CRISPR-Cas technologies have emerged as powerful and versatile tools in gene therapy. In addition to the widely used SpCas9 system, alternative platforms including modified amino acid sequences, size-optimized variants, and other Cas enzymes from diverse bacterial species have been developed to apply this technology in various genetic contexts. In addition, base editors and prime editors for precise gene editing, the Cas13 system targeting RNA, and CRISPRa/i systems have enabled diverse and adaptable approaches for genome and RNA editing, as well as for regulating gene expression. Typically, CRISPR-Cas components are transported to the target in the form of DNA, RNA, or ribonucleoprotein complexes using various delivery methods, such as electroporation, adeno-associated viruses, and lipid nanoparticles. To amplify therapeutic efficiency, continued developments in targeted delivery technologies are required, with increased safety and stability of therapeutic biomolecules. CRISPR-based therapeutics hold an inexhaustible potential for the treatment of many diseases, including rare congenital diseases, by making permanent corrections at the genomic DNA level. In this review, we present various CRISPR-based tools, their delivery systems, and clinical progress in the CRISPR-Cas technology, highlighting its innovative prospects for gene therapy.

Dengue, caused by four serotypes of dengue viruses (DENV-1 to DENV-4), is the most prevalent and widely mosquito-borne viral disease affecting humans. Dengue virus (DENV) infection has been reported in over 100 countries, and approximately half of the world's population is now at risk. The paucity of universally licensed DENV vaccines highlights the urgent need to address this public health concern. Action and attention to antibody-dependent enhancement increase the difficulty of vaccine development. With the worsening dengue fever epidemic, Dengvaxia^® (CYD-TDV) and Qdenga^® (TAK-003) have been approved for use in specific populations in affected areas. However, these vaccines do not provide a balanced immune response to all four DENV serotypes and the vaccination cannot cover all populations. There is still a need to develop a safe, broad-spectrum, and effective vaccine to address the increasing number of dengue cases worldwide. This review provides an overview of the existing DENV vaccines, as well as potential candidates for future studies on DENV vaccine development, and discusses the challenges and possible solutions in the field.

The increasing environmental concerns regarding conventional plastics have led to a growing demand for sustainable alternatives, such as biodegradable plastics. Yeast cell factories, specifically Saccharomyces cerevisiae and Yarrowia lipolytica, have emerged as promising platforms for bioplastic production due to their scalability, robustness, and ease of manipulation. This review highlights synthetic biology approaches aimed at developing yeast cell factories to produce key biodegradable plastics, including polylactic acid (PLA), polyhydroxyalkanoates (PHAs), and poly (butylene adipate-co-terephthalate) (PBAT). We explore recent advancements in engineered yeast strains that utilize various synthetic biology strategies, such as the incorporation of new genetic elements at the gene, pathway, and cellular system levels. The combined efforts of metabolic engineering, protein engineering, and adaptive evolution have enhanced strain efficiency and maximized product yields. Additionally, this review addresses the importance of integrating computational tools and machine learning into the Design-Build-Test-Learn cycle for strain development. This integration aims to facilitate strain development while minimizing effort and maximizing performance. However, challenges remain in improving strain robustness and scaling up industrial production processes. By combining advanced synthetic biology techniques with computational approaches, yeast cell factories hold significant potential for the sustainable and scalable production of bioplastics, thus contributing to a greener bioeconomy.

Protein solubility is a critical factor in the production of recombinant proteins, which are widely used in various industries, including pharmaceuticals, diagnostics, and biotechnology. Predicting protein solubility remains a challenging task due to the complexity of protein structures and the multitude of factors influencing solubility. Recent advances in computational methods, particularly those based on machine learning, have provided powerful tools for predicting protein solubility, thereby reducing the need for extensive experimental trials. This review provides an overview of current computational approaches to predict protein solubility. We discuss the datasets, features, and algorithms employed in these models. The review aims to bridge the gap between computational predictions and experimental validations, fostering the development of more accurate and reliable solubility prediction models that can significantly enhance recombinant protein production.

Streptomyces are a crucial source of bioactive secondary metabolites with significant clinical applications. Recent studies of bacterial and metagenome-assembled genomes have revealed that Streptomyces harbors a substantial number of uncharacterized silent secondary metabolite biosynthetic gene clusters (BGCs). These BGCs represent a vast diversity of biosynthetic pathways for natural product synthesis, indicating significant untapped potential for discovering new metabolites. To exploit this potential, genome mining using comprehensive strategies that leverage extensive genomic databases can be conducted. By linking BGCs to their encoded products and integrating genetic manipulation techniques, researchers can greatly enhance the identification of new secondary metabolites with therapeutic relevance. In this context, we present a step-by-step guide for using the antiSMASH pipeline to identify secondary metabolite-coding BGCs within the complete genome of a novel Streptomyces strain. This protocol also outlines gene manipulation methods that can be applied to Streptomyces to activate cryptic clusters of interest and validate the functions of biosynthetic genes. By following these guidelines, researchers can pave the way for discovering and characterizing valuable natural products.

The escalating antibiotic resistance crisis poses a significant challenge to global public health, threatening the efficacy of current treatments and driving the emergence of multidrug-resistant pathogens. Among the various factors associated with bacterial antibiotic resistance, small regulatory RNAs (sRNAs) have emerged as pivotal post-transcriptional regulators which orchestrate bacterial adaptation to antibiotic pressure via diverse mechanisms. This review consolidates the current knowledge on sRNA-mediated mechanisms, focusing on drug uptake, drug efflux systems, lipopolysaccharides, cell wall modification, biofilm formation, and mutagenesis. Recent advances in transcriptomics and functional analyses have revealed novel sRNAs and their regulatory networks, expanding our understanding of resistance mechanisms. These findings highlight the potential of targeting sRNA-mediated pathways as an innovative therapeutic strategy to combat antibiotic resistance, and offer promising avenues for managing challenging bacterial infections.

The widespread use of antibiotics in aquaculture has led to the emergence of multidrug-resistant pathogens and environmental concerns, highlighting the need for sustainable, eco-friendly alternatives. In this study, we isolated and characterized three novel bacteriophages from aquaculture effluents in Korean shrimp farms that target the key Vibrio pathogens, Vibrio harveyi, and Vibrio parahaemolyticus. Bacteriophages were isolated through environmental enrichment and serial purification using double-layer agar assays. Transmission electron microscopy revealed that the phages infecting V. harveyi, designated as vB_VhaS-MS01 and vB_VhaS-MS03, exhibited typical Siphoviridae morphology with long contractile tails and icosahedral heads, whereas the phage isolated from V. parahaemolyticus (vB_VpaP-MS02) displayed Podoviridae characteristics with an icosahedral head and short tail.

Whole-genome sequencing produced complete, circularized genomes of 81,710 bp for vB_VhaS-MS01, 81,874 bp for vB_VhaS-MS03, and 76,865 bp for vB_VpaP-MS02, each showing a modular genome organization typical of Caudoviricetes. Genomic and phylogenetic analyses based on the terminase large subunit gene revealed that although vB_VhaS-MS01 and vB_VhaS-MS03 were closely related, vB_VpaP-MS02 exhibited a distinct genomic architecture that reflects its unique morphology and host specificity. Collectively, these comparative analyses demonstrated that all three phages possess genetic sequences markedly different from those of previously reported bacteriophages, thereby establishing their novelty. One-step growth and multiplicity of infection (MOI) experiments demonstrated significant differences in replication kinetics, such as burst size and lytic efficiency, among the phages, with vB_VhaS-MS03 maintaining the most effective bacterial control, even at an MOI of 0.01. Additionally, host range assays showed that vB_VhaS-MS03 possessed a broader spectrum of activity, supporting its potential use as a stand-alone agent or key component of phage cocktails. These findings highlight the potential of region-specific phage therapy as a targeted and sustainable alternative to antibiotics for controlling Vibrio infections in aquaculture.

