The link between impaired gamma oscillations and Alzheimer's disease (AD) has inspired therapies using rhythmic physical stimuli. However, given that cognition requires cross-frequency interactions like theta-gamma coupling, single-frequency stimulation may yield limited benefits. This study therefore applied a compound pulsed magnetic field (cPMF) with theta rhythm-modulated gamma frequency to evaluate its efficacy and mechanisms against AD pathology compared with single gamma-frequency pulsed magnetic field (sPMF). Local field potential results showed that cPMF outperformed sPMF by significantly enhancing hippocampal oscillations and particularly rescuing the impaired theta-gamma phase-amplitude coupling in AD mice, which was positively correlated with improved cognitive performance in behavioral tests. Correspondingly, cPMF treatment enhanced blood flow perfusion in the prefrontal and cerebral cortices of AD mice, which may contribute to amyloid-β clearance and neuroinflammation attenuation. At the molecular level, cPMF rescued AD-related transcriptional alterations by upregulating key genes involved in cholinergic signaling (Chat, Chrm1), glymphatic function (Aqp4), and synaptic plasticity (Gria1, an AMPA receptor subunit). These findings indicated that cPMF stimulation achieved multi-level restorative effects by enhancing neuronal activity, promoting cerebral perfusion, facilitating amyloid-β clearance, and rectifying aberrant gene expression, ultimately leading to cognitive improvement in AD mice. This cPMF stimulation paradigm highlights the therapeutic potential of targeting endogenous oscillatory interactions for treating neurological disorders.

Abstract Camptothecin derivatives are first-line anticancer drugs used worldwide for the treatment of diverse malignant tumors. However, the biosynthetic pathway of camptothecin has remained elusive for five decades. Here, we fully map its entire biosynthetic route. We discovered five key missing enzymes (OpCAR, OpSDR11, OpCS, OpGH1, and OpSTR) via the combination of MALDI mass spectrometry imaging, single-cell RNA sequencing and co-expression analysis. Meanwhile, we demonstrated a free flavin mononucleotide triggered the non-enzymatic 6-5-6 to 6-6-5 fused-ring skeleton rearrangement, filling the last gap in camptothecin biosynthesis. Finally, we validated this identified pathway and achieved the de novo biosynthesis of camptothecin in Saccharomyces cerevisiae . These discoveries uncover the long-standing mystery underlying camptothecin and pave the way for manufacturing camptothecin and its derivatives through synthetic biology approaches.

ABSTRACT Taxol (paclitaxel) is a frontline anticancer drug widely applied for the treatment of breast, ovarian and lung cancers. Currently, its supply mainly relies on the semi-synthesis using baccatin III from Taxus plants. Heterologous biosynthesis of baccatin III in microorganisms offers a promising solution to alleviate global Taxol supply shortage, but remains challenging due to pathway complexity. Here, we report a novel taxusin-mediated biosynthetic pathway for baccatin III production via the identification of C13 deacetylase, elucidation of the exact sequence underlying C1 hydroxylation, and stepwise enzymatic functional validation. Through protein engineering of the promiscuous C1 and C5 hydroxylases, coupled with the distribution of pathway modules in Saccharomyces cerevisiae and Escherichia coli , we achieved the de novo biosynthesis of baccatin III. Collectively, our findings remodel the current biosynthetic framework governing the formation of Taxol precursors and highlight the great potential of microbial cell factories for the production of complex plant-derived therapeutic compounds. Highlights • Discovery of C13 deacetylase reveals a novel biosynthetic route to baccatin III via taxusin • Stepwise verification of the complete biosynthetic route to baccatin III through taxusin and baccatin VI • Single-site mutation reversed the product selectivity of T1OH and converted T5OH into a specific taxoid C5 hydroxylase • Complete biosynthesis of baccatin III in engineered microbes

Persister cells constitute a subpopulation of dormant cells that are transiently tolerant to antibiotics and associated with chronic infection. We have known (p)ppGpp synthetase Rsh promoted persister cells formation during rifampicin exposure in Brucella, but the metabolic regulation mechanism by which Rsh promotes persister cells formation is unknown. In this study, we firstly characterized the proteome and metabolome of persister cells formation during rifampicin exposure between B. abortus A19 (WT) and Δrsh strains. We found that sulfur metabolism-related protein (NAD(P)/FAD-dependent sulfite reductase, cysI) was significantly down-regulated in Δrsh compared with WT strain during persister cells formation (P < 0.01). The Rsh significantly affected hydrogen sulfide consumption by hydrogen sulfide measurement (P < 0.01). And then we found Rsh positively regulated CysI that its promoter region was 122 bp of upstream of cysI gene. In addition, the cysI knockout strain (ΔcysI) was constructed which significantly reduced persister cells formation (P < 0.01) and also reduced consumption of hydrogen sulfide compared with WT strain. Taken together, we firstly reported that (p)ppGpp synthetase Rsh promotes persister cells formation through regulating sulfur metabolism mediated by sulfite reductase CysI in B. abortus. Our results provide a potential target and new strategy for clearing persister cells as well as prevention of Brucella infection.

