Saccharopolyspora erythraea is an industrially important actinobacteria and a model in polyketide biosynthesis studies. However, its genetic toolbox remains limited. Here, we evaluated the properties of integrative and replicative vectors in Sacch. erythraea DSM40517. Three actinophage-based integrative vectors (φC31, φBT1, VWB) were efficiently transferred into Sacch. erythraea. The attB sites for these vector systems were identified. In case of φBT1- and VWB-based vectors, unique integration sites exists within the chromosome of the strain. In case of pSET152 (φC31), at least four different loci were identified with different efficiency of recombination. Our data revealed that all three integrative vectors are excised from the chromosome of the strain with varying frequencies, highlighting the need for selective pressures for their stable maintenance. Commonly used replicative vectors with Streptomyces replicons (pIJ101 and pSG5) have low transfer efficiency in Sacch. erythraea. To solve this problem, we constructed a shuttle vector pYS191 based on the replicon of plasmid pJV1. This vector was efficiently transferred into Sacch. erythraea and other actinobacteria including Streptomyces, Saccharothrix and Couchioplanes species. pYS191 is maintained at a low copy number under selective conditions, and is rapidly lost without antibiotic pressure. Neither integrative nor replicative vectors significantly affected erythromycin production. This study provides a comprehensive assessment of vector system in Sacch. erythraea and reports a new replicative vector based on rarely exploited pJV1 replicon for transient gene expression and genome engineering. These results expand the genetic toolbox for Saccharopolyspora and facilitate its use in natural products research and industrial biotechnology. KEY POINTS: • Actinobacterial integrative vectors are suited for gene cloning in Sacch. erythraea. • attB sites for three integrative vectors identified within Sacch. erythraea genome. • A new replicative vector pYS191 for use in Sacch. erythraea was constructed.

Conventional sulfuric acid production is tied heavily to the fossil fuel industry, where it is typically obtained from the desulfurization of fuels. Here, the biological production of sulfuric acid by Acidithiobacillus thiooxidans in a reactor setup was optimized using design of experiments methodology. The effects of initial sulfur dosage, phosphate (PO43-) concentration, and inoculum size were evaluated with respect to sulfate (SO42-) concentration, optical density, and pH. A significant effect was observed for sulfur dosage; the highest sulfur supplementation, 35 g L-1, yielded the highest SO42- concentration and lowest pH after 14 days of cultivation. A mean sulfur-to-SO42- conversion rate of 81 ± 9% was determined across all trials conducted with35gL-1 of sulfur. Among all trials, a maximum SO42- molarity of 1.08 M was achieved, with a corresponding pH of 0.18. No significanteffects were determined for PO43- and inoculum volume. The feasibility of using an industrial by-product as a sulfur source was demonstrated, in addition to establishing a strong correlation between the measured SO42- concentration and conductivity of the biological lixiviant. KEY POINTS: • Sulfur dosage correlated positively with SO42- concentration. • DoE revealed no significant effect for PO43- concentration and inoculum size. • Strong correlation between SO42- concentration and conductivity was established.

Fusarium graminearum poses a major threat to global cereal production, necessitating sustainable control measures. This study elucidates the antagonistic mechanism of the biocontrol agent Bacillus velezensis EU07 against F. graminearum through combined transcriptomic and morphological analyses. Exposure to EU07 compromised fungal cellular integrity, causing severe hyphal distortion. RNA-seq profiling revealed that EU07 triggers a profound transcriptomic collapse rather than a standard stress response. Over 35% of the altered fungal transcriptome caused critical suppression, targeting essential metabolic hubs, secretome effectors, and master transcription factors to inhibit growth and loss of pathogenicity. In a compensatory defensive shift, the pathogen massively reallocated its transcriptional resources. This defence was characterised by a ~ 30-fold enrichment of transmembrane amino acid transporters for putative toxin efflux and an 80-fold enrichment of glutathione S-transferases to mitigate severe oxidative stress. Furthermore, EU07 suppressed core vulnerability nodes, including proline dehydrogenase (PRODH) and apolipophorin, disrupting the pathogen's osmotic resilience and membrane integrity. Molecular docking analysis indicated that the Bacillus lipopeptide iturin A directly targets apolipophorin with high affinity (- 7.2 kcal/mol). Our findings indicated that B. velezensis EU07 overwhelms F. graminearum through simultaneous metabolic starvation and loss of virulence, revealing highly vulnerable fungal targets for next-generation RNAi-based biocontrol.