This review explores current advancements in microbiome functional analysis enabled by next-generation sequencing technologies, which have transformed our understanding of microbial communities from mere taxonomic composition to their functional potential. We examine approaches that move beyond species identification to characterize microbial activities, interactions, and their roles in host health and disease. Genome-scale metabolic models allow for in-depth simulations of metabolic networks, enabling researchers to predict microbial metabolism, growth, and interspecies interactions in diverse environments. Additionally, computational methods for predicting metabolite profiles offer indirect insights into microbial metabolic outputs, which is crucial for identifying biomarkers and potential therapeutic targets. Functional pathway analysis tools further reveal microbial contributions to metabolic pathways, highlighting alterations in response to environmental changes and disease states. Together, these methods offer a powerful framework for understanding the complex metabolic interactions within microbial communities and their impact on host physiology. While significant progress has been made, challenges remain in the accuracy of predictive models and the completeness of reference databases, which limit the applicability of these methods in under-characterized ecosystems. The integration of these computational tools with multi-omic data holds promise for personalized approaches in precision medicine, allowing for targeted interventions that modulate the microbiome to improve health outcomes. This review highlights recent advances in microbiome functional analysis, providing a roadmap for future research and translational applications in human health and environmental microbiology.

Precise and tunable gene expression is crucial for various biotechnological applications, including protein overexpression, fine-tuned metabolic pathway engineering, and dynamic gene regulation. Untranslated regions (UTRs) of mRNAs have emerged as key regulatory elements that modulate transcription and translation. In this review, we explore recent advances in UTR engineering strategies for bacterial gene expression optimization. We discuss approaches for enhancing protein expression through AU-rich elements, RG4 structures, and synthetic dual UTRs, as well as ProQC systems that improve translation fidelity. Additionally, we examine strategies for fine-tuning gene expression using UTR libraries and synthetic terminators that balance metabolic flux. Finally, we highlight riboswitches and toehold switches, which enable dynamic gene regulation in response to environmental or metabolic cues. The integration of these UTR-based regulatory tools provides a versatile and modular framework for optimizing bacterial gene expression, enhancing metabolic engineering, and advancing synthetic biology applications.

Extracellular vesicles derived from probiotics have received considerable attention for their pivotal role in bacterial‒host communication. These nanosized, bilayer-encapsulated vesicles carry diverse bioactive molecules, such as proteins, lipids, nucleic acids, and metabolites. Currently, ample evidence has emerged that probiotic extracellular vesicles may modulate several processes of host physiological hemostasis and offer therapeutic benefits. This review examines the biogenesis, composition, and immunomodulatory functions of probiotic-derived extracellular vesicles in probiotic–host interactions, highlighting the therapeutic potential of probiotic extracellular vesicles in the diagnosis and treatment of conditions such as cancer and inflammatory bowel disease. We further summarize the techniques for the separation and purification of extracellular vesicles, providing a methodological foundation for future research and applications. Although the field of probiotic extracellular vesicle research is still in its infancy, the prospects for their application in the biomedical field are broad, potentially emerging as a novel therapeutic approach.

The increase of sequence data in public nucleotide databases has made DNA sequence-based identification an indispensable tool for fungal identification. However, the large proportion of mislabeled sequence data in public databases leads to frequent misidentifications. Inaccurate identification is causing severe problems, especially for industrial and clinical fungi, and edible mushrooms. Existing species identification pipelines require separate validation of a dataset obtained from public databases containing mislabeled taxonomic identifications. To address this issue, we developed FunVIP, a fully automated phylogeny-based fungal validation and identification pipeline (https://github.com/Changwanseo/FunVIP). FunVIP employs phylogeny-based identification with validation, where the result is achievable only with a query, database, and a single command. FunVIP command comprises nine steps within a workflow: input management, sequence-set organization, alignment, trimming, concatenation, model selection, tree inference, tree interpretation, and report generation. Users may acquire identification results, phylogenetic tree evidence, and reports of conflicts and issues detected in multiple checkpoints during the analysis. The conflicting sample validation performance of FunVIP was demonstrated by re-iterating the manual revision of a fungal genus with a database with mislabeled sequences, Fuscoporia. We also compared the identification performance of FunVIP with BLAST and q2-feature-classifier with two mass double-revised fungal datasets, Sanghuangporus and Aspergillus section Terrei. Therefore, with its automatic validation ability and high identification performance, FunVIP proves to be a highly promising tool for achieving easy and accurate fungal identification.

Two Gram-stain-negative, strictly aerobic, non-motile, rod-shaped bacteria, designated D3-12ᵀ and G2-2ᵀ, were isolated from the phycosphere of marine red algae. Both strains exhibited catalase- and oxidase-positive activities. Strain D3-12ᵀ grew optimally at 30°C, pH 7.0, and 2.0–3.0% (w/v) NaCl, while strain G2-2ᵀ showed optimal growth at 30°C, pH 7.0, and 2.0% NaCl. Ubiquinone-10 was the sole respiratory quinone in both strains. The major fatty acids (> 5%) in strain D3-12ᵀ were feature 8 (C_18:1 ω7c and/or C_18:1 ω6c), 11-methyl-C_18:1 ω7c, and C_16:0, while strain G2-2ᵀ contained summed feature 8 and C_16:0. The predominant polar lipids in strain D3-12ᵀ were phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine, whereas strain G2-2ᵀ contained phosphatidylglycerol and diphosphatidylglycerol. The genomic DNA G + C content was 59.9% for strain D3-12ᵀ and 60.2% for strain G2-2ᵀ. Phylogenetic analyses based on 16S rRNA and whole-genome sequences placed both strains into distinct lineages within the family Roseobacteraceae, separate from previously described genera. Genome-based relatedness metrics, including average nucleotide identity, digital DNA-DNA hybridization, average amino acid identity, and percentage of conserved proteins, further confirmed that these strains represent novel genera. Based on phenotypic, chemotaxonomic, and molecular characteristics, strains D3-12ᵀ and G2-2ᵀ are proposed as novel genera: Phycobium rhodophyticola gen. nov., sp. nov. (D3-12ᵀ = KACC 22712ᵀ = JCM 35528ᵀ) and Aliiphycobium algicola gen. nov., sp. nov. (G2-2ᵀ = KACC 22602ᵀ = JCM 35752ᵀ). Additionally, metabolic features relevant to interactions with marine algae, including genes associated with carbohydrate-active enzymes, vitamin biosynthesis, phenylacetic acid production, and bacterioferritin synthesis, were bioinformatically investigated.

The innate immune system relies on innate immune sensors, such as pattern recognition receptors (PRRs), to detect pathogens and initiate immune responses, crucial for controlling infections but also implicated in inflammatory diseases. These innate immune sensors, including Toll-like receptors (TLRs), nod-like receptors (NLRs), RIG-I-like receptors (RLRs), absent in melanoma 2 (AIM2), and Z-DNA binding protein 1 (ZBP1) trigger signaling pathways that produce cytokines, modulating inflammation and cell death. Traditional therapies focus on directly targeting pathogens; however, host-targeting therapeutic strategies have emerged as innovative approaches to modulate innate immune sensor activity. These strategies aim to fine-tune the immune response, either enhancing antiviral defenses or mitigating hyperinflammation to prevent tissue damage. This review explores innate immune sensor-based therapeutic approaches, including inhibitors, agonists, and antagonists, that enhance antiviral defense or suppress harmful inflammation, highlighting innate immune sensors as promising targets in infectious and inflammatory disease treatment.

Antimicrobial resistance (AMR) poses a serious threat to public health, with the emergence of extended-spectrum beta-lactamases (ESBLs) in Enterobacteriaceae, particularly Escherichia coli, raising significant concerns. This study aims to elucidate the drivers of antimicrobial resistance, and the global spread of cefotaxime-resistant E. coli (CREC) strains. Whole-genome sequencing (WGS) was performed to explore genome-level characteristics, and phylogenetic analysis was conducted to compare twenty CREC strains from this study, which were isolated from broiler chicken farms in Bangladesh, with a global collection (n = 456) of CREC strains from multiple countries and hosts. The MIC analysis showed over 70% of strains isolated from broiler chickens exhibiting MIC values ≥ 256 mg/L for cefotaxime. Notably, 85% of the studied farms (17/20) tested positive for CREC by the end of the production cycle, with CREC counts increasing from 0.83 ± 1.75 log10 CFU/g feces on day 1 to 5.24 ± 0.72 log10 CFU/g feces by day 28. WGS revealed the presence of multiple resistance genes, including bla_CTX-M, which was found in 30% of the strains. Phylogenetic comparison showed that the Bangladeshi strains were closely related to strains from diverse geographical regions and host species. This study provides a comprehensive understanding of the molecular epidemiology of CREC. The close phylogenetic relationships between Bangladeshi and global strains demonstrate the widespread presence of cefotaxime-resistant bacteria and emphasize the importance of monitoring AMR in food-producing animals to mitigate the spread of resistant strains.

