Applied Microbiology and Biotechnology最新文献

Recurrent bovine mastitis is a global concern that causes substantial economic losses and is exacerbated by pathogen internalization into mammary epithelial cells, and the emergence of antimicrobial resistance. These challenges necessitate the development of alternative antimicrobial strategies with multimodal activity. In this study, the naturally occurring molecule octanoic acid (OA) was evaluated for its antimicrobial efficacy and multitargeted mode of action against mastitis-associated pathogens. OA exhibited rapid bactericidal activity within 1 h and significantly reduced bacterial pathogenicity by attenuating toxin activity and inhibiting pathogen adhesion and internalization into epithelial cells. Transcriptomic analysis of Staphylococcus aureus revealed extensive OA-induced transcriptional alterations across multiple functional categories, including virulence regulation, stress response, metabolism, DNA replication and repair, membrane-associated functions, and transport systems, suggesting a broad cellular response to OA exposure. OA treatment also upregulated endogenous antimicrobial peptide (AMP) gene expression in MAC-T cells and did not induce detectable resistance even after 30 serial passages. Membrane perturbation was supported by molecular dynamics simulations and validated experimentally using DiBAC assays. In vivo toxicity assessment using Galleria mellonella demonstrated no observable toxicity up to 1000 mM OA. In addition, quantum chemical, physicochemical, and ADME/Tox analyses provided predictive insights into the chemical stability, drug-likeness, and safety profile of OA. Collectively, these findings suggest that OA exerts a multifaceted antimicrobial effect and represents a promising candidate for the development of next-generation antimicrobials targeting recurrent and resistant infections.

• Octanoic acid (OA) rapidly kills mastitis pathogens via multimodal mechanisms.

• OA prevents adhesion and internalization and mitigates toxicity in vitro and in silico.

• OA alters mRNA expression profiles, revealing key antimicrobial pathways.

Phyllosphere microbes survive in an open and complex environment. Previous studies have characterized seasonal changes in host nutrient content as key factors affecting the balance of colonized phyllosphere microbial communities (PMCs). Meanwhile, climate factors (such as temperature and precipitation) could also influence plant growth and the composition of PMCs. However, the interacting effects of climate factors and seasonal variations in nutritional components on PMCs remain poorly understood. By comparing the partial correlation of climate factors and nutrient contents of grass with PMCs, we found that changes in the crude fiber (CF) content of grasses were negatively correlated with the archaeal community diversity. Conversely, the crude protein (CP) content in grasses was negatively correlated with both the richness and diversity of the fungal community (Pearson’s test, p < 0.05). The redundancy analysis (RDA) and multiple regression on (dis)similarity matrices (MRM) further confirmed that the content of CF was the primary factor influencing the distribution of the archaeal community, and CF content also significantly affected the distribution of the fungal community (Spearman’s test, p < 0.05). The Mantel test and regression analysis revealed a positive correlation between changes in CF and NDF content and the nearest taxon index (NTI). These findings suggest that changes in nutrient component content have a stronger effect on archaeal and fungal communities than on bacterial communities within PMCs, reflecting a more stable state of bacterial communities. This study demonstrated that the grass nutrient content plays a crucial role in dynamically shaping phyllosphere microbial communities.

• The changes in grass nutrient content significantly affected the structures and assembly of phyllosphere microbial community (PMCs) compared to the impact of climate change on PMCs.

• The contents of CF and CP were significantly correlated with the alpha diversity and community composition of archaea and fungi.

• Deterministic processes with heterogeneous selection governed the archaeal community.

In endangered species conservation, fecal samples are a vital non-invasive tool for gut microbiota analysis. Yet, the influence of external exposure time on microbial composition and function remains unclear, constraining data accuracy and reliability. To address this, we investigated the time-gradient effect in the globally endangered forest musk deer (Moschus berezovskii). Using non-invasive sampling under standardized captive conditions, fecal samples were collected at six storage times: (0, 1, 2, 4, 6, 8 days). Gut microbiota composition, diversity, enterotypes, and functional differences were assessed through 16S rRNA gene sequencing on the Illumina MiSeq platform. In total, 147,013 valid ASVs (amplicon sequence variants) were obtained showing significant shifts in microbial composition with storage time. Dominant phyla included Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Increasing storage time led to declining α-diversity, reduced community stability, and more unique genera. PCoA (principal coordinates analysis) and NMDS (non-metric multidimensional scaling) indicated progressive separation of experimental groups from control groups, with Anosim and Adonis confirming progressive separation with storage time. Structurally, Firmicutes decreased while Proteobacteria, specifically the Acinetobacter genus, increased with storage time. Community assembly shifted from deterministic to stochastic processes, reflecting stronger environmental disturbance effects. These results demonstrate that the gut microbiota composition, diversity, and ecological functions in forest musk deer feces are highly sensitive to storage time. Thus, preservation duration must be strictly controlled as a critical variable in microbiome studies. This work establishes methodological standards for non-invasive fecal metagenomics in endangered species, providing theoretical insights and practical guidance for improving scientific rigor in conservation-related microbiome research.

