Applied Microbiology and Biotechnology最新文献

The anaerobic bacterium Clostridium cellulovorans is a promising candidate for the sustainable production of biofuels and platform chemicals due to its cellulolytic properties. However, the genomic engineering of the species is hampered because of its poor genetic accessibility and the lack of genetic tools. To overcome this limitation, a protocol for triparental conjugation was established that enables the reliable transfer of vectors for markerless chromosomal modification into C. cellulovorans. The availability of reporter genes is another requirement for strain engineering and biotechnological applications. In this work, the oxygen-free fluorescence absorption-shift tag (FAST) system was used to characterize promoter strength in C. cellulovorans. Selected promoters were used to establish a CRISPR/Cas system for markerless chromosomal modifications. For stringent control of expression of Cas9, a theophylline-dependent riboswitch was used, and additionally, the anti-CRISPR protein AcrIIA4 was used to reduce the basal activity of the Cas9 in the off-state of the riboswitch. Finally, the newly established CRISPR/Cas system was used for the markerless deletion of the genes encoding two restriction endonucleases of a type II restriction-modification (RS) system from the chromosome of C. cellulovorans. In comparison to the WT, the conjugation efficiency when using the deletion mutant as the recipient strain was improved by about one order of magnitude, without the need for prior C. cellulovorans-specific in vivo methylation of the conjugative plasmid in the E. coli donor strain. KEY POINTS: • Quantification of heterologous promoters enables rational choice for genetic engineering. • CRISPR/Cas with riboswitch and anti-CRISPR allows efficient gene deletion in C. cellulovorans. • Conjugation protocol and type II REase deletion enhance genetic accessibility.

The rapid advancement of nanotechnology, particularly in the realm of pharmaceutical sciences, has significantly transformed the potential for treating life-threatening diseases. A pivotal aspect of this evolution is the emergence of "green nanotechnology," which emphasizes the environmentally sustainable synthesis of raw materials through biological processes. This review focuses on the biological synthesis and application of zinc oxide (ZnO) nanoparticles (NPs) from probiotic bacteria, particularly those sourced from wastewater. Microorganisms from wastewater tolerate harmful elements and enzymatically convert toxic heavy metals into eco-friendly materials. These probiotic bacteria are instrumental in the synthesis of ZnO NPs and exhibit remarkable antimicrobial properties with diverse industrial applications. As the challenge of drug-resistant pathogens escalates, innovative strategies for combating microbial infections are essential. This review explores the intersection of nanotechnology, microbiology, and antibacterial resistance, highlighting the importance of selecting suitable probiotic bacteria for synthesizing ZnO NPs with potent antibacterial activity. Additionally, the review addresses the biofunctionalization of NPs and their applications in environmental remediation and therapeutic innovations, including wound healing, antibacterial, and anticancer treatments. Eco-friendly NP synthesis relies on the identification of these suitable microbial "nano-factories." Targeting probiotic bacteria from wastewater can uncover new microbial NP synthesis capabilities, advancing environmentally friendly NP production methods. KEY POINTS: • Innovative strategies are needed to combat drug-resistant pathogens like MRSA. • Wastewater-derived probiotic bacteria are an eco-friendly method for ZnO synthesis. • ZnO NPs show significant antimicrobial activity against various pathogens.

The fermentative production of valuable chemicals from lignocellulosic feedstocks has attracted considerable attention. Although Saccharomyces cerevisiae is a promising microbial host, it lacks the ability to efficiently metabolize xylose, a major component of lignocellulosic feedstocks. The xylose oxidative pathway offers advantages such as simplified metabolic regulation and fewer enzymatic steps. Specifically, the pathway involves the conversion of xylose into 2-keto-3-deoxy-xylonate, which can be channeled into two distinct pathways, the Dahms pathway and the Weimberg pathway. However, the growth of yeast on xylose as the sole carbon source through the xylose oxidative pathway has not been achieved, limiting its utilization. We successfully engineered S. cerevisiae to metabolize xylose as its sole carbon source via the xylose oxidative pathways, achieved by enhancing enzyme activities through iron metabolism engineering and rational enzyme selection. We found that increasing the supply of the iron-sulfur cluster to activate the bottleneck enzyme XylD by BOL2 disruption and tTYW1 overexpression facilitated the growth of xylose and the production of ethylene glycol at 1.5 g/L via the Dahms pathway. Furthermore, phylogenetic analysis of xylonate dehydratases led to the identification of a highly active homologous enzyme. A strain possessing the Dahms pathway with this highly active enzyme exhibited reduced xylonate accumulation. Furthermore, the introduction of enzymes based on phylogenetic tree analysis allowed for the utilization of xylose as the sole carbon source through the Weimberg pathway. This study highlights the potential of iron metabolism engineering and phylogenetic enzyme selection for the development of non-native metabolic pathways in yeast. KEY POINTS: • A 1.5 g/L ethylene glycol was produced via the Dahms pathway in S. cerevisiae. • Enzyme activation enabled growth on xylose via both the Dahms and Weimberg pathways. • Tested enzymes in this study may expand the application of xylose oxidative pathway.

Microbial fuel cell (MFC) technology has received increased interest as a suitable approach for treating wastewater while producing electricity. However, there remains a lack of studies investigating the impact of inoculum type and hydraulic retention time (HRT) on the efficiency of MFCs in treating industrial saline wastewater. The effect of three different inocula (activated sludge from a fish-canning industry and two domestic wastewater treatment plants, WWTPs) on electrochemical and physicochemical parameters and the anodic microbiome of a two-chambered continuous-flow MFC was studied. For each inoculum, three different HRTs were tested (1 day, 3 days, and 6 days). The inoculum from the fish canning industry significantly increased voltage production (with a maximum value of 802 mV), power density (with a maximum value of 78 mW m^-2), coulombic efficiency (with a maximum value of 19.3%), and organic removal rate (ORR) compared to the inocula from domestic WWTPs. This effect was linked to greater absolute and relative abundances of electroactive microorganisms (e.g., Geobacter, Desulfovibrio, and Rhodobacter) and predicted electron transfer genes in the anode microbiome likely due to better adaption to salinity conditions. The ORR and current production were also enhanced at shorter HRTs (1 day vs. 3 and 6 days) across all inocula. This effect was related to a greater abundance and diversity of bacterial communities at HRT of 1 day compared to longer HRTs. Our findings have important bioengineering implications and can help improve the performance of MFCs treating saline effluents such as those from the seafood industry. KEY POINTS: • Inoculum type and HRT impact organic matter removal and current production. • Changes in bioenergy generation were linked to the electroactive anodic microbiome. • Shorter HRT favored increases in the performance of the MFC.

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. KEY POINTS: • 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. KEY POINTS: • 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. KEY POINTS: •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. KEY POINTS: • 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. KEY POINTS: • 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. KEY POINTS: • 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.