Alzheimer's disease (AD) imposes a heavy burden on families and society. Rhythmic magnetic stimulation has emerged as a promising non-invasive therapy to mitigate AD-related cognitive decline. In this study, we applied a rhythmic unipolar compound pulsed magnetic field (cPMF; carrier frequency: 40 Hz, repetition rate: 5 Hz, magnetic flux density: 0-20 mT) incorporating both theta and gamma rhythms to evaluate its effects on behavior and neural oscillations in AD mice and to explore the underlying mechanisms. 5xFAD mice received unipolar cPMF stimulation for 1 h/d over 8 consecutive weeks. Learning and memory were assessed using the novel object recognition (NOR) and the Morris water maze (MWM) tests. In NOR test, unipolar cPMF-treated mice showed a higher cognitive index in test phase 2, and in MWM test, exhibited shorter escape latencies in the training trial and spent less time to first cross the precise former platform location with a higher crossing frequency over this target area in the probe trial. Local field potentials (LFPs) in the hippocampal CA1 area were recorded via in vivo electrophysiology. LFP analysis showed that unipolar cPMF treatment enhanced power of cognitive-related neural oscillations and strengthened theta-gamma phase-amplitude coupling. RNA sequencing analysis further indicated that unipolar cPMF-treated mice exhibited differential gene expression in molecular function and multiple neurotransmitter synaptic signaling pathways. In conclusion, unipolar cPMF might improve cognitive function in 5xFAD mice by modulating cognitive-related neural oscillations. These findings could provide experimental support for the low-intensity pulsed magnetic stimulation as a potential therapeutic strategy for AD.

Unlike hyperthermia after intratumoral injection, the method of integrated magnetic targeted hyperthermia (iMTH) guides magnetic medium to the target site and then directly performs in-situ heating, showing great potential for effective treatment of deep-seated tumors in the body. Magnetotactic bacteria (MTB), having chain-like arranged magnetic nanoparticles within its body and active movement along an external magnetic field, are considered as a very fitted material for iMTH. However, the amount of MTB concentrated on the deep-seated tumor posed a significant challenge for the successful implementation of iMTH. Herein, we aim to validate the strategy of integrating magnetic targeting and hyperthermia. An in-situ liver tumor model in mouse was developed as deep-seated tumors. After administering the polar MTB MO-1 intravenously via the tail vein, a focusing magnetic field navigated these bacteria to effectively accumulate at the deep-seated tumor site. Immediately afterwards, this targeted aggregation of MO-1 cells triggered a localized magnetic hyperthermia directly at the cancer site under an applied alternating magnetic field. Our findings demonstrated that this hyperthermia induced by the bacteria led to the death of liver cancer cells, thereby effectively curbing the progression and growth of the cancer. These promising results suggested that an iMTH approach was developed, harnessing the power of MTB. This method stands as an exciting and potential therapeutic strategy for the treatment of deep-seated tumors, offering new hope in the fight against cancer.

Magnetotactic bacteria (MTB), known for their precision in navigating along magnetic fields, also exhibit lightsensitive behaviors. In Magnetospirillum magneticum AMB-1, the photoreceptor Amb2291 is involved in phototaxis regulation and magnetosome synthesis, particularly under oxidative stress. The magnetoreceptor Amb0994 modulates flagellar activity in response to magnetic field changes. Our study used a magneto-optical system to analyze the U-turn motility of north-seeking AMB-1 wild type (WT), amb2291 and amb0994 mutants under reversed magnetic fields and controlled light conditions. The results showed that WT strains consistently executed U-turns in response to magnetic fields, regardless of light variations. The diameters of U-turn of amb0994 mutant were smaller than those of the WT control. When illuminated with blue light in a direction opposite to the magnetic field, Delta amb0994 exhibited slower U-turns with diameters similar to WT. In contrast, the Delta amb2291 strain exhibited exaggerated U-turn movements under blue light, characterized by larger movement diameters and times compared to the WT, particularly whatever the light propagation direction is the same or opposite to the magnetic field in the initial state of motility. Gene expression analysis revealed that long-term exposure to blue light and magnetic fields led to a significant upregulation of amb2291 in Delta amb0994 mutant strains and amb0994 in Delta amb2291 mutant strains. These indicate a potential cooperative role of amb2291 and amb0994 in modulating bacterial motility under blue light. This research enhances our understanding of photoreception in MTB and its impact on magnetotaxis, shedding light on how environmental factors interact with microorganisms.