This study presents a novel multiplex assay based on capillary electrophoresis (CE) for the simultaneous detection of three Mycobacterium tuberculosis complex (MTBC) genes: IS6110, rpoB, and HSP65. Unlike conventional molecular diagnostic methods that target only a single gene, which may lead to misdiagnosis or missed diagnosis, this CE-based multiplex approach provides comprehensive detection to reduce diagnostic errors. Specificity testing with 76 microorganisms representing common respiratory pathogens confirmed 100% analytical specificity with no cross-reactivity, while sensitivity analysis demonstrated detection limits ranging from 10 to 20 copies/mL for all three target genes. In a prospective clinical validation study of 1067 patients suspected of pulmonary tuberculosis, the multiplex assay showed 77.4% sensitivity (CI 74.9%–79.9%), 99.6% specificity (CI 99.2%–100%), 96.0% positive predictive value (CI 94.8%–97.2%), and 97.1% negative predictive value (CI 96.1%–98.1%). Notably, the study identified 6 MTBC strains (4.8% of TB patients) with IS6110 deletions through whole-genome sequencing, which would result in false-negative results for any commercial PCR kits targeting IS6110. This integrated multiplex approach enhances diagnostic accuracy by simultaneously targeting multiple genes; then it offers the potential to reduce misdiagnosis and missed diagnosis of tuberculosis. In summary, the multiplex assay provides a more comprehensive alternative to current single-target molecular methods for MTBC detection.

• The multiplex assay provides one-run results for IS6110, rpoB, and HSP65.

• The multiplex assay is a more comprehensive method to detect MTBC.

• This approach can reduce misdiagnosis and missed diagnosis of TB.

Arsenic (As) is a toxic metalloid widespread in the environment, and many organisms have evolved mechanisms to mitigate its toxic effects. Bioinformatic analyses revealed that acr3 genes are predominantly distributed in mushrooms, highlighting their evolutionary and functional importance in eukaryotic arsenic metabolism. In this study, two homologous genes, HbACR3 and HsACR3, from the mushrooms Hebeloma bulbiferum and Hebeloma sinapizans were identified and functionally characterized. Both encode 399-amino-acid membrane proteins showing 99% sequence identity to each other and substantial similarity to previously characterized ACR3-type arsenite transporters from plants, yeasts, and bacteria. Heterologous expression of HbACR3 and HsACR3 in a Saccharomyces cerevisiae arr3Δ mutant restored resistance to arsenite and arsenate and significantly reduced intracellular arsenic accumulation. Fluorescence microscopy of GFP-tagged HbACR3 and HsACR3 confirmed their localization to the plasma membrane, consistent with an efflux transport function. Exposure of H. bulbiferum and H. sinapizans mycelia to arsenate led to a significant but differential transcriptional upregulation of both genes. This work provides new insight into the evolution, distribution, and physiological significance of ACR3 transporters in eukaryotic arsenic homeostasis.

Biocatalytic approaches have gained increasing attention as sustainable alternatives to metal-catalyzed asymmetric reductions of ketones to obtain enantiopure alcohols, important intermediates for pharmaceutical synthesis. For example, enzyme-catalyzed reduction of substituted benzophenone analogs to produce chiral diaryl methanols has attracted interest, as they are the key intermediates in the synthesis of antihistamines. However, benzophenone analogs are difficult to be reduced by enzymes due to steric hindrance. Moreover, the similarities between the two groups adjacent to the carbonyl group make achieving high enantioselectivity in reduction challenging. In this study, we examined the reduction of benzophenone and its analogs by Geotrichum candidum acetophenone reductase (GcAPRD). However, the wild type did not exhibit activity toward benzophenone due to the substrate’s bulkiness. Then, two mutants of GcAPRD (Trp288Ala and Phe56Ile/Trp288Ala) were applied to catalyze the reduction of benzophenone, resulting in high reduction yield (≥ 80%). In addition, both mutants exhibited catalytic activity toward methyl- and halogen-substituted benzophenones, especially toward 3- and 4-substituted substrates. Regarding enantioselectivity, Trp288Ala generally reduced both 3- and 4-substituted substrates to (R)-alcohols with up to 97% ee. In contrast, Phe56Ile/Trp288Ala reduced 3-substituted substrates to (R)-alcohols with up to 89% ee but reduced 4-substituted substrates to (S)-alcohols with up to 92% ee. At last, the reduction mechanism was investigated using molecular docking simulations.

• GcAPRD mutants exhibited catalytic performance toward benzophenone analogs.

• GcAPRD Phe56Ile/Trp288Ala exhibited substituent-dependent enantioselectivity.

• Introducing Phe56Ile into GcAPRD Trp288Ala resulted in a clear enantiopreference.

Saccharomyces cerevisiae is an established production host for therapeutic proteins; many of those are small proteins such as insulin or glucagon-like peptide-1 (GLP-1) analogs. Contrastingly, proteins of higher molecular weight, foremost antibodies, did not reach the market due, among other factors, to limiting productivity. Here we addressed the loss of product to protein degradation through a combination of genetic engineering of the host and medium optimization. We screened target genes that either directly or indirectly can lead to proteolytic degradation. We identified four deletions that are beneficial for expression: PEP1 and VPS30, which both can channel proteins to the vacuole for degradation; MON2, which can lead to the re-uptake of secreted proteins; and ALG3, which can affect the permeability of the cell wall. In parallel, we developed a small-scale fed-batch cultivation system for 24-well deep well plate cultivations and using an amino acid-rich medium. To stabilize secreted proteins, we screened chemical chaperones and osmolytes. We fortified the medium with arginine, 4-phenylbutyrate (4-PBA), and Tween-20. Using the engineered yeast strain, which features VPS30, PEP1, and ALG3 deletions, and the small-scale fed-batch system, we obtained 2.5 µg/mL of a secreted chimeric fusion of a nanobody to the crystallizable fragment (Fc) of a human immunoglobulin. Instrumental to the increase in the final titer were the reduced losses. This was achieved by a combination of complementary measures: improving diffusion through the cell wall, achieved through genetic engineering, and reducing losses to proteolytic degradation through medium optimization and genetic engineering. Moreover, we showed that the engineered strain and cultivation set-up are suitable for the production of different antibodies.

• Chemical chaperones and amino acid-rich medium increased secreted protein titers.

• Medium and host engineering are instrumental for improving productivity.

• Small-scale cultivation system enables production levels suitable for characterization.