Xuefeng Lu

The RNA interference (RNAi) machinery regulates diverse cellular processes in fungi, but its functions in industrial Aspergillus terreus remain unknown. Here, we systematically investigated two industrial strains: the itaconic acid-producing A. terreus LYT10 and the lovastatin-producing A. terreus HZ01. Both strains possess RNAi machinery core components, yet the phenotypic consequences of RNAi disruption differed between the two strains. In strain LYT10, RNAi deficiency had negligible effects on growth, conidiation and itaconic acid production. Conversely, in strain HZ01, it caused severe growth impairment, dysfunctional conidiation, reduced lovastatin production, and activated by-product geodin biosynthesis. Notably, the dicer-like 2 (dcl2) deletion mutant exhibited the most severe phenotype among all mutants. Transcriptomic analysis linked these phenotypic changes to strong repression of the lovastatin biosynthetic gene cluster, downregulation of acetyl-CoA flux, and partial induction of the geodin biosynthetic gene cluster in strain HZ01. Small RNA profiling identified 14 DCL2-dependent microRNA-like RNAs (milRNAs) in the Δdcl2 mutant. The milRNA-mRNA network analysis revealed that these DCL2-dependent milRNAs target genes involved in the regulation of glucose uptake, maintenance of fatty acid metabolic homeostasis, and suppression of geodin biosynthesis, thereby indirectly influencing lovastatin biosynthesis. Collectively, the RNAi machinery via DCL2 and milRNAs to exert precise post-transcriptional regulation on target genes expression, ensuring normal development and efficient production of lovastatin in industrial A. terreus HZ01. This study reveals that the regulatory roles of RNAi machinery differ between the two industrial A. terreus strains and elucidates a milRNA-mediated regulatory mechanism in strain HZ01, offering new insights into fungal RNAi biology and its biotechnological applications.

Background: Microbial communities of multicellular, heterocyst-forming cyanobacteria assimilate organic nitrogen from dinitrogen gas through nitrogen fixation and are important photosynthetic primary producers. This makes them ecologically significant, particularly in freshwater, wetlands, and agricultural ecosystems. Cyanophages, viruses that specifically infect cyanobacteria play a key role in lysing them, terminating cyanobacterial blooms, and turning over the nutrients accumulated by these communities, with tremendous relevance for the biogeochemical cycles in these environments. However, only a limited number of cyanophages infecting N ₂ -fixing cyanobacteria have been genome sequenced to date, and it has remained unknown how their interaction proceeds with the individual cells within the filaments forming the backbone of these communities. Results: We isolated and characterized three Caudoviricetes cyanophages (A-Lf14, A-Alj1, and A-Hlh1) specifically infecting the N ₂ -fixing cyanobacterium Anabaena sp. PCC 7120. Super-resolution microscopy revealed heterogeneous infection outcomes among adjacent cells within individual host filaments, indicating asynchronous infection progression at the single-cell level. Comparative genomics revealed a conserved ~30 kb invertible genomic region flanked by inverted repeats, a structural configuration with limited precedent in cyanophages. Using diagnostic PCR, Sanger sequencing, and read-mapping, we validated that two isomers stably coexist within phage populations. In contrast to the previously characterized cyanophage A-1(L), the three new phages encode a non-cyanobacterial alkaline phosphatase (PhoD). The expression of phoD was highly upregulated during infection, suggesting a phage-driven mechanism for phosphorus acquisition. Conversely, these phages lack the early-expressed tnpB endonuclease gene carried by A-1(L). Transcription of tnpB was strongly upregulated for both strands ( tnpB _fwd and tnpB _rev ) during A-1(L) infection, while infection by the three novel phages modestly induced the transcription of five identical host-encoded tnpB copies. In a highly unusual setting, both tnpB strands are protein-coding over their entire length, leading to the expression of a 347-residue protein from the tnpB _rev strand. Conclusions: This study reveals that genomic isomerism is a source of structural plasticity in freshwater cyanophages and uncovers functionally distinct auxiliary gene repertoires that link phage infection strategies to nutrient status. Our findings provide mechanistic insight into how cyanophages diversify their genomes, interact with their hosts and show that a phage gene can be protein-coding on both strands.