Biofilms are complex adhesive structures that establish chronic infection and allow robust protection from external stressors such as antibiotics. Cellulose as one of the compositions of bacteria biofilm which protect bacteria from stress, host immune responses and resistance to antibiotics. Bacterial stress responses are regulated via guanosine pentaphosphate and tetraphosphate (p)ppGpp. This molecule has been a target of research efforts to counteract biofilm formation in pathogenic bacteria. However, a role for (p)ppGpp synthetase Rel influencing in biofilms and its cellulose formation has not been identified in Brucella. Firstly, rel mutant significantly decreased biofilm biomass and rendered biofilms more susceptible to most antibiotics. The rel mutant also showed greatly decreased biofilm architectures including exopolysaccharide, extracellular DNA, and lipid. Remarkably, we found rel mutant significantly decreased biofilm cellulose formation. We further combined proteomic analysis to explore the key proteins involved in cellulose regulation of Rel in Brucella biofilm formation. 287 differentially expressed proteins (DEPs) were identified and enriched in diverse metabolic pathway between WT and Δrel strains including purine and sulfur metabolism, transcription factors and glycosyltransferases which may be related to cellulose formation. The Q-PCR showed that mRNA levels of only glycosyltransferase (WP_006161578.1) of the 12 down-DFPs had significantly upregulated in rel mutant contrast to WT strain and β-galactosidase assay showed a negative regulatory in rel mutant. Furthermore, Rel-dependent biofilms cellulose was also restored and accompanied by an increase in glycosyltransferase (WP_006161578.1) when glucose was added in TSB medium. Overall, this work expands the role of (p)ppGpp synthetase Rel as an important regulator in biofilm and cellulose formation that is tightly linked with pathogenicity and chronic persistent infections in Brucella.

Tumor heterogeneity poses numerous challenges for targeted drug therapy. Although tumor cell-derived nanovesicles (NVs) have emerged as an intriguing method for tumor targeting, how to exert the antitumor effect after targeting remains a key concern. Magnetotactic bacteria (MTB) synthesize chain-like magnetite (Fe3O4) crystals with inherent magnetic moments, which could generate significant torque under a desired magnetic field and move along the magnetic field using their own flagella. Herein, a composite of MTB AMB-1 and NVs was fabricated via electrostatic adsorption where AMB-1 could transport NVs to the tumor site by a guiding magnetic field, while NVs also assist AMB-1 in binding to tumor cells. Subsequently, under the influence of a swing magnetic field (sMF), MTB exert physical stimuli on the cells, inducing the changes of mitochondrial membrane potential and cellular reactive oxygen species (ROS). Finally, it is revealed that the NVs-loaded AMB-1 induced a decrease in cellular viability and significantly inhibited the growth of tumors in vivo under the sMF. Therefore, by remote control of the guidance and stimuli production, the NVs-loaded AMB-1 was highly promising to advance the development of targeted therapeutic strategies for tumors under the context of tumor heterogeneity.

Brucella spp. are facultative intracellular bacteria that infect and induce brucellosis in a diverse range of mammalian hosts. The disease causes major global economic losses and also is a worldwide threat to public health security. Characterization of bacterial and host factors that promote intracellular survival of Brucella is key for the prevention and control of brucellosis. In this study, we identified proteins involved in intracellular survival of Brucella abortus A19 in RAW264.7 macrophage cells by liquid chromatography-mass spectrometry of macrophages with or without B. abortus infection. The functions of these proteins, the signaling pathways in which the proteins participate, the domain entries enriched by the proteins, and the subcellular localization of the differentially-expressed proteins were deciphered. Differential protein expression revealed that Slc2a1, which is a key Glycolytic protein, was significantly upregulated in infected macrophage cells. This observation was confirmed by qRT-PCR and Western blotting studies. The role of Slc2a1 in the intracellular survival of B. abortus was probed by overexpressing and knocking down SLC2A1 in RAW264.7 cells. Overproduction of the protein promoted intracellular proliferation of B. abortus whereas knockdown of SLC2A1 inhibited proliferation of the bacterium. Finally, we determined that the Secreted Effector Protein A (SepA) effector of B. abortus enhanced SLC2A1 expression in macrophage cells. Thus, B. abortus stimulates host SLC2A1 expression via the SepA effector protein to aid bacterial survival in the macrophage environment which suggests that SepA may be a novel antibacterial target to combat Brucella infection.