Pectin-rich biomass, derived from fruit and citrus processing waste, presents a promising yet underutilized resource for sustainable biofuel and biochemical production. Its low lignin content and high concentrations of fermentable sugars, including D-galacturonic acid, L-arabinose, and D-xylose, make it an attractive feedstock. Unlike lignocellulosic biomass, pectin-rich hydrolysates require milder pretreatment, improving sugar recovery efficiency. However, industrial strains such as Saccharomyces cerevisiae exhibit strong glucose preference, limiting the efficient co-fermentation of mixed sugars. While prior reviews have broadly addressed lignocellulosic biomass utilization, this mini-review uniquely centers on the specific metabolic challenges and opportunities associated with pectin-rich feedstocks. In addition to incorporating established strategies for the co-utilization of cellobiose and xylose, we highlight recent advances that allow S. cerevisiae to metabolize carbon sources specifically from pectin-rich biomass, such as L-arabinose and D-galacturonic acid—monomers not prevalent in traditional lignocellulosic biomass. By integrating discussions on sugar transport engineering, redox balancing, and pathway optimization, this review offers a comprehensive framework to overcome glucose repression and support efficient co-fermentation of carbon sources from conventional and pectin-rich biomass. Drawing on these advances, we outline practical strategies to enhance fermentation performance and expand the valorization of food processing residues in biomanufacturing.

Bandavirus dabieense, a single-stranded RNA virus, is the causative agent of severe fever with thrombocytopenia syndrome (SFTS), a disease associated with high fatality rates. Early and accurate diagnosis is essential for improving clinical outcomes, particularly given the limited therapeutic options and high mortality rates associated with SFTS. However, while highly sensitive, conventional diagnostic methods such as PCR and qRT-PCR require specialized laboratory facilities and trained personnel, making them impractical for rapid detection in resource-limited settings. To address these challenges, we developed a rapid and highly sensitive assay for Bandavirus dabieense detection by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) with CRISPR/Cas12a technology. LAMP primers and guide RNA sequences were designed to target the L gene, ensuring broad detection across viral genotypes. The optimized assay demonstrated a detection limit of 5 RNA copies per reaction, showing more sensitivity than qRT-PCR, and exhibited 100% concordance with qRT-PCR results in clinical samples. Given its speed, accuracy, and field applicability, this LAMP-CRISPR/Cas12a-based assay represents a promising diagnostic tool for early SFTSV detection, particularly in resource-constrained environments where conventional molecular diagnostics are not readily available.

Two Gram-stain-positive, facultatively anaerobic, rod-shaped, and non-motile lactic acid bacterial strains, designated as strains CBA3605^T and CBA3606^T, were isolated from kimchi, a traditional Korean fermented food. Both strains were oxidase- and catalase-negative, non-spore-forming, non-hemolytic, and non-gas-producing. Optimal growth conditions for the two strains were observed at 30°C, pH 5.0, and 0% NaCl. The two genomes were composed of a circular chromosome and three plasmids and the DNA G + C content of 43.0%, respectively. Strains CBA3605^T and CBA3606^T were most closely related to Lactiplantibacillus (Lp.) pingfangensis 382-1^T with 16S rRNA sequence similarity of 99.4% and 99.1%, respectively. However, the orthologous average nucleotide identities between CBA3605^T and CBA3606^T were 91.7%, and those with strain 382-1^T were 76.9% and 76.5%, respectively. Digital DNA–DNA hybridization values between CBA3605^T and CBA3606^T were 45.0%, and those with strain 382-1^T were 21.4% and 21.0%, respectively. The major fatty acids detected in both strains included C_16:0, C_18:1 ω9c, and summed features 7 (C_19:1 ω7c, C_19:1 ω6c, C_19:0 cyclo ω10c, and/or C_19:0 ω6c). The peptidoglycan of both strains CBA3605^T and CBA3606^T contained meso-diaminopimelic acid and was classified as A4α type (L-Lys–D-Asp). In polar lipid analyses, only strain CBA3605^T contained aminophosphoglycolipid, which was absent in CBA3606^T, although both strains harbored same major polar lipids (diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine). Based on phenotypic, phylogenetic, genomic, biochemical, and chemotaxonomic analyses, strains CBA3605^T and CBA3606^T represent two novel species of the genus Lactiplantibacillus, for which the names Lactiplantibacillus koreensis sp. nov. and Lactiplantibacillus kimchii sp. nov. are proposed, with CBA3605^T (= KACC 81073BP^T = JCM 37965^T), and CBA3606^T (= KACC 81074BP^T = JCM 37966^T) as the type strains.

A Gram-stain-negative, aerobic, non-motile, rod-shaped, and orange-pigmented bacterium, designated CJ426^T, was isolated from ginseng soil in Anseong, Korea. Strain CJ426^T grew optimally on Reasoner’s 2A agar at 30°C and pH 7.0 in the absence of NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain CJ426^T belonged to the family Chitinophagaceae and had the highest sequence similarity with Niabella hibiscisoli KACC 18857^T (98.7%). The 16S rRNA gene sequence similarities with other members of the genus Niabella ranged from 92.3% to 98.1%. Phylogenomic analyses and overall genomic relatedness indices, including average nucleotide identity, average amino acid identity, and the percentage of conserved proteins values, supported the classification of strain CJ426^T as a representative of a novel genus within the family Chitinophagaceae. Furthermore, genome-based analyses suggested that five members of the genus Niabella, including N. aquatica, N. defluvii, N. ginsengisoli, N. hibiscisoli, and, N. yanshanensis, should be separated from other Niabella species and be assigned as a novel genus. The major isoprenoid quinone of strain CJ426^T was menaquinone-7 (MK-7). The predominant polar lipids were phosphatidylethanolamine and six unidentified aminolipids. The major fatty acids were iso-C_15:0, iso-C_15:1 G, and iso-C_17:0 3-OH. The genome of strain CJ426^T was 6.3 Mbp in size, consisting of three contigs, with a G + C content of 41.9%. Based on a polyphasic taxonomic approach, strain CJ426^T represents a novel genus and species within the family Chitinophagaceae, for which the name Paraniabella aurantiaca gen. nov., sp. nov. is proposed. The type strain is CJ426^T (= KACC 23908^T = JCM 37728^T).

Systemic sclerosis (SSc) is a chronic autoimmune disorder characterised by skin fibrosis and internal organ involvement. Disruptions in the microbial communities on the skin may contribute to the onset of autoimmune diseases that affect the skin. However, current research on the skin microbiome in SSc is lacking. This study aimed to investigate skin microbiome associated with disease severity in SSc. Skin swabs were collected from the upper limbs of 46 healthy controls (HCs) and 36 patients with SSc. Metagenomic analysis based on the 16S rRNA gene was conducted and stratified by cutaneous subtype and modified Rodnan skin score (mRSS) severity. Significant differences in skin bacterial communities were observed between the HCs and patients with SSc, with further significant variations based on subtype and mRSS severity. The identified biomarkers were Bacteroides and Faecalibacterium for patients with diffuse cutaneous SSc with high mRSS (≥ 10) and Mycobacterium and Parabacteroides for those with low mRSS (< 10). Gardnerella, Abies, Lactobacillus, and Roseburia were the biomarkers in patients with limited cutaneous SSc (lcSS) and high mRSS, whereas Coprococcus predominated in patients with lcSS and low mRSS. Cutaneous subtype analysis identified Pediococcus as a biomarker in the HCs, whereas mRSS analysis revealed the presence of Pseudomonas in conjunction with Pediococcus. In conclusion, patients with SSc exhibit distinct skin microbiota compared with healthy controls. Bacterial composition varies by systemic sclerosis cutaneous subtype and skin thickness.