The cleavage of the C10–C4a bond in anthraquinones is a key step in generating ring-opened quinone derivatives in filamentous fungi. GedF, a short-chain dehydrogenase/reductase (SDR) from Aspergillus terreus, together with the dioxygenase GedK, mediates this transformation, yet the enzymatic mechanism of GedF remains unclear. Here, isotope labeling experiments confirm that reduction of Questin-to-Questin hydroquinone incorporates one proton from NADPH and one from water. Structural modeling, molecular docking, and site-directed mutagenesis reveal that GedF employs a noncanonical catalytic architecture featuring a conserved Ser–Tyr catalytic core instead of the classical Asn-Ser-Tyr-Lys tetrad typical of SDRs. Notably, mutagenesis and comparative analysis indicate that a positively charged residue is required for catalysis but is not strictly position-conserved, consistent with a role in maintaining the catalytic microenvironment and facilitating proton transfer. Phylogenetic and sequence analyses show that GedF belongs to the NAD(P)H-dependent SDR clade, and that variation in the positioning of basic residues occurs among homologues while preserving the conserved Ser–Tyr catalytic core. These findings elucidate the catalytic mechanism of GedF and uncover an alternative SDR catalytic strategy involved in anthraquinone ring-opening biosynthesis in filamentous fungi.

Cyanobacteria are essential primary producers in aquatic ecosystems. Silver nanoclusters (Ag NCs) are emerging ultrasmall nanomaterials with broad applications; however, they inevitably enter aquatic environments. This study investigated the nanotoxicity of Ag NCs on model cyanobacterium Synechococcus elongatus PCC 7942 (Syn7942), revealing concentration-dependent growth inhibition with a 72 h half-maximal effective concentration of 1.19 mg L⁻¹. Ag NCs internalized into Syn7942 cells severely impaired the photosynthetic machinery, evidenced by reduced chlorophyll a content and suppressed photochemical reactions, leading to impaired electron transport. This photosynthetic dysfunction resulted in substantial reactive oxygen species generation and lipid peroxidation, as indicated by elevated levels of malondialdehydes. Although the activities of key antioxidant enzymes were enhanced, oxidative damage ultimately overwhelmed the cellular defense capacity. Furthermore, quantitative analysis of silver ions (Ag⁺) release coupled with comparative toxicity assays of Ag⁺ and Ag NCs confirmed that the toxicity originates from the Ag NCs themselves, rather than from the released Ag⁺. Transcriptional level analysis revealed that Ag NCs altered the expression of genes involved in photosynthesis and metal detoxification. Collectively, this study reveals a unique toxicity mechanism for Ag NCs distinct from that for Ag NPs and Ag⁺, highlighting the potential ecological risks posed by ultrasmall nanomaterials.

Plant isoflavonoids constitute a class of secondary metabolites that hold great potential in nutraceutical and pharmaceutical industries, considering their prominent human health-promoting properties. As a result, there is increasing interest in establishing alternative sources to these bioactive molecules to cope with the uneconomic and non-sustainable nature of traditional crop-based production. Here, we present the construction of a yeast platform that can de novo produce isoflavonoid calycosin-7-glucoside (CG), one of the quality markers for important medicinal Astragalus herbs. Through identifying active plant catalytic enzymes, optimizing carbon sources, utilizing and providing key precursor daidzein, and engineering biosynthesis of key metabolic cofactors, production of 47.60 mg/L CG from simple carbon sources was achieved in shake flask fermentations. In addition, we showcased the feasibility of modifying the substrate preference of selected plant glycosyltransferase by rational protein engineering. Our work provides an important step toward developing industrial-scale biomanufacturing of CG and may be extended to engineer complete biosynthesis of other value-added isoflavonoids in microbes.

