2. Fluorescence correlation spectroscopy (FCS) revealed that IC selectively binds to dsDNA, demonstrated by a significant reduction in the diffusion coefficient, whereas no binding was observed with ssDNA or RNA. Mechanistically, IC permeates the phage capsid, leading to genome ejection and capsid deformation, as confirmed by TEM imaging. Under optimal conditions (50 °C, 220 rpm), IC achieved up to a 7-log reduction in phage titer, with kinetic theory supporting the enhanced collision frequency induced by agitation. Additionally, IC protected E. coli cultures from phage-induced lysis without affecting bacterial growth or protein production, as demonstrated by GFP expression assays. IC’s effectiveness and environmental safety, combined with its FDA approval and cost-effectiveness, make it a promising antiphage agent for industrial applications.

• Indigo carmine effectively inactivates a broad spectrum of bacteriophages, offering protection to bacteria in industrial cultures.

• A novel application of indigo carmine as a food-grade, environmentally safe, and FDA-approved antiphage agent protecting bacterial cultures.

• Antiphage activity arises from indigo carmine’s interaction with DNA within the phage capsid without harming bacterial cells or compromising protein production in bacterial cultures.

The construction of artificial reefs (ARs) is an effective way to restore habitats and increase and breed fishery resources in marine ranches. However, studies on the impacts of ARs on the structure, function, and assembly patterns of the bacterial community (BC), which is important in biogeochemical cycles, are lacking. The compositions, diversities, assembly patterns, predicted functions, and key environmental factors of the attached and free-living microbial communities in five-year ARs (O-ARs) and one-year ARs (N-ARs) in Fangchenggang, China, were analyzed via 16S rRNA gene sequencing. Proteobacteria was the dominant taxon in all the samples, with an average relative abundance of 44.48%, followed by Bacteroidetes (17.42%) and Cyanobacteria (15.19%). The composition of bacterial phyla was similar between O-ARs and N-ARs, but the relative abundance of Cyanobacteria was greater in the water column (38.56%) than on the AR surface (mean of 7.40%). The results revealed that the Shannon‒Wiener diversity indices were 5.64 and 5.45 for O-ARs and N-ARs, respectively. Principal coordinate analysis (PCoA) revealed different distributions of O-ARs and N-ARs in the microbial community. Additionally, network analysis revealed that the bacterial community was more complex and stable in O-ARs than in N-ARs, indicating that the 5-year AR presented a more diverse and stable microbial community overall. The KEGG database was used to predict that nitrogen metabolism, carbon metabolism, and membrane transport were the dominant microbial functions, accounting for 29.93% of the total functional abundances. The results of the neutral community model revealed that stochastic processes (67.2%) dominated the assembly of BCs. Interestingly, deterministic processes may be increasingly important in community aggregation over time. Moreover, a null model revealed that dispersal limitation was the most important process among the stochastic processes, accounting for 57.14% of the total. In addition, redundancy analysis (RDA) revealed that hydrological factors obviously impacted the structure and function of the microbial community. Our results showed that the construction of ARs slightly promotes local diversities in the structure and function of the microbial community, indicating it requires a longer time to enhance the diversity of the microbial community on artificial reefs.

• Artificial reefs facilitate the diversity and functions of the microbial community

• Stochastic processes dominate the assembly of the microbial community in artificial reefs

• Nitrogen and carbon metabolism dominate microbial functions in artificial reefs

The relationship of microbial community and cometabolic consumption of 2-chlorophenol (2-CP) in a nitrifying sequencing batch reactor (SBR) was studied. The assessment of the population dynamics of the nitrifying sludge during the cometabolic 2-CP consumption with increasing ammonium (NH₄⁺) concentrations in the SBR showed the presence of 39 different species of which 10 were always present in all cycles. Fifty-five percent of the species found were grouped as Proteobacteria (45% as β-proteobacteria and 10% as γ-proteobacteria class), 30% as Acidobacteria, and 15% as Deinococcus-Thermus phyla. NH₄⁺ and cometabolic 2-CP consumption could be related to the presence and permanence of ammonium-oxidizing bacteria (AOB) species and heterotrophic bacteria, while the complete nitrification to the presence of nitrite-oxidizing bacteria (NOB) species. A correlation analysis showed that the complete and stable nitrifying performance (NH₄⁺ consumption efficiencies (ENH₄⁺-N) > 99% and nitrate production yields (YNO₃⁻-N) between 0.93 and 0.99), as well as the increase in specific rates (ammonium (qNH₄⁺-N) and 2-CP (q2-CP-C) consumption and nitrate production (qNO₃⁻-N)), was associated with the homogeneity of the bacterial community (J index = 0.99). The increase in the proportion of individuals of AOB species such as Nitrosomonas oligotropha and Nitrosomonas marina was associated with the increase in qNH₄⁺-N (r ≥ 0.69) and q2-CP-C (r ≥ 0.64) and, therefore, with the 2-CP cometabolic consumption in the SBR. Finally, the increase in the proportion of individuals of heterotrophic species such as Dokdonella ginsengisoli, Deinococcus peraridilitoris, Truepera radiovictrix, and Stenotrophobacter terrae was associated with the increase in q2-CP-C (r ≥ 0.59).