Gout is an inflammatory arthritis resulting from the deposition of monosodium urate crystals. Urate-lowering therapies for gout have limitations, including side effects and limited efficacy, highlighting the need for novel therapeutic approaches to improve patient outcomes. In this context, our research team conducted a microbiome analysis of fecal samples from healthy individuals and gout patients, identifying Bifidobacterium as a key biomarker. Subsequently, we isolated and identified this strain, B. longum PMC72, and demonstrated its efficacy in a gout mouse model. In potassium oxonate (PO)-induced hyperuricemia mice, PMC72 significantly alleviated nausea, gait disturbances, ankle inflammation, and improved renal health. These effects were associated with marked reductions in oxidative stress markers, including serum uric acid, blood urea nitrogen, hepatic xanthine oxidase, and malondialdehyde (MDA) levels in serum, liver, and joint samples, as well as the downregulation of inflammation and uric acid transport-related gene expression in kidney samples. These benefits were comparable to those treated with Febuxostat, a standard urate-lowering therapy for gout. Furthermore, gut microbiome analysis revealed that PMC72 restored dysbiosis induced by hyperuricemia, contrasting with the reduced microbial diversity observed with febuxostat alone, and showed a complete recovery to eubiosis when combined with Febuxostat. These findings position PMC72 as a promising microbial therapeutic candidate for gout management, demonstrating significant development potential and serving as a benchmark for reverse translational microbiome-based therapeutic research.

Bladder cancer is the most common malignancy of the urinary tract and is a major health burden globally. Recent advances in microbiome research have revealed that the urinary tract harbors a resident microbial community, overturning the long-held belief in its sterility. Increasing evidence suggests that microbial dysbiosis and microbially derived metabolites contribute to bladder cancer carcinogenesis, progression, and therapeutic responses. Distinct microbial signatures have been observed in bladder cancer patients, with notable differences across disease stages and between primary and recurrent cases. Mechanistic studies have demonstrated that microbe-associated metabolites and toxins can drive DNA damage, chronic inflammation, extracellular matrix remodeling, and epithelial–mesenchymal transition. In addition, biofilm formation allows bacteria to evade immune responses and promotes persistent inflammation, creating a tumor-permissive niche. Beyond pathogenesis, microbial activity also influences therapeutic outcomes; for instance, some microbial pathways can inactivate frontline chemotherapy, while others generate metabolites with anti-tumor properties. Collectively, these patterns define a microbiota–metabolite–immunity axis, presenting opportunities for precision oncology. Targeting microbial pathways, profiling urinary microbiota, and harnessing beneficial metabolites offer promising advancements in biomarker discovery, prognostic refinement, and the development of novel therapeutic strategies for bladder cancer.

Bacteria-free reverse genetics techniques are crucial for the efficient generation of recombinant viruses, bypassing the need for labor-intensive bacterial cloning. These methods are particularly relevant for studying the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19. This study compared the efficiency of three bacteria-free approaches—circular polymerase extension reaction (CPER) with and without nick sealing and infectious sub-genomic amplicons (ISA)—to bacterial artificial chromosome (BAC)-based technology for rescuing SARS-CoV-2. Significant differences in viral titers following transfection were observed between methods. CPER with nick sealing generated virus titers comparable to those of the BAC-based method and 10 times higher than those of the standard CPER. In contrast, ISA demonstrated extremely low efficiency, as cytopathic effects were detected only after two passages. All rescued viruses exhibited replication kinetics consistent with those of the original strain, with no significant deviation in replication capacity. Furthermore, the utility of CPER and ISA in genetically modifying SARS-CoV-2 was demonstrated by successfully inserting the gene encoding green fluorescent protein into the genome. Overall, this study underscores the potential of bacteria-free methods, such as CPER and ISA, in advancing SARS-CoV-2 research while highlighting their significant differences in efficiency.

Microbial biosynthesis using yeast species offers numerous advantages to produce industrially relevant biofuels and biochemicals. Conventional metabolic engineering approaches in yeast focus on biosynthetic pathways in the cytoplasm, but these approaches are disturbed by various undesired factors including metabolic crosstalk, competing pathways and insufficient precursors. Given that eukaryotic cells contain subcellular organelles with distinct physicochemical properties, an emerging strategy to overcome cytosolic pathway engineering bottlenecks is through repurposing these organelles as specialized microbial cell factories for enhanced production of valuable chemicals. Here, we review recent progress and significant outcomes of harnessing organelle engineering for biofuels and biochemicals production in both conventional and non-conventional yeasts. We highlight key engineering strategies for the compartmentalization of biosynthetic pathways within specific organelles such as mitochondria, peroxisomes, and endoplasmic reticulum; involved in engineering of signal peptide, cofactor and energy enhancement, organelle biogenesis and dual subcellular engineering. Finally, we discuss the potential and challenges of organelle engineering for future studies and propose an automated pipeline to fully exploit this approach.

Two novel, Gram-stain-negative, anaerobic, and non-motile bacterial strains, designated KFT8^T and CG01^T, were isolated from the feces of healthy individuals without diagnosed diseases and characterized using a polyphasic approach. Phylogenetic analysis revealed that both strains belong to the genus Bacteroides, with < 99.0% similarity in their 16S rRNA gene sequences to B. facilis NSJ-77^T and B. nordii JCM 12987^T. Within the genus Bacteroides, strain KFT8^T exhibited the highest Orthologous Average Nucleotide Identity value of 94.7% and a digital DNA-DNA hybridization value of 63.7% with B. ovatus ATCC 8483^T, whereas strain CG01^T showed the highest values of 95.3% and 63.3%, respectively, with B. nordii JCM 12987^T. The values between the two novel strains were 74.8% and 21.4%, respectively, which are below the species delineation thresholds, supporting their classification as novel species. The major fatty acid of strain KFT8^T was C_18:1 ω9c, whereas strain CG01^T predominantly contained summed feature 11 (comprising iso-C_17:0 3OH and/or C_18:2 DMA). The only respiratory quinone was MK-11, the major polar lipid was phosphatidylethanolamine. Both strains produced succinic acid and acetic acid as common metabolic end-products of fermentation, while lactic acid and formic acid were detected individually in each strain. Based on polyphasic characterization, strains KFT8^T (= KCTC 15614^T = JCM 36011^T) and CG01^T (= KCTC 15613^T = JCM 36010^T) represent two novel species within the genus Bacteroides, for which the names Bacteroides celer sp. nov. and Bacteroides mucinivorans sp. nov. are proposed, respectively. Additionally, genome-based analyses and phenotypic comparisons revealed that B. koreensis and B. kribbi represent the same strain, showing genomic relatedness to B. ovatus that exceeds the threshold for species delineation. Consequently, we propose the reclassification of B. koreensis Shin et al. 2017 and B. kribbi Shin et al. 2017 as later heterotypic synonyms of B. ovatus Eggerth and Gagnon 1933 (Approved Lists 1980).

The global spread of COVID-19 has underscored the urgent need for advanced tools to study emerging coronaviruses. Reverse genetics systems have become indispensable for dissecting viral gene functions, developing live-attenuated vaccine candidates, and identifying antiviral targets. In this study, we describe a robust and efficient reverse genetics platform for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The system is based on the assembly of a full-length infectious cDNA clone from seven overlapping fragments, each flanked by homologous sequences to facilitate seamless assembly using the Gibson assembly method. Individual cloning of each fragment into plasmids enables modular manipulation of the viral genome, allowing rapid site-directed mutagenesis by fragment exchange. Infectious recombinant virus was successfully recovered from the assembled cDNA, exhibiting uniform plaque morphology and genetic homogeneity compared to clinical isolates. Additionally, fluorescent reporter viruses were generated to enable real-time visualization of infection, and the effects of different mammalian promoters on viral rescue were evaluated. This reverse genetics platform enables efficient generation and manipulation of recombinant SARS-CoV-2, providing a valuable resource for virological research and the development of preventive and therapeutic antiviral measures.

