Microbial Biotechnology最新文献

Insulin glargine, a long-acting insulin analog, is essential for diabetes treatment. However, its industrial production remains challenging due to limitations in conventional expression systems. Here, we employed the industrial filamentous fungus Trichoderma reesei as an alternative expression host for insulin glargine production, leveraging its superior protein secretion capacity. Initially, expression constructs containing the constitutive Pcdna1 promoter and CBH1 signal peptide (SP1) showed successful transcription but failed to achieve extracellular secretion, presumably due to induced endoplasmic reticulum (ER) stress. Consequently, we implemented a fusion protein strategy utilising three distinct carrier proteins (CBH1, CBH2, and LA-20) to enhance glargine secretion. Notably, the CBH1 fusion not only enabled detectable glargine secretion but also significantly alleviated the ER stress. Furthermore, replacement of SP1 with the Aspergillus niger β-glucoamylase signal peptide achieved a fourfold enhancement in glargine secretion and further reduced cellular stress responses. Following these systematic optimizations, a final yield of 58.95 mIU/L glargine was achieved in shake-flask cultures. Thus, the combined strategy described here could achieve extracellular production of glargine in T. reesei, suggesting that it is a promising host for secretory production of therapeutic recombinant proteins, particularly complex analogs like glargine.

High-quality silage enhances palatability and feed intake; however, the effects of co-fermentation with flavouring agents and lactic acid bacteria (LAB) on its flavour quality, core microbiota, and taste-active amino acids remain unclear. This study investigated the effects of fermentation using Lactiplantibacillus plantarum (LP) alone or in combination with phenethyl acetate (LPP) on the flavour profile of alfalfa silage and its subsequent influence on feed intake. Both LP and LPP significantly improved fermentation quality versus control (CK), with markedly higher feed intake-LP > CK and LPP > LP. Key flavour compounds, including dimethyl trisulfide, 4-ethylphenol and β-damascenone, were significantly increased in the LP alone group. Contrarily, essential taste-related amino acids including aspartic acid, alanine, proline, histidine, isoleucine, and valine were decreased, except for arginine. These metabolic shifts collectively contributed to enhanced feed intake. The addition of LPP increased phenylethyl alcohol, benzyl alcohol and hexanal, and decreased arginine, contributing to enhanced palatability. Aryl alcohol dehydrogenase, proline aminopeptidase, histidine dehydrogenase, and branched-chain amino acid transaminase from LP played a crucial role in the formation of phenylethyl alcohol, proline, histidine and isoleucine during fermentation. These results provide insights into how LAB and flavouring agents jointly regulate flavour development in high-quality alfalfa silage.

Insufficient product yield remains a major bottleneck in the development of microbial cell factories for fine chemical production. In vivo enzyme colocalisation on synthetic scaffolds has emerged as a promising strategy to enhance metabolic efficiency, yet current approaches are often labour-intensive and inefficient. Here, we present the development of a 'scaffoldomics' platform, a generic synthetic framework designed to enable combinatorial scaffolding of biosynthetic pathways for the generation of diverse multi-enzyme complexes. This system enables the assembly of up to four pathway enzymes onto a protein scaffold. As a proof of concept, the platform was applied to the biosynthesis of naringenin, a key flavonoid intermediate. Integration of a chromosomally encoded naringenin biosensor in Escherichia coli allowed for real-time detection and pathway evaluation. The biosensor response curve was established and confirmed functional naringenin production. Moreover, comparative experiments demonstrated that the addition of a scaffold to docking enzymes significantly enhanced yield up to a ~19-fold, indicating a positive colocalisation effect. These results highlight the utility of the scaffoldomics platform as a powerful tool for further efficient combinatorial design, construction, and optimisation of biosynthetic pathways in synthetic biology.

The nervous system and the immune system are integral components of the tumour microenvironment, and neuroimmune mechanisms play critical roles in tumour metastasis, immune evasion and metabolic reprogramming. However, the relationship between the gut microbiota and cancer neuro-immunity remains poorly understood. This knowledge gap hampers our understanding of how these systems contribute to tumour progression and therapeutic resistance. This article systematically explores the interactions among the gut microbiota, the nervous system and the immune system in the regulation of tumour progression, with a particular focus on elucidating the pathways by which the gut microbiota and its metabolites modulate tumour phenotypes via neuroimmune mechanisms, and summarises the regulatory mechanisms through which cancer neuro-immunity shapes gut microbiota composition. Additionally, this article summarises the interplay between immunotherapy and cancer neuro-immunity, and explores the potential of microbiota-based interventions, such as faecal microbiota transplantation, probiotics, prebiotics and synbiotics, to enhance the efficacy of immunotherapy through neuroimmune mechanisms.