This research identifies Ectopseudomonas oleovorans CECT 5344 R1D as a novel and highly efficient biocatalyst for the sustainable production of 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). By employing benchtop Nuclear Magnetic Resonance (NMR) spectroscopy with a recirculating flow system, we achieved real-time monitoring and a detailed stoichiometric characterisation of the 5-hydroxymethylfurfural (HMF) detoxification pathway. This work contributes to the specialised field of real-time metabolic tracking of living biocatalysts, a niche where in situ NMR data is still scarce compared to end-point analytical methods. Our findings demonstrate that this strain, while not utilising HMF for biomass growth, maintains exceptional metabolic stability and quantitative molar yields over 125h of operation, requiring significantly lower cell densities than other reported Pseudomonas strains. The in-line NMR setup enabled the definitive identification of 2,5-bis(hydroxymethyl)furan (BHMF) as a transient intermediate, clarifying previous metabolic misidentifications and revealing a highly specific, non-consuming "cell factory" behaviour. Furthermore, the system allowed for the assessment of bacterial tolerance thresholds and the exploration of the enzymatic substrate scope against other environmentally relevant aldehydes. This work underscores the synergy between a robust, living biocatalyst and a precise monitoring platform, providing an optimised workflow for bioprocess development in sustainable aldehyde valorisation. KEY POINTS: • Real-time monitoring of a microbial process via in-line benchtop NMR. • Versatile biodetoxification of various natural and synthetic aldehydes. •Green chemistry alternative to petrochemicals using renewable feedstocks.

Postbiotics have recently emerged as a promising alternative to probiotics in aquaculture due to their improved safety, stability, and ease of application; however, the biological effects of postbiotics derived from Vibrio proteolyticus remain largely unexplored. In this study, we evaluated the impact of dietary supplementation with ethanol-inactivated V. proteolyticus DCF12.2 on the intestinal microbiota composition and transcriptomic profile of zebrafish (Danio rerio). Fish were fed a control diet (CTRL group) or a diet supplemented with 1% lyophilized ethanol-inactivated V. proteolyticus DCF12.2 (VP group) for 21days. Alpha and beta diversity analyses exhibited no significant differences in overall gut microbial composition between dietary groups, nor at the phylum or genus level. However, differential abundance analysis at the amplicon sequence variant (ASV) level identified significant differences in ASVs affiliated with the genera Aeromonas, Delftia, Ralstonia, Shewanella, and Stenotrophomonas, which were enriched in fish receiving the VP diet (p < 0.0001). Despite these ASV-level shifts, predicted microbial metabolic functions did not differ significantly between treatments. In contrast, transcriptomic analysis via Gene Set Enrichment Analysis (GSEA) detected a clear host response: a significant downregulation of the cytokine-cytokine receptor interaction pathway and the upregulation of pathways related to nutrient metabolism, oxidative phosphorylation, and cellular biosynthesis in fish fed the VP diet (adjusted p < 0.05). These results suggest that dietary supplementation with postbiotics from V. proteolyticus DCF12.2 modulates intestinal metabolic and immune pathways without inducing major restructuring of the gut microbiota composition. Collectively, these findings provide new insights into host-postbiotic interactions and support the potential application of postbiotics from V. proteolyticus DCF12.2 as functional feed additives for sustainable aquaculture. KEY POINTS: •Postbiotic diet did not alter gut microbial diversity but induced ASV-level differences •Postbiotic diet upregulated nutrient metabolism, oxidative phosphorylation, and cellular biosynthesis •Cytokine-cytokine receptor signaling was downregulated by the postbiotic diet.

Dominance of Bacillus species on conventional agar often hampers the recovery of diverse soil bacteria ("Bacillus dominance"). To address this, we developed an agar medium containing leaf mold extract (LME) and evaluated its effectiveness for soil microorganism isolation compared to conventional yeast malt extract agar (YME agar). The 16S rRNA gene sequencing revealed that YME agar cultures were dominated by Bacillus-related bacteria, while LME agar enabled the growth of a broader range of taxa, especially actinomycetes like Streptomyces. LME agar supported higher bacterial diversity even in samples with high Bacillus abundance. Some LME isolates had very low 16S rRNA gene similarity to known species or could not be amplified with universal primers, suggesting potentially novel taxa. One unidentified isolate proliferated only in liquid LME, indicating a unique nutrient requirement. We further screened LME-derived isolates (569 strains) for therapeutic activity using an insect (the silkworm, Bombyx mori) model infected with three pathogenic fungi (Rhizopus arrhizus, Aspergillus fumigatus, Candida albicans). Distinct extracts showed significant efficacy in each infection model, and the overall hit rate was 6.2%. Notably, the extract of the rare species Ancylobacter sonchi showed marked efficacy against R. arrhizus infection, underscoring LME agar's potential for accessing novel bioresources. These results suggest that LME agar suppresses Bacillus dominance and expands access to diverse, rare, and previously uncultured soil bacteria, providing a powerful platform for microbial library construction and novel drug discovery. KEY POINTS: • LME agar prevents Bacillus dominance, enabling recovery of diverse microbial colonies. • LME agar accesses rarer and more diverse microbiota than nutrient-rich media. • A strain from LME agar showed therapeutic activity in silkworms infected withR. arrhizus.