The 2015 Zika virus (ZIKV) outbreak in Brazil and its global spread underscored the urgent need for effective and broadly protective vaccines. While C57BL/6 and BALB/c mice are widely used in preclinical vaccine research, direct comparisons of their ability to elicit ZIKV-specific neutralizing antibodies (nAbs) remain limited. This study aimed to systematically evaluate and compare the immunogenic potential of these two common mouse strains across diverse vaccine platforms, focusing on their capacity to generate functional neutralizing antibody responses. We assessed nAb and IgG responses following four vaccination strategies: (1) DNA vaccine encoding prMEΔTM followed by E protein domain III boost, (2) recombinant EΔTM protein expressed using baculovirus system, (3) formalin-inactivated ZIKV, and (4) live ZIKV. Although both strains generated detectable ZIKV- and E protein-specific IgG, the magnitude and quality of responses varied by vaccine platform and strain. Notably, C57BL/6 mice consistently mounted significantly higher nAb titers than BALB/c mice across all immunization groups, including subunit- and whole-virus-based vaccines. In contrast, BALB/c mice showed lower or undetectable nAb responses, despite comparable or higher total IgG levels in some cases. These findings show that host genetic background is a critical determinant of vaccine-induced neutralization and underscore the importance of selecting appropriate animal models in ZIKV vaccine development. C57BL/6 mice, due to their robust nAb responses, represent a reliable model for evaluating vaccine immunogenicity. Conversely, the limited nAb responses in BALB/c mice position them as a potential low-responder model, offering a stringent system to test the potency and breadth of protective immunity under suboptimal conditions.

Synbiotics have become a new-age treatment tool for limiting the progression of metabolic dysfunction-associated steatotic liver disease; however, inclusive comparisons of various synbiotic treatments are still lacking. Here, we have explored and evaluated multiple synbiotic combinations incorporating three distinctive prebiotics, lactitol, lactulose and fructooligosaccharides. Of the synbiotic treatments evaluated, a combination of fructooligosaccharides and probiotics (FOS+Pro) exhibited superior protection against western diet-induced liver degeneration. This synbiotic (FOS+Pro) combination resulted in the lowest body weight gains, liver weights and liver/body weight ratios. The FOS+Pro synbiotic combination substantially alleviated liver histopathological markers and reduced serum AST and cholesterol levels. FOS+Pro ameliorated hepatic inflammation by lowering expression of proinflammatory markers including TNF-α, IL-1β, IL-6, and CCL2. FOS+Pro significantly improved steatosis by restricting the expression of lipid metabolic regulators (ACC1, FAS) and lipid transporters (CD36) in the liver. These findings are critical in suggesting that synbiotic treatments are capable of restraining western diet-induced metabolic dysfunction in the liver. Additionally, this study demonstrated that adding probiotic strains amplified the effectiveness of fructooligosaccharides but not all prebiotics.

Porins in the outer membrane (OM) of Gram-negative bacteria play two main functions: passage of various extracellular molecules and maintenance of membrane integrity. OmpC, a non-specific porin, is involved in both functions; however, the exact mechanism of maintenance of membrane integrity remains unknown. In this study, we found that inhibiting cardiolipin biosynthesis partially restored the growth defect of the ompC mutant under envelope stress. Among the three enzymes involved in cardiolipin biosynthesis, ClsABC, this effect is primarily associated with ClsA. Notably, the deletion of ClsA also suppressed the similar phenotypes of an Escherichia coli mutant lacking YhdP, a transmembrane protein involved in phospholipid transport from the inner membrane to the OM. Collectively, these results imply that OmpC may contribute to membrane integrity, partially through mechanisms linked to transport or biosynthesis of phospholipids such as cardiolipin.

The global rise in obesity and its associated metabolic complications underscores the urgent need for safe and effective interventions. This study investigated the anti-obesity efficacy of a probiotic mixture containing Bifidobacterium breve BR3 and Lactiplantibacillus plantarum LP3 in C57BL/6 mice with high-fat diet (HFD)-induced obesity. After obesity was established by feeding a 60% kcal HFD, the probiotic mixture was administered orally for 4 weeks. Compared with the control group, mice receiving the L. plantarum LP3 and B. breve BR3 mixture exhibited significant reductions in body weight and total fat mass, as assessed by Dual-energy X-ray Absorptiometry (DXA) and Echo Magnetic Resonance Imaging (EchoMRI). The probiotic treatment also lowered serum Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), and glucose levels, and attenuated lipid accumulation in both hepatic and epididymal adipose tissues. Transcriptomic profiling revealed upregulation of lipolytic genes (Sirt1, Pparα) and downregulation of lipogenic genes (Srebp1c, Fas), suggesting that the probiotic mixture promotes lipid catabolism while suppressing lipid synthesis. Additionally, serum adipokine levels were favorably modulated, indicating improved metabolic homeostasis. Gut microbiota analysis demonstrated an increased relative abundance of beneficial genera, including Akkermansia and Bacteroides, highlighting a microbiome-mediated contribution to the observed metabolic benefits. Overall, our findings indicate that the combined administration of Lactiplantibacillus plantarum LP3 and Bifidobacterium breve BR3 exerts multi-faceted anti-obesity effects by enhancing lipolysis, regulating lipid metabolism, and restoring a healthy gut microbial balance. This probiotic mixture represents a promising therapeutic approach for managing obesity and related metabolic disorders.

Minicells, which are anucleate cells generated by irregular cell division, are emerging as promising drug delivery systems owing to advances in synthetic biology. However, their development is largely limited to a few model bacteria, highlighting the need to explore minicell platforms in alternative hosts. Lactiplantibacillus plantarum (L. plantarum), a probiotic bacterium classified as Generally Recognized as Safe, is an ideal candidate for such exploration. Minicell-producing L. plantarum was engineered by deleting the putative minD gene via plasmid-mediated homologous recombination, which inactivates cell division to form spherical minicells. Anucleate cells were isolated through differential centrifugation and filtration, followed by additional drug treatment to completely eliminate progenitor cells. Microscopy and flow cytometry analyses of the purified sample confirmed the absence of progenitor cells by DAPI staining. This protocol effectively produces bacterial minicells from L. plantarum for use in various biotechnological applications, including therapeutic agent delivery.

Marine organisms often form symbiotic relationships with various microorganisms to adapt and thrive in harsh environments. These symbiotic microbes contribute to host survival by providing nutrition, modulating the hosts’ immune system, and supporting overall physiological stability. Advances in high-throughput sequencing technologies have enabled a deeper understanding of the structure and function of symbiotic microbial communities, as well as host-microbe interactions. Notably, symbiotic bacteria associated with marine invertebrates such as corals and sponges are recognized as a potential source of useful bioactive compounds, including antibiotics and enzymes. However, obtaining high-quality microbial DNA from host tissues still remains a technical challenge due to the presence of unknown substances. This study focuses on optimizing sample preparation and DNA extraction procedures and additional purification to improve the recovery of microbial DNA while minimizing host DNA contamination. Comparison between several methods was conducted using sponge samples to evaluate DNA quality and microbial recovery. A sample designated as 2110BU-001 was collected from the east coast of the Republic of Korea and used for culture-independent microbial cell isolation. Total bacterial DNA was extracted by using a manual Phenol-Chloroform protocol and three commercial kits. DNA extracted using the standard manual method showed both the highest yield and the largest fragment size. However, PCR (Polymerase chain reaction) test showed that quality of manually extracted DNA was not enough for sequencing. Therefore, the quality of DNA was improved through additional purification steps. Briefly, host eukaryotic cells were removed by mechanical process and almost only bacterial DNA was successfully obtained by combination of manual extraction method and further purification processes. The established protocol was successfully introduced to extraction of metagenomic DNA from mussel and jellyfish microbiomes, indicating that it can be widely applied to various marine organisms.