Candidal vaginitis is a prevalent inflammatory condition of the reproductive tract that has a substantial impact on women's health. Conventional antibiotic therapy frequently results in recurrence due to the non-selective elimination of vaginal microbiota and subsequent dysbiosis. In order to overcome this limitation, the focus was directed towards traditional Chinese medicine, which has antimicrobial properties and it was here that pseudolaric acid B (PAB) was identified as a promising active monomer through the utilisation of virtual screening and bioinformatics approaches. In vitro experiments confirmed that PAB inhibits biofilm formation, reduces ergosterol biosynthesis by downregulating the key gene ERG11, and enhances fungal cell membrane permeability, ultimately resulting in fungal cell death. In vivo studies in a mouse model of Candida albicans-induced vaginitis demonstrated that PAB exhibits superior therapeutic efficacy compared to conventional antibiotics. This is characterised by the restoration of normal tissue architecture, a reduction in inflammatory responses and a significant enrichment of beneficial vaginal microbiota as revealed by 16S rRNA sequencing. Collectively, these results highlight PAB as a promising dual-mechanism antifungal agent which acts, at least in part, by disrupting fungal ergosterol biosynthesis and directly eradicating pathogens, while restoring vaginal microecological homeostasis.

Skunavirus is the most frequently encountered Lactococcus-infecting (bacterio)phage genus in dairy fermentations and may cause milk acidification failure or inefficiencies. During phage infection, DNA packaging into the phage prohead is a crucial step for the successful maturation of phage progeny. In the Skunavirus genus of lactococcal phages, a predicted HNH endonuclease-encoding gene adjacent to two putative DNA packaging genes is highly conserved. In the current study, we show that this gene, designated here as hrdP (HNH endonuclease related to DNA packaging), is essential for the production of infective Skunavirus sk1 virions. HrdP exhibits non-specific endonuclease activity, while deletion of hrdP from the sk1 genome results in loss of plaque-forming ability without hindering its DNA replication or protein production. Transmission electron microscopy analysis showed that the hrdP deletion mutant in sk1 is unable to produce intact virions. However, the proheads in this mutant displayed conformational changes associated with capsid expansion, suggesting that DNA packaging had initiated and progressed to a certain extent. This study provides valuable insights into the specific role of HrdP in Skunavirus members, highlighting it as a potential target for developing anti-phage strategies to enhance starter culture robustness.

Infertility affects ~1 in 6 people of reproductive age and remains difficult to treat because causes are heterogeneous and diagnostics are incomplete. Recent evidence reframes the female reproductive tract as a low-biomass but biologically active microbial ecosystem. Dysbiosis, typically loss of protective Lactobacillus species (notably L. crispatus) with overgrowth of anaerobic pathobionts, is associated with implantation failure and recurrent pregnancy loss. Framing conditions such as chronic endometritis and reproducible low-Lactobacillus endometrial profiles as dysbiosis-related disorders clarifies opportunities for prevention, companion diagnostics and microbiome-directed therapies. This narrative review contrasts receptive (Lactobacillus-dominant) versus dysbiotic states and summarises mechanisms linking microbiota to fertility: microbial metabolites (lactic acid, short-chain fatty acids) support epithelial barrier function and immune tolerance, whereas dysbiosis provokes inflammation that impairs implantation. Although observational data consistently associate Lactobacillus dominance with better outcomes, evidence quality is low-to-moderate due to retrospective designs, methodological heterogeneity, and a lack of adequately powered, diagnostic-stratified randomised trials. The review highlights precision microbial therapeutics under development, single-strain next-generation probiotics, synthetic consortia, engineered live biotherapeutics, postbiotics, targeted phage/endolysins and vaginal microbiota transplantation, and proposes a diagnostic-driven roadmap that matches microbiome endotypes and clinical contexts (e.g., preconception vs. immediate embryo transfer) to specific interventions. Regulatory and safety issues for reproductive biologics are also considered. The reproductive microbiome is a promising translational frontier but currently offers a consistent signal rather than definitive proof of benefit. To translate promise into practice requires standardised low-biomass sampling/reporting, mechanistic validation in human-relevant models and diagnostic-stratified randomised trials with staged endpoints, alongside strategies to address engraftment, formulation and regulatory pathways.