Coordinating phosphate acquisition with overall nutritional status is critical for cyanobacterial survival in oligotrophic environments, yet the molecular basis for substrate selectivity in phosphate-binding proteins (PBPs) remains incompletely understood. Here we reveal two evolutionarily distinct PBP architectures in Synechocystis sp. PCC 6803 through integrative structural and biochemical analyses. High-resolution crystal structures (1.76-1.9 Å) of PstS1 and SphX bound to inorganic phosphate (Pi) demonstrate that D-type PBPs utilize a conserved aspartate-mediated low-barrier hydrogen bond (LBHB) for high-affinity Pi binding (nanomolar Kd), whereas S-type SphX employs a previously uncharacterized Asp-Arg-Thr catalytic triad with reduced affinity (micromolar Kd). Despite forming comparable hydrogen bonding networks (14 versus 15 bonds), SphX exhibits 26-fold lower Pi affinity due to threonine-mediated proton acceptance replacing the LBHB mechanism. Systematic mutagenesis confirms the functional importance of each triad residue and indicates partial compensation by neighboring residues. Phylogenetic analysis demonstrates that these architectural variants are conserved across cyanobacterial lineages, representing adaptive solutions to diverse nutrient-limited environments. Our findings establish a mechanistic framework for understanding PBP functional diversification and provide molecular insights relevant to optimizing cyanobacterial productivity in biotechnology applications.

The cyanobacterial circadian clock orchestrates a wide range of physiological processes. However, its involvement in extracellular vesicle (EV) biogenesis remains largely unexplored. In this study, we investigated the role of the master circadian output regulator RpaA in modulating EV production in Synechococcus elongatus PCC 7942. Deletion of rpaA simultaneously abolished the bioluminescence rhythmicity and rhythmic EV production, whereas overexpression of rpaA enhances the output of circadian clock signals in a dose-dependent manner and markedly increases the bioluminescence intensity and EV production while maintaining the normal circadian oscillation patterns. Transcriptional analyses indicated that rpaA positively regulates lpxD to promote membrane synthesis and differentially inhibits glgC transcription to optimize carbon allocation. These findings reveal a previously unrecognized link between circadian output signaling and EV biogenesis and suggest that rpaA overexpression is an effective strategy for improving the yield of EVs. It also provides novel insights and regulatory targets for facilitating the synthesis of other high-value metabolites in cyanobacterial cell factories, and holds certain practical significance for advancing cyanobacterial synthetic biology research and the application of nanocarrier technology.

Photosynthesis, the main energy source for life on Earth, confronts escalating challenges of high-light–high-temperature stress (HLHT). Our previous study identified a mutation in ATP synthase, F₁-α-C252Y, that significantly enhances the HLHT tolerance of Synechococcus elongatus PCC 7942 (Sye7942), although the underlying mechanism remains obscure. In this study, we found that this mutation led to elevated levels of the b subunit of F_o, F₁ subunits, and the ATP synthase within cells, without affecting ATP synthetic activity, indicating improved intracellular ATP synthesis activity. Additionally, the mutation altered the transcriptome of Sye7942, impacting the expression of genes involved in crucial processes, such as the electron transport chain, carbon fixation, and regulatory factors, which are crucial for cyanobacteria’s adaptation to stresses. Correspondingly, the mutant exhibited enhanced photosynthesis, accelerated growth, and increased glycogen under HLHT conditions, showing improved adaptation. The higher intracellular ATP synthesis activity, along with enhanced photosynthetic activity, suggests increased ATP production in the mutant under HLHT. Enhancing ATP production and remodeling the cellular transcriptome appear to be key strategies employed by the C252Y mutation for Sye7942 acclimating to HLHT. These findings provide valuable insights for enhancing photosynthetic efficiency and stress resilience in cyanobacteria and other photosynthetic organisms facing HLHT challenges.