Mycobacterium avium complex (MAC) organisms are widespread environmental pathogens associated with chronic pulmonary infections. Although M. avium is known to invade epithelial cells, the molecular mechanisms underlying this process remain incompletely understood. In this study, we identified a novel role for MAVRS09815 (formerly MAV2054), a family 2A encapsulin nanocompartment shell protein, in mediating bacterial adhesion, epithelial cell invasion, and in vivo virulence. We engineered a recombinant M. smegmatis strain expressing MAV2054 (Ms_2054) and an M. avium MAV2054 deletion mutant (Δ2054). Ms_2054 exhibited enhanced epithelial invasion, whereas Δ2054 showed reduced intracellular survival. Recombinant MAV2054 protein was bound directly to human epithelial cells in a dose-dependent manner. Pretreatment of host cells with cytochalasin D or vinblastine significantly inhibited bacterial internalization, indicating that MAV2054-mediated invasion is cytoskeleton-dependent. Confocal and scanning electron microscopy revealed MAV2054-dependent membrane rearrangements during infection. Pull-down assays demonstrated that MAV2054 activates Cdc42, a key regulator of actin polymerization, with reduced activation observed in Δ2054-infected cells. In a murine intratracheal infection model, the Δ2054 exhibited significantly reduced bacterial burdens and lung inflammation compared to the wild type. These findings demonstrate that MAV2054 enhances M. avium virulence by promoting epithelial cell invasion through Cdc42-dependent cytoskeletal remodeling. This study reveals a previously unrecognized role for an encapsulin-like protein in host-pathogen interactions and highlights its potential as a therapeutic target in MAC infections.

Dual specificity phosphatases (DUSPs), a subfamily of the protein tyrosine phosphatase (PTP) family, dephosphorylate not only phosphotyrosine but also phosphoserine and phosphothreonine residues. Beyond the 26 members of this family in humans, DUSPs represent the only type of PTPs found across a wide range of microorganisms, including bacteria, archaea, and viruses. This review presents a comprehensive structural analysis of human and microbial DUSPs. These proteins commonly share core features, such as a typical DUSP fold, shallow active site pocket, signature active site motif known as the P-loop, and conserved aspartate residue that acts as a general acid/base. However, DUSPs from diverse microorganisms also display unique structural and functional characteristics. Pseudomonas aeruginosa TpbA is the only bacterial DUSP identified to date, while a second candidate was proposed in this review. Archaeal DUSPs are hyperthermostable, contain a unique motif in their P-loops, and employ dual general acid/base residues. Poxviral DUSPs are characterized by the formation of domain-swapped homodimers. The presence of DUSPs across all domains of life and viruses, along with their low specificity for phosphorylated amino acids and structural similarity to classical PTPs, suggests that DUSPs represent the ancestral form of PTPs.

Existing microbial engineering strategies—encompassing metabolic engineering, systems biology, and systems metabolic engineering—have significantly enhanced the potential of microbial cell factories as sustainable alternatives to the petrochemical industry by optimizing metabolic pathways. Recently, systems metabolic engineering, which integrates tools from synthetic biology, enzyme engineering, omics technology, and evolutionary engineering, has been successfully developed. By leveraging modern engineering strategies within the Design-Build-Test-Learn (DBTL) cycle framework, these advancements have revolutionized the biosynthesis of valuable compounds. This review highlights recent progress in the metabolic engineering of Corynebacterium glutamicum, a versatile microbial platform, achieved through various approaches from traditional metabolic engineering to advanced systems metabolic engineering, all within the DBTL cycle. A particular focus is placed C5 platform chemicals derived from L-lysine, one of the key amino acid production pathways of C. glutamicum. The development of DBTL cycle-based metabolic engineering strategies for this process is discussed.

Strains Mo2-6^T, S9, KG4-3^T, and 50Mo3-2, identified as coagulase-negative, Gram-stain-positive, halotolerant, non-motile coccoid bacteria, were isolated from traditional Korean soybean foods. Strains Mo2-6^T and S9 were both catalase- and oxidase-negative, whereas KG4-3^T and 50Mo3-2 were catalase-positive but oxidase-negative. The optimal growth conditions for Mo2-6^T and S9 were 30°C, 2% NaCl, and pH 7.0, while KG4-3^T and 50Mo3-2 grew best at 35°C, 2% NaCl, and pH 7.0. All strains contained menaquinone-7 as the predominant isoprenoid quinone, with anteiso-C_15:0 and iso-C_15:0 as the major cellular fatty acids (> 10%). Additionally, anteiso-C_13:0 was a major fatty acid in strain KG4-3^T. The DNA G + C contents of strains Mo2-6^T, S9, KG4-3^T, and 50Mo3-2 were 33.4%, 33.3%, 32.5%, and 32.7%, respectively. Phylogenetic analyses based on the 16S rRNA gene and whole-genome sequences revealed that strains Mo2-6^T and S9, as well as KG4-3^T and 50Mo3-2, formed distinct lineages within the genus Staphylococcus. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses confirmed that strains Mo2-6^T and S9, as well as KG4-3^T and 50Mo3-2, belonged to the same species. Meanwhile, dDDH and ANI values between strains Mo2-6^T and KG4-3^T, as well as comparisons with other Staphylococcus type strains, were below the species delineation thresholds, indicating they represent novel species. Based on phenotypic, chemotaxonomic, and molecular data, we propose strain Mo2-6^T as the type strain of Staphylococcus parequorum sp. nov. (=KACC 23685^T =JCM 37038^T) and strain KG4-3^T as the type strain of Staphylococcus halotolerans sp. nov. (=KACC 23684^T =JCM 37037^T).

The oral administration of synthetic drugs can effectively reduce blood lipid levels, but adverse reactions may occur. Because of this, the hypolipidemic ability of natural products has been increasingly investigated. We evaluate the safety and hypolipidemic characteristics of a water-soluble blue pigment extracted using HPD-400 resin from the fungus Quambalaria cyanescens. Hypolipidemic ability was examined by constructing a hyperlipidemia model with different doses of blue pigment (50, 100, and 200 mg/kg. mouse body weight) for 28 d. Blue pigment purity increased from 20.32% to 70.70% following treatment with HPD-400 resin. Acute toxicity tests revealed blue pigment sourced from Q. cyanescens to have no toxic effects on mouse body weight, mortality, or behavioral characteristics. Subacute toxicity tests revealed no significant differences in food intake, body weight, or organ weights between treatment groups and controls. Histopathological examination of the liver and kidney tissues of mice administered blue pigment were normal, and serum enzyme activities and blood constituents were also within normal ranges. Blue pigment can significantly reduce the weight of mice, reduce liver and kidney damage and fat accumulation. It can also reduce total cholesterol, triglyceride and low density lipoprotein cholesterol in serum and liver tissue, and increase the level of high density lipoprotein cholesterol. Reduce the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatinine, urea and uric acid in serum. Increase the activities of total superoxide dismutase, glutathione peroxidase and catalase in serum and liver tissue, reduce the content of malondialdehyde, and up-regulate liver lipase and lipoprotein lipase. Our work proves that blue pigment is nontoxic, has the function of reducing blood lipid, and can alleviate obesity-related symptoms by regulating lipid metabolism and oxidative stress.

Tella is a traditional beverage widely accepted by consumers, despite the lack of product consistency owing to its reliance on natural fermentation. This study aimed to identify potential industrial lactic acid bacteria (LAB) starter cultures based on their technological properties. Seven LAB strains isolated from Tella were characterized for their carbohydrate utilization, salt content, temperature, and acid tolerances, growth and acidification rates, and metabolite profiles. Most strains efficiently utilized various carbohydrates, with Lactiplantibacillus plantarum TDM41 showing exceptional versatility. The strains exhibited similar growth characteristics. Principal component analysis of stress tolerance properties revealed that L. plantarum TDM41, Pediococcus pentosaceus TAA01, and Leuconostoc mesenteroides TDB22 exhibited superior tolerance ability. Strong acidification properties were detected in the L. plantarum TDM41, P. pentosaceus TAA01, and Leuconostoc mesenteroides TDB22 strains after 24 h incubation at 30°C. L. plantarum TDM41 displayed the fastest acidification rate throughout the analysis period. All LAB strains produced significant amounts of diverse organic acids, including lactic acid, citric acid, acetic acid, malic acid, and succinic acid, with lactic acid being the primary acid produced by each strain. Overall, strains L. plantarum TDM41 and P. pentosaceus TAA01 prove to be potential candidates for Tella industrial starter cultures and similar cereal products owing to their robust technological properties.