Faecal microbiota transplantation (FMT) is highly effective for recurrent Clostridioides difficile infection but yields inconsistent benefits in chronic indications. As a crude whole-microbiota transplant, FMT contains numerous undefined active components, complicating efforts to ensure treatment predictability and stability. Therefore, we propose Advance Microbiota Transplantation (AMT), a comprehensive, phase-based hypothesis that employs an addition-subtraction strategy throughout the pre-, peri- and post-transplant stages. AMT comprises donor and recipient pre-treatment, procedural optimisation and post-transplant adjuvant interventions to mitigate donor variability, ecological resistance, procedural heterogeneity and unstable engraftment. Through a systematic synthesis of current evidence-based FMT research, we explored how the addition-subtraction strategy can be operationalised to shape the AMT concept and define testable, phase-specific levers, thereby providing a foundation for future clinical translation. In parallel, we appraised the reporting quality using the Preferred Reporting Items for Microbiotherapy (PRIM) and identified six persistently under-reported items that limit the interpretability, comparability, and reproducibility of FMT research. This review aims to facilitate the integration of AMT into clinical practice.

While Candida albicans alcohol dehydrogenase I (Adh1) conventionally functions as an alcohol dehydrogenase, this study builds upon previous work to redefine its novel role in regulating hyphal morphogenesis and elucidates the underlying mechanisms. Adh1 knockout strains exhibited hyperfilamentation, suggesting that Adh1 directly regulates true hyphal development, independent of its canonical metabolic activity. Leveraging this phenotype, a 'reverse screening strategy' identified 2-hydroxyanthraquinone (HAQ) through high-throughput screening as a potent inhibitor of biofilm formation and hyphal growth by targeting Adh1. Biochemical and structural analyses confirmed HAQ's direct binding to Adh1's F224/A254/Q257 interface. Mechanistically, affinity purification-mass spectrometry revealed Adh1 modulates the SNF1 signalling axis by accelerating SNF1 dephosphorylation via interactions with Bmh1/Ssb1 regulators, thereby inhibiting hyphal conversion. HAQ disrupted these interactions, reducing SNF1 phosphorylation levels in an Adh1-dependent manner. This work establishes Adh1 as both an endogenous SNF1 pathway suppressor and an exogenous drug target, while demonstrating the efficacy of phenotype-driven discovery pipelines. The findings provide a novel antifungal strategy targeting virulence-regulating metabolic enzymes and validate HAQ as a lead compound for therapeutic development against C. albicans pathogenicity.

Trichoderma species are widely recognised as biocontrol agents, yet the transcriptional mechanisms underlying their antagonism against foliar pathogens remain largely unexplored. In this study, we identified 32 bZIP genes in Trichoderma harzianum Tha739 and demonstrated that bZIP63.5 exhibited the strongest upregulation under Alternaria alternata metabolite stress. Overexpression and knockdown strains of bZIP63.5 were constructed, and phenotypic assays revealed that bZIP63.5 significantly enhanced the antifungal activity of T. harzianum through direct confrontation, volatile organic compound production, and secreted metabolites. Using yeast one-hybrid and electrophoretic mobility shift assays, we identified that bZIP63.5 binds to known cis-acting elements (TGTCACA, TGTCA, GTGA, TGAC) as well as a novel motif (TACGGAC). Yeast two-hybrid screening further revealed that bZIP63.5 interacts with multiple proteins, including histone H4 and heat shock protein HSP70, suggesting its involvement in chromatin regulation and stress response. Transcriptome analysis of the bZIP63.5 overexpression strain showed that it directly upregulates a set of zinc-finger transcription factors, which in turn activate downstream detoxification genes (ABC transporters, cytochrome P450s) and defence-related enzymes (glycosyl hydrolases, peroxidases). ChIP-PCR confirmed the direct binding of bZIP63.5 to the promoters of these zinc-finger TFs. Functional validation through overexpression and knockout of three core zinc-finger TFs (Zn2CyS6ZF56.5, C2H2ZF36.8, C2H2ZF40.8) demonstrated their essential roles in enhancing the biocontrol efficacy of T. harzianum against A. alternata. This study elucidates a hierarchical transcriptional network centered on bZIP63.5 that coordinates detoxification and defence responses in T. harzianum, providing novel insights into the molecular mechanisms of fungal biocontrol and potential targets for improving disease management in forestry systems.