Huzhou University

Phloretin has garnered significant interest due to its substantial value in the pharmaceutical, cosmetic, and food industries. However, its supply via plant extraction remains constrained by low yield and complex processing. In this study, an engineered Saccharomyces cerevisiae strain was constructed for high-level de novo phloretin biosynthesis via multi-module carbon-flux rewiring. In the downstream phloretin-forming module, an integrated three-route network was constructed, consisting of two direct phloretin-forming routes and a phloretic acid recycling route. Specifically, introduction of naringenin chalcone reductase re-channeled flux at the chalcone node, while enzyme screening and copy-number optimization of 4-coumarate:coenzyme A ligase and chalcone synthase reduced phloretic acid accumulation, effectively recovering carbon otherwise lost to byproduct formation. In the aromatic amino-acid supply module, key pathway steps were reinforced and feedback regulation was relieved, with careful balancing of key pathway genes to prevent metabolic imbalance. In the malonyl-coenzyme A supply module, cytosolic acetyl-coenzyme A generation was enhanced and organelle-to-cytosol carbon partitioning was rewired, thereby expanding the cytosolic malonyl-coenzyme A pool required for chalcone synthase-mediated condensation. The resulting engineered strain achieved phloretin titers of 460.6 mg/L in shake-flask cultures and 1.817 g/L in 5-L fed-batch fermentation, representing the highest reported de novo titer in yeast to date. This study demonstrates that coordinated control of branch-point flux, byproduct recycling, and precursor supply is essential for high-level microbial phloretin production and provides a scalable framework for microbial production of phloretin and related derivatives.

Osteoarthritis (OA) is a chronic joint disease, and early diagnosis of OA is crucial for its treatment, which is more beneficial for osteoarthritis patients to relieve symptoms, delay disease progression, and improve quality of life. Hypochlorous acid plays a "double-edged sword" role in OA, and can serve as a biomarker for OA. Here, based on the advantages of NIR fluorescent probe and two-photon fluorescent probe, a hypochlorous acid-activatable two-photon excited NIR fluorescent probe (TP-NIR-HClO) based on spirolactam ring-opening strategy was developed for imaging OA this paper. TP-NIR-HClO showed a highly selective two-photon excited NIR fluorescence "off-on" response for HClO in solution, with a high signal-to-background ratio (the maximum SBR was approximately 150), a detection limit (30 nM), and a linear response range (150 nM to 6 μM). Moreover, TP-NIR-HClO was successfully applied for two-photon imaging of HClO in cells and tissues, with a maximum imaging depth of 225 μM. Finally, TP-NIR-HClO was successfully applied for imaging osteoarthritis in mice. All the results confirm that TP-NIR-HClO is a useful tool for imaging HClO in biosystem and studying the pathology and the diagnosis of osteoarthritis.

Fireflies are regarded as one of the most charismatic beetles due to their bioluminescence and ecological importance as bioindicators of freshwater quality. However, molecular mechanisms of larval development and sexual dimorphism in aquatic species remain poorly understood. Here, we performed multi-stage transcriptomic analysis of the aquatic firefly Aquatica leii across larval instars from L2 to L6, together with adult females and males, with three biological replicates per stage. Using time-series expression clustering, differential expression analysis, and weighted gene co-expression network analysis (WGCNA), we characterized the transcriptional dynamics of continuous larval development and the onset of sex-biased gene expression. We identified a critical transcriptional transition occurred at L5-L6, marked by downregulation of early morphogenetic genes and upregulation of juvenile hormone metabolism, oxidoreductase activity, and muscle contraction genes, indicating a shift from growth to metamorphic preparation. WGCNA identified a module strongly correlated with L6 (R = 0.97) enriched for the same functions, confirming a coordinated late-larval program. Notably, genes exhibiting sex-biased expression in adults were already expressed during late larval stages (L5 and L6), and 123 genes progressively upregulated from L2 to L6 showed enrichment in chitin biosynthesis, heart contraction, and ion transport; among these, six genes maintained high expression in adults with clear male-biased (Alei052192, Alei006658, and Alei087054) or female-biased (Alei003725, Alei096818, and Alei074026) patterns. These findings establish that transcriptional foundations for sexual dimorphism and adult tissue formation are laid during late larval stages, providing the first multi-stage transcriptomic resource for aquatic firefly conservation and breeding.