Antarctic fungi can effectively adapt to extreme environments, which leads to the production of unique bioactive compounds. Studies on the discovery of fungi in the diverse environments of Antarctica and their potential applications are increasing, yet remain limited. In this study, fungi were isolated from various substrates on the Fildes Peninsula in Antarctica and screened for their antibiosis activity against two significant plant pathogenic fungi, Botrytis cinerea and Fusarium culmorum. Phylogenetic analysis using multiple genetic markers revealed that the isolated Antarctic fungal strains are diverse, some of which are novel, emphasizing the underexplored biodiversity of Antarctic fungi. These findings suggest that these fungi have potential for the development of new antifungal agents that can be applied in agriculture to manage fungal plant pathogens. Furthermore, the antibiosis activities of the isolated Antarctic fungi were evaluated using a dual-culture assay. The results indicated that several strains from the genera Cyathicula, Penicillium, and Pseudeurotium significantly inhibited pathogen growth, with Penicillium pancosmium showing the highest inhibitory activity against Botrytis cinerea. Similarly, Aspergillus and Tolypocladium strains exhibited strong antagonistic effects against Fusarium culmorum. This study enhances our understanding of Antarctic fungal diversity and highlights its potential for biotechnological applications.

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) were thought to be carried-out by anaerobic bacteria constrained to anoxic conditions as they use nitrate (NO₃^-) as a terminal electron acceptor instead of molecular O₂. Three soil bacilli, Neobacillus spp. strains PS2-9 and PS3-12 and Bacillus salipaludis PS3-36, were isolated from rice paddy field soil in Korea. The bacterial strains were selected as possible candidates performing aerobic denitrification and DNRA as they were observed to reduce NO₃^- and produce extracellular NH₄⁺ regardless of oxygen presence at the initial screening. Whole genome sequencing revealed that these strains possessed all the denitrification and DNRA functional genes in their genomes, including the nirK, nosZ, nirB, and nrfA genes, which were simultaneously cotranscribed under aerobic condition. The ratio between the assimilatory and dissimilatory NO₃^- reduction pathways depended on the availability of a nitrogen source for cell growth, other than NO₃^-. Based on the phenotypic and transcriptional analyses of the NO₃^- reductions, all three of the facultative anaerobic strains reduced NO₃^- likely in both assimilatory and dissimilatory pathways under both aerobic and anoxic conditions. To our knowledge, this is the first report that describes coexistence of NO₃^- assimilation, denitrification, and DNRA in a Bacillus or Neobacillus strain under aerobic condition. These strains may play a pivotal role in the soil nitrogen cycle.

Collagenase and keratinase are two important proteolytic enzymes with recognized applications in biotechnology and medicine, particularly in the enzymatic removal of necrotic tissue and the control of infection. In the present work, a soil isolate of Bacillus subtilis strain AB2 (PX453297.1) was optimized for enzyme production under different nutritional and physicochemical conditions. The enzymes were recovered by ammonium sulphate precipitation and dialysis, examined by SDS-PAGE and zymography, and further assessed for pH and temperature optima, stability, the influence of metal ions, and kinetic parameters. Maximum collagenase activity (4.41 ± 0.22 U/ml) was observed at 37°C and pH 7.5 in a glucose–peptone medium, whereas keratinase production was enhanced between 37 and 40°C at pH 7.5 in lactose–peptone medium. Protein bands of approximately 55 and 33 kDa were detected, representing 6.2- and 5.5-fold purification. Collagenase showed an alkaline optimum (pH 10.0, 37–45°C) with K_m 0.31% and V_max 1.92 U/ml, while keratinase exhibited dual optima (pH 3.0 and ~7.0) with K_m 0.27% and V_max 0.84 U/ml. Biofilm assays revealed that collagenase reduced pre-formed biomass by 62–68% and viable counts by 1.1–1.7 log₁₀, clearly outperforming keratinase (41–57%, 0.7–1.2 log₁₀). When combined with conventional antibiotics, both enzymes potentiated activity, with notable synergy between collagenase and oxacillin against Staphylococcus aureus (FICI 0.31–0.37), ciprofloxacin against Pseudomonas aeruginosa (FICI 0.37–0.50), and meropenem against Klebsiella pneumoniae (FICI 0.28–0.44). These results indicate that B. subtilis AB2 produces collagenase and keratinase with distinct biochemical characteristics and strong antibiofilm properties, underscoring their promise as adjuncts in chronic wound care as well as in industrial applications.

Akkermansia muciniphila (AKK, A. muciniphila) fortifies the intestinal barrier, inhibits the colonization of pathogenic bacteria, and protects the host’s health. Nevertheless, the existing literature offers inadequate evidence to ascertain whether A. muciniphila can effectively treat Candida albicans (C. albicans) infections in vitro, and the underlying mechanisms remain ambiguous. This study, animal models were established through gavage with clinical isolates of C. albicans to induce gastrointestinal tract colonization and subsequent translocation infection. The models were subsequently administered A. muciniphila. We examined the analysis of 16S rRNA gene sequencing, metabolomics of colonic contents, and transcriptomics of colonic tissue. The intestinal barrier, inflammatory responses, and immune cell infiltration are analyzed. This study revealed that A. muciniphila markedly mitigated C. albicans translocation infection and modified the intestinal microbial community structure and metabolic attributes in model mice. After administering A. muciniphila to the translocation infection group, there was a notable increase in the prevalence of bacteria that produce short-chain fatty acids, including Eubacterium_F. Moreover, there was a significant increase in the levels of specific pathogens, including Faecalibaculum, Turicibacter, and Turicimonas. The study demonstrated that A. muciniphila treatment can improve the composition of intestinal microbiota and metabolites, augment the tight junctions of colonic tissue and diminish systemic inflammatory response. This presents an innovative therapeutic approach for the potential treatment of intestinal C. albicans infection using A. muciniphila.

Atopic dermatitis (AD) is a widespread inflammatory skin condition that affects the population worldwide. Given the implication of microbiota in AD pathogenesis, we investigated whether human-derived Lactobacillus strains could modulate AD. In this study, we identified Lactobacillus crispatus KBL693 as a probiotic candidate for AD treatment. In vitro, KBL693 suppressed mast cell degranulation and IL-4 production by T cells, suggesting its ability to attenuate key type 2 immune responses. Consistent outcomes were observed in a murine AD model, where oral administration of KBL693 alleviated disease symptoms and reduced hallmark type 2 immune markers, including plasma IgE as well as IL-4, IL-5, and IL-13 levels in skin lesions. In addition to downregulating these AD-associated immune responses, KBL693 promoted regulatory T cell (Treg) expansion in mesenteric lymph nodes, indicating its potential to restore immune balance. Collectively, these findings highlight the therapeutic potential of KBL693 for AD through enhancement of Tregs and suppression of type 2 immune responses.

Pathogenic fungi pose major threats to both global food security and human health, yet the molecular basis of their virulence remains only partially understood. Beyond genetic and transcriptional control, emerging evidence highlights protein glycosylation as a key post-translational modification that governs fungal development, stress adaptation, and host interactions. Glycosylation regulates protein folding, stability, trafficking, and immune evasion, thereby shaping infection processes across diverse pathogens. While extensively studied in model organisms, our understanding of glycosylation in pathogenic fungi remains fragmented and lacks a coherent framework linking glycosylation dynamics to fungal development and pathogenicity. This review synthesizes recent advances from proteomic, transcriptomic, and glycomic studies in pathogenic fungi, focusing on interspecific variation in glycogenes and enzymes, hierarchical regulatory networks, and glycoprotein-mediated mechanisms of virulence. Finally, we outline current challenges and highlight glycosylation-targeted strategies as promising avenues for antifungal intervention.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies have emerged as powerful tools for precise genome editing, leading to a revolution in genetic research and biotechnology across diverse organisms including microalgae. Since the 1950s, microalgal production has evolved from initial cultivation under controlled conditions to advanced metabolic engineering to meet industrial demands. However, effective genetic modification in microalgae has faced significant challenges, including issues with transformation efficiency, limited target selection, and genetic differences between species, as interspecies genetic variation limits the use of genetic tools from one species to another. This review summarized recent advancements in CRISPR systems applied to microalgae, with a focus on improving gene editing precision and efficiency, while addressing organism-specific challenges. We also discuss notable successes in utilizing the class 2 CRISPR-associated (Cas) proteins, including Cas9 and Cas12a, as well as emerging CRISPR-based approaches tailored to overcome microalgal cellular barriers. Additionally, we propose future perspectives for utilizing CRISPR/Cas strategies in microalgal biotechnology.

