Ectomycorrhizal fungi form symbiotic relationships with a wide range of terrestrial plants, acquiring carbohydrates for themselves and promoting nutrient uptake in their host plants. However, some ectomycorrhizal fungi cannot effectively obtain the thiamine necessary for growth from their host or synthesize it themselves. Ectomycorrhizal fungi can recruit hypha-associated microorganisms, which play a vital role in promoting nutrient absorption and ectomycorrhizal root formation, ultimately colonizing within fruiting bodies to form a unique bacterial microbiota. In this study, non-targeted metabolomics and whole-genome sequencing were employed to investigate the colonization characteristics of the hyphae-associated bacterium Bacillus altitudinis B4 on the mycelial surface of ectomycorrhizal fungus Suillus clintonianus, as well as the synergistic promotion of thiamine synthesis and absorption by B. altitudinis B4 and the fungal mycelium, respectively. The results suggested that S. clintonianus first secreted ureidosuccinic acid and pregnenolone, recruiting the hyphae-associated bacterium B. altitudinis B4 to the mycelial surface. Subsequently, the ureidosuccinic acid secreted by S. clintonianus further stimulated B. altitudinis B4 to enhance thiamine production by increasing its biomass and upregulating the expression of related functional genes. Finally, S. clintonianus absorbed the thiamine secreted by the B. altitudinis B4, promoting fungal growth and increasing the colonization rate in association with Pinus massoniana. This study elucidates the thiamine acquisition mechanisms of ectomycorrhizal fungi, highlighting the critical role of bacterial partners in fungal nutrition and host-fungal interactions.

Conventional genetic engineering approaches for bacterial metabolic pathway manipulation, although highly applicable, still face limitations including metabolic burden, irreversibility, and dependency on host cellular machinery. Cell-penetrating peptide-peptide nucleic acid conjugates (CPP-PNAs), known for their applicability as antibacterial tools and in the elucidation of protein function, offer a promising alternative to overcome such limitations. Since the application of CPP-PNA in metabolic engineering and pathway elucidation remains largely unexplored, we developed and validated a CPP-PNA platform using Synechocystis sp. PCC 6803 as a model system to demonstrate targeted metabolic pathway evaluation. High compatibility and dose-dependent permeation efficiency in strain PCC 6803 was first observed when the amphipathic CPP (KFF)₃K was employed, achieving clear cell growth inhibition at 10 µM and above. Specific targeting of D-lactate dehydrogenase (Ddh) using CPP-Syn6803ddh conjugates achieved near-complete protein translation knockdown within 24 h, as confirmed by Western blot analysis. Metabolomics analysis using LC-MS on predetermined metabolites revealed that CPP–PNA treatment produced metabolic effects comparable to stable genetic knockout strains, with both approaches showing a significant 2.5-fold increase in pyruvate accumulation compared to wild-type controls. Further elucidating the reason for pyruvate accumulation, we observed compensatory activation of the glyoxalase pathway at 48 h post-treatment, resulting in 3-fold increased D-lactate production presumably through methylglyoxal detoxification. Validating this observation, RT-qPCR analysis confirmed 2-3-fold upregulation of the glyII gene, encoding for the glyoxalase II (GlyII) enzyme, in both CPP-PNA treated and knockout strains, while double CPP-PNA inhibition experiments targeting both Ddh and glyoxalase pathways suppressed D-lactate accumulation. This study establishes CPP-PNAs as efficient tools for rapid, and simple metabolic pathway investigation. The approach produces results comparable to conventional genetic knockouts while offering dose-dependent control and avoiding permanent genomic alterations. Our findings reveal unexpected metabolic complexity in Synechocystis sp. PCC 6803 D-lactate synthesis under light conditions and demonstrate the utility of CPP-PNA for uncovering compensatory pathway activation. This platform represents a valuable addition to bacterial genetic engineering, addressing some of the critical limitations faced by conventional approaches, while showing potential for further understanding the biochemistry of metabolite-producing bacteria.