• Thirty-nine bacterial species were identified in the nitrifying sludge population of the SBR.

• β-Proteobacteria and Acidobacteria were the prevalent (85%) bacterial groups.

• AOB and heterotrophic bacteria participate in NH₄⁺ and cometabolic 2-CP consumption.

Aloe vera (L.) Burm.f. is a traditional Chinese medicine known for treating various ailments, including fungal infections. Aloin is one of the major components from A. vera, but its antifungal mechanism and therapeutic potential against oral candidiasis are not clear. This study aimed to examine the mechanism of aloin against Candida albicans and its inhibitory activity against oral candidiasis. In this study, we for the first time found that aloin could induce the formation of abnormal hyphae with smaller hyphal diameters and fewer branching points in C. albicans including 11 clinical isolates without growth inhibition. The transcriptome and further cell wall contents analysis indicated that aloin remodeled the cell wall to increase the contents of β-1,3-glucan and furtherly showed an antagonistic effect with micafungin. Aloin also significantly inhibited the cell damage of oral epithelial cells and oral candidiasis in mice infected by C. albicans due to its inhibitory actions on the hyphal development and expressions of virulence factors, including candidalysin (coded by ECE1). Our results suggest that aloin is a promising antifungal agent for controlling candidiasis and targeting hyphal development and pathogenesis represents a practical strategy for developing new antifungal drugs.

• Aloin remodels the C. albicans cell wall to form avirulent hyphae.

• Aloin inhibits C. albicans infections in oral epithelial cells and mouse mucosa without toxicity.

• Aloin is a promising antifungal agent with therapeutic potential against C. albicans infections.

Metagenomes present a source for novel enzymes, but under 1% of environmental microbes are cultivatable. Because of its useful properties, Escherichia coli has been used as a host organism in functional genomic screens. However, due to differing expression machineries in the expression host compared to the source organism of the DNA sequences, screening outcomes can be biased. Here, we focused on one of the limiting processes—translation initiation. To that end, we created an operon-like screening system in E. coli to select mutants of the ribosomal protein S1 with more relaxed sequence requirements for 5’-untranslated regions of mRNAs. We created two mutation libraries of the ribosomal protein S1, one covering domains 3 and 4 (D3-D4) and the second covering domains 3 to 5 (D3-D5). Most mutants from library D3-D4 proofed to be specific for a particular UTR sequence and improved only expression from a single construct. Only mutant 3 from library D3-D4 led to increased expression of four different reporters improving fluorescence levels by up to 21%. Mutants isolated from D3-D5 library led up to 90% higher expression compared to the control, though the mutants with highest improvements exhibited a specialist phenotype. The most promising mutant, mutant 4, exhibited a generalist phenotype and showed increased expression in all six reporter strains compared to the control. This could indicate the potential for a more promiscuous translation initiation of metagenomic sequences in E. coli although at the price of smaller increases compared to specialist mutants.

• An operon-like selection system allowed to isolate generalist and specialist S1 mutants.

• S1 mutants improved translation of mRNAs with 5'-UTRs from metagenomic sequences.

• Use of S1 mutants could increase coverage from metagenomic libraries in functional screens.

Second-generation (2G) bioethanol production, derived from lignocellulosic biomass, has emerged as a sustainable alternative to fossil fuels by addressing growing energy demands and environmental concerns. Fungal sugar transporters (STs) play a critical role in this process, enabling the uptake of monosaccharides such as glucose and xylose, which are released during the enzymatic hydrolysis of biomass. This mini-review explores recent advances in the structural and functional characterization of STs in filamentous fungi and yeasts, highlighting their roles in processes such as cellulase induction, carbon catabolite repression, and sugar signaling pathways. The review also emphasizes the potential of genetic engineering to enhance the specificity and efficiency of these transporters, overcoming challenges such as substrate competition and limited pentose metabolism in industrial strains. By integrating the latest research findings, this work underscores the pivotal role of fungal STs in optimizing lignocellulosic bioethanol production and advancing the bioeconomy. Future prospects for engineering transport systems and their implications for industrial biotechnology are also discussed.