Spo0A, the master regulator of sporulation initiation in Bacillus subtilis, controls over 500 genes directly or indirectly in early sporulation stages. Although the effects of Spo0A disruption on sporulation have been extensively studied, a comprehensive understanding of the genomic response throughout growth phases remain elusive. Here, we examined the transcriptomic changes in Spo0A mutant strain, R211E, and wild-type across a time-course RNA-seq to identify impacted biological processes and pathways. The R211E strain, which exhibits sporulation deficiency, was constructed using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)9 system, highlighting the critical role of proper Cas9 dosing in gene editing. Functional analysis of 3,010 differentially expressed genes (DEGs) showed significant alterations in sporulation, quorum sensing, metabolism, and biofilm formation. The R211E disrupted the Spo0A-AbrB regulatory pathway, reducing biofilm formation and enhancing flagellar gene expression. Up-regulated metabolic pathways, including glycolysis, histidine, and purine biosynthesis, increased cell numbers during vegetative growth. Further, the mutant displayed elevated vegetative autolysin expression, resulting in reduced cell viability in the stationary phase. We also introduce the novel potential of R211E in a recombinant protein expression system that facilitated protein release into the supernatant, providing valuable insight for future research in metabolic engineering and efficient production systems in B. subtilis.

Two rod-shaped, Gram-positive, spore-forming, motile, and strictly anaerobic bacteria, FM7315^T and FM7330^T were isolated from Myeolchi-jeot, a traditional Korean fermented anchovy. Phylogenetic and phylogenomic analyses based on the 16S rRNA gene and genome sequences revealed that strains FM7315^T and FM7330^T represent novel species within the genus Haloimpatiens. The genome sizes of strains FM7315^T and FM7330^T were 3,052,517 bp and 4,194,114 bp, respectively, with G + C contents of 29.7 mol% and 28.0 mol%, respectively. Strain FM7315^T exhibited growth at 20–37°C, 0–2% NaCl, and pH range of 5.0–8.0, whereas strain FM7330^T grew at 25–45°C, 0–4% NaCl, and pH range of 5.0–9.0. Strain FM7315^T contains C_14:0, C_16:0, C_18:1 ω9c, Summed Feature 3 (C_16:1 ω7c/C_16:1 ω6c), and Summed Feature 8 (C_18:1 ω7c/C_18:1 ω6c) as major fatty acids, along with diphosphatidylglycerol, phosphatidylglycerol, glycolipid, two aminophospholipids, and five unidentified lipids. Strain FM7330^T contains C_16:0, C_17:1 ω8c, and C_18:1 ω9c as major fatty acids, along with diphosphatidylglycerol, two phosphatidylglycerols, four aminophospholipids, and six unidentified lipids. Based on their phenotypic, chemotaxonomic, and molecular characteristics, strains FM7315^T and FM7330^T represent two novel species of the genus Haloimpatiens, for which the names Haloimpatiens sporogenes sp. nov. (FM7315^T = KCTC 25939^T = JCM 37574^T) and Haloimpatiens myeolchijeotgali sp. nov. (FM7330^T = KCTC 25938^T = JCM 37575^T) have been proposed.

Prebiotics are indigestible dietary components that improve host health by stimulating the growth and metabolic activity of beneficial intestinal microbes. The whole grains are rich in non-digestible carbohydrates, which may confer prebiotic potential. Among them, millet and quinoa have gained attention as dietary alternatives due to the growing popularity of gluten-free diets. In this study, we examined the effects of proso millet and quinoa on the human gut microbiota using an in vitro fecal incubation model. Both grains altered alpha diversity metrics, including microbial richness, evenness, and phylogenetic diversity. Beta diversity analysis showed that the proso millet and quinoa treatment groups exhibited distinct clustering patterns compared to the control, highlighting their impact on microbial community structure. Taxonomic analysis showed an increase in beneficial genera, including Bifidobacterium, and a decrease in taxa such as Enterobacteriaceae and Flavonifractor. To assess metabolic changes associated with microbial fermentation, short-chain fatty acid (SCFA) intensities were measured. The intensities of acetic acid, propionic acid, and butyric acid were significantly higher in the proso millet- and quinoa-treated groups compared to the control group. Spearman correlation analysis showed that the abundances of Bifidobacterium and Blautia were significantly positively associated with SCFA intensities. Furthermore, predicted functional pathway analysis identified enrichment of carbohydrate-related pathways in proso millet and quinoa treatments. Quinoa supplementation led to a broader enhancement of metabolic pathways, including glycolysis/gluconeogenesis, starch and sucrose metabolism, and pentose phosphate pathways, whereas proso millet enriched galactose metabolism, and starch and sucrose metabolism. These findings suggest that proso millet and quinoa influence gut microbial diversity, composition, and function.

Pseudomonas aeruginosa (P. aeruginosa) is resistant to several drugs as well as antibiotics and is thus classified as multidrug resistant and extensively drug resistant. These bacteria have a secretion system called the "type 3 secretion system (T3SS)", which facilitates infection by delivering an effector protein. ExoenzymeS (ExoS) is known to induce cell death and activate caspase-1. In particular, patients infected with P. aeruginosa develop diseases associated with high mortality, such as pneumonia, because no drug targets an ExoS or T3SS. We selected natural compounds to treat T3SS-mediated pneumonia and chose alizarin, a red dye. We confirmed the effects of alizarin on T3SS by bacterial PCR and ELISA. It was confirmed that alizarin regulates ExoS by inhibiting exsA but also popD and pscF. Furthermore, in infected H292 cells, it not only attenuates inflammation by inhibiting lipopolysaccharide (LPS)-induced phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 but also interferes with the level of ExoS delivered into the host and modulates caspase-1. We confirmed this result and determined that it led to decreases in proinflammatory cytokines such as Interleukin-1beta (IL-1β), Interleukin-6 (IL-6), and Interleukin-18 (IL-18). Therefore, we suggest that alizarin is a suitable drug for treating pneumonia caused by P. aeruginosa because it helps to attenuate inflammation by regulating T3SS and NF-κB signaling.

A thermophilic strain of Paecilomyces variotii (MR1), capable of surviving temperatures above 40°C, was isolated from a paper mill and investigated as a host for heterologous protein production. To prevent environmental dissemination of spores, UV mutagenesis was employed to create a conidia-deficient strain, UM7. This strain underwent gene editing using Cas9-gRNA ribonucleoprotein (RNP) with HR donor DNA fragments, incorporating promoter sequences amplified from the genomic DNA of P. variotii (P_H4, P_P2, P_S8, P_tub, P_tef1, and P_gpdA), along with a signal sequence-tagged eGFP, flanked by 5’-upstream (336 bp) and 3’-downstream (363 bp) regions of pyrG. Co-transformation of HR donor DNA with RNP into protoplasts yielded 48 mutant pyrG transformants capable of surviving in the presence of 5-fluoroorotic acid (5-FOA). Sequence analysis identified 16 of the 48 pyrG-disrupted mutants carrying complete HR donor DNAs with the six different promoter sequences, indicating successful homology-directed repair (HDR). Evaluation of promoter strength revealed that P_gpdA was the most effective for intracellular GFP production; however, it resulted in negligible extracellular GFP signal under all promoter conditions. A newly edited strain with an HDR integration module connecting P_gpdA directly to eGFP, without the signal sequence, exhibited enhanced GFP expression in both mycelial cells and culture broth, suggesting that the signal peptide negatively affect protein expression and secretion. This work represents the first successful RNP-mediated gene editing in P. variotii, contributing to the application of this thermophilic fungus in protein production.