The use of nucleic acid-based nanostructures as synthetic biological tools to interface with and regulate cell processes remains challenging. A major obstacle lies in nuclear delivery and retention within live eukaryotic cells. Here, we present a platform of single-stranded RNAs that can co-transcriptionally fold into defined nanostructures and assemble into rings, ribbons, and nanonet-like architectures. We validate the formation of these structures in vitro using atomic force microscopy. Then, we demonstrate the functional integration of fluorescent aptamers and RNA sensing capability within the single chain by co-folding with these structures. Notably, we show that the RNA nanonets can be co-transcriptionally produced and assembled directly inside the nucleus of live human cells. We use confocal live-cell imaging and transmission electron microscopy to reveal well-defined nanostructure patterns retained in the nucleus. Together, these results establish a genetically encoded, self-assembling RNA nanostructure system with programmable geometry and localization, providing a foundation for the development of RNA-based nanodevices to examine biological properties in live cells and tissues. The use of nucleic acid-based nanostructures as synthetic biological tools remains challenging. Here they present synthetic RNA structures that fold and self-assemble inside human cell nuclei, offering a platform for imaging, sensing, and future therapeutic applications.

Continuous two-stage E. coli fermentations offer potential for high-efficiency bioprocesses but are often limited by plasmid instability, genetic mutations, and unintended expression during seed phases. In this study, we aimed to overcome these limitations by employing a plasmid-dependent auxotrophic selection system, specifically using a thymidine auxotrophy (thyA deletion), in conjunction with a series of plasmid modifications to enhance stability and expression control. We engineered the E. coli strain enGenes-eXpress V2 ΔthyA and constructed modified plasmids containing thyA along with regulatory elements to maintain plasmid stability and minimize basal expression. The modified strains were evaluated in continuous two-stage fermentations under carbon-limited conditions. Our results indicate significant reduction in plasmid loss, improved population homogeneity, and suppressed basal expression in non-induced phases. The addition of elements such as the cer (ColE1 resolution) site and a modified T7 promoter further enhanced plasmid stability and reduced basal expression levels. High-throughput screenings in microbioreactor setups confirmed that optimized constructs maintained a homogeneous producing population and suppressed non-producing cells over extended periods, which was validated by fed-batch cultivations and single-cell analyses. Finally, the two most promising constructs demonstrated high robustness in continuous two-stage chemostat fermentations lasting over 1000 h, maintaining stable GFP titers, plasmid concentrations, and cell dry mass throughout the process. Our findings demonstrate that the auxotrophic marker thyA-based selection system, combined with strategic plasmid modifications, can substantially improve the genetic stability and productivity of E. coli in continuous bioprocesses. This approach provides a robust platform for sustainable, antibiotic- free production in industrial biotechnology, highlighting its potential for scale-up in long-term continuous fermentations.

The dominant bacteria enriched in the Fusarium wilt plants’ rhizosphere are of increasing interest, as they adapt well to the diseased rhizosphere. However, general information about these bacteria is still lacking. Here, we perform a meta-analysis of Fusarium wilt plants rhizosphere and comprehensive studies to obtain information about the robust variation in the rhizosphere microbiome of Fusarium wilt plants. We demonstrate that Fusarium infection reproducibly changes the rhizosphere bacterial community composition. The rhizosphere microbiomes of Fusarium wilt plants are characterized by the enrichment of Flavobacterium, gene cassettes involved in antioxidant functions related to sulfur metabolism and the root secreted tocopherol acetate. We further isolate antagonistic Flavobacterium anhuiense from the diseased tomato rhizosphere, and reveal that the growth of F. anhuiense and the expression of genes related to carbohydrate metabolism in this strain are significantly stimulated by tocopherol acetate. Furthermore, the inhibitory effect of F. anhuiense against F. oxysporum and F. anhuiense population enhancement by tocopherol acetate are confirmed in planta. The robust variation in the rhizosphere microbiome elucidates key principles governing the general assembly mechanism of the microbiome in the Fusarium wilt plants’ rhizosphere. This study reveals how tomato plants recruit specific bacteria to combat Fusarium wilt. A key bacterium is identified to thrive on root-derived compounds and suppress the fungal pathogen, offering insights into plant-microbe defense alliances.