Soil salinization poses a significant threat to global agriculture, necessitating the development of sustainable strategies to increase crop resilience. Plant growth-promoting rhizobacteria (PGPR) offer a promising solution; however, their effectiveness in saline soils is often limited by their own salt sensitivity. The role of the extracytoplasmic function of the sigma factor AlgU in the salt stress adaptation of the nitrogen-fixing PGPR Pseudomonas stutzeri A1501 was investigated in this study. Through the construction of isogenic algU knockout (ΔalgU) and overexpression (OE-algU) strains, combined with phenotypic assays, transcriptomic profiling, and plant experiments, we demonstrate that AlgU acts as a master regulator of salinity tolerance. AlgU increased bacterial survival under acute salt shock, promoted biofilm formation, and, crucially, protected nitrogenase activity from salt inhibition. RNA-seq analysis revealed that AlgU orchestrates a comprehensive transcriptional reprogramming, upregulating the expression of genes involved in exopolysaccharide synthesis, osmoprotection (otsA), and central carbon metabolism (zwf, fumC). This coordinated response ensures an adequate supply of energy and reducing equivalents while maintaining cellular homeostasis. Consequently, inoculation with the OE-algU strain significantly alleviated salt stress in maize, improving seedling growth in pot experiments and outperforming the wild-type strain by increasing grain yield in saline-alkali field trials. Our findings establish AlgU as a key genetic determinant for engineering salt-tolerant PGPR, providing a mechanistic framework for the development of effective microbial inoculants to improve crop productivity in saline soils. KEY POINTS: AlgU orchestrates biofilm formation, osmoprotection, and energy metabolism to confer salt tolerance in P. stutzeri.Engineering an AlgU overexpression strain increases maize yield under saline-alkali field conditions.AlgU safeguards nitrogenase activity under salt stress by reprogramming energy and osmoprotective pathways, thus linking bacterial resilience to improved crop yield.

Rainbow trout β-defensin 3 (rtDefb3) is identified as a small cationic antimicrobial peptide, but its recombinant expression and functional characterization have not been reported. In this study, the mature rtDefb3 Komagataella phaffii coding sequence was cloned from trout liver cDNA and heterologously expressed in GS115 using the pPIC9K with an α-factor secretion signal. Expression condition was optimized by adjusting methanol concentration (0.5-1.25%), induction temperature (26-30°C), and induction duration (24-168h). Optimized induction with 1.0% methanol at 30 ℃ for 96h produced ~ 7mg/mL of secreted peptide, which was purified to > 90% purity using one-step Ni-IDA affinity chromatography. The identity and purity of the recombinant rtDefb3 were confirmed by Tricine-SDS-PAGE, HPLC (~ 97% purity), and MALDI-TOF mass spectrometry. Functional assays revealed potent broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. These findings demonstrate, for the first time, the successful production of active rtDefb3 in Komagataella phaffii, establishing a scalable expression platform for fish β-defensins. The recombinant rtDefb3 peptide holds promise as a natural antimicrobial agent for aquaculture and potential therapeutic applications, addressing the urgent demand for alternatives to conventional antibiotics. KEY POINTS: • First successful recombinant production of rainbow trout β-defensin 3 in Komagataella phaffii • via optimized induction strategy. • High-yield secretory expression (~ 7mg/mL) with > 90% purity confirmed by HPLC and biochemical analysis. • Recombinant rtDefb3 displayed potent broad-spectrum antibacterial activity, offering a repeatable and scalable system and functional validation.

Our study evaluated the bioaugmentation potential of the Pseudomonas qingdaonensis ZCR6 strain to support the phytoremediation of multicontaminated soil. This metal-resistant strain expresses several plant growth-promoting features and petroleum hydrocarbon degradative potential in biochemical tests. A microcosm experiment with maize (Zea mays) treated with a suspension of live or thermally inactivated ZCR6 cells was performed (representing a scenario of unsuccessful bioaugmentation). Although the ZCR6 colonized the rhizosphere and plant tissues, its application did not impact the removal of hydrocarbons compared to the control (17.4% vs. 19.6%, respectively). In contrast, treatment of soil with thermally inactivated bacterial biomass significantly increased hydrocarbon removal efficiency by approximately 8%, compared to the nontreated control. The Zn accumulation in the roots of maize treated with either live or thermally inactivated cells of ZCR6 was about 30% higher compared to the control. The soil transcriptomic analysis revealed downregulation of bacterial genes involved in hydrocarbon degradation after soil inoculation with live ZCR6, whereas plants showed upregulation of stress-related genes. No increase in the expression of bacterial genes encoding plant growth-promoting mechanisms was observed in any of the treatments. Our research has shown that the utility of each strain considered for bioaugmentation must be properly verified prior to in situ application. This should also include testing scenarios assuming a lack of survival and/or activity of the introduced strain and assessing its actual impact on the metabolic activity of plants and soil microorganisms by applying advanced transcriptomic approaches. KEY POINTS: • Bioaugmentation significantly downregulated bacterial genes of degrading pathways. • Soil inoculation increased transcription of genes involved in maize stress response. • Treatments didn't increase the expression of genes crucial for phytoremediation.