The sesquiterpene lactone parthenolide is a promising anticancer drug. Its biosynthesis via a microbial cell factory has been considered as a sustainable alternative to plant extraction. Herein, systematic metabolic engineering approaches, as well as the introduction of a novel noncanonical tricarboxylic acid (TCA) cycle, were employed to enhance the production of the key precursor germacrene A. By identifying two new dehydrogenases and controlling the expression of parthenolide synthase, we further achieved the elimination of byproducts and enhanced parthenolide production. A two-stage fermentation approach and in situ product extraction using macroreticular resin were further applied to relieve the nocuous effect of costunolide and parthenolide on the growth of yeast cell factories, ultimately achieving a titer of 549.7 mg/L for parthenolide and 972.7 mg/L for costunolide in a 10 L fermenter, which represents the highest reported titer obtained by microbial fermentation. The strategies should also contribute to the microbial cell factory-construction for other natural products exhibiting toxicity.

Ergot alkaloids (EAs) are a class of indole derivatives used as prescription drugs for the treatment of neurological diseases. Due to the limited production of EAs by Claviceps and the enantioselective difficulties encountered in chemical synthesis, a sustainable supply of EAs remains challenging. Recently, numerous attempts have been made to produce EAs using heterologous hosts. However, these efforts have only resulted in the production of the precursor, lysergic acid (LA), with low efficiency. Here, we report the de novo high-efficient biosynthesis of LA and a series of LA-derived EAs in Aspergillus oryzae cell factories. Based on genome sequencing of the EA-producing strain, C. purpurea 22.07, an EA biosynthetic gene cluster was annotated and characterized. After introducing and optimizing the agroclavine (AG) biosynthetic pathway in A. oryzae, we constructed an efficient chassis strain for AG production. We then confirmed the function of the annotated CloA ' to catalyze the successive oxidation of AG into LA and isolysergic acid (ILA) in this AG-producing chassis and realize their de novo production with titers of 52.68 +/- 1.49 and 6.32 +/- 2.08 mg L-1, respectively. The subsequent introduction of the downstream non-ribosomal peptide synthetase genes LpsB ' and LpsC ' enabled the complete biosynthesis of ergometrine and a series of its analogs, achieving a total titer of more than 160 mg L-1. The unexpected biosynthesis of isolysergyl-glycine and lysergyl-glycine revealed a novel function of LpsC ', which utilizes glycine as a substrate. Our work successfully realized the complete biosynthesis of a series of EAs in an industrially feasible fungus, which will open new avenues for manufacturing EAs in a green and sustainable manner.

Catalytic bioparts are fundamental to the design, construction and optimization of biological systems for specific metabolic pathways. However, the functional characterization information of these bioparts is frequently dispersed across multiple databases and literature sources, posing significant challenges to the effective design and optimization of specific chassis or cell factories. We developed the Registry and Database of Bioparts for Synthetic Biology (RDBSB), a comprehensive resource encompassing 83 193 curated catalytic bioparts with experimental evidences. RDBSB offers their detailed qualitative and quantitative catalytic information, including critical parameters such as activities, substrates, optimal pH and temperature, and chassis specificity. The platform features an interactive search engine, visualization tools and analysis utilities such as biopart finder, structure prediction and pathway design tools. Additionally, RDBSB promotes community engagement through a catalytic bioparts submission system to facilitate rapid data sharing and utilization. To date, RDBSB has supported the contribution of >1000 catalytic bioparts. We anticipate that the database will significantly enhance the resources available for pathway design in synthetic biology and serve essential tools for researchers. RDBSB is freely available at https://www.biosino.org/rdbsb/.

Magnetogenetics has shown great potential for cell function and neuromodulation using heat or force effects under different magnetic fields; however, there is still a contradiction between experimental effects and underlying mechanisms by theoretical computation. In this study, we aimed to investigate the role of reactive oxygen species (ROS) in mechanical force-dependent regulation from a physicochemical perspective. The transient receptor potential vanilloid 4 (TRPV4) cation channels fused to ferritin (T4F) were overexpressed in HEK293T cells and exposed to static magnetic fields (sMF, 1.4-5.0 mT; gradient: 1.62 mT/cm). An elevation of ROS levels was found under sMF in T4F-overexpressing cells, which could lead to lipid oxidation. Compared with the overexpression of TRPV4, ferritin in T4F promoted the generation of ROS under the stimulation of sMF, probably related to the release of iron ions from ferritin. Then, the resulting ROS regulated the opening of the TRPV4 channel, which was attenuated by the direct addition of ROS inhibitors or an iron ion chelator, highlighting a close relationship among iron release, ROS production, and TRPV4 channel activation. Taken together, these findings indicate that the produced ROS under sMF act on the TRPV4 channel, regulating the influx of calcium ions. The study would provide a scientific basis for the application of magnetic regulation in cellular or neural regulation and disease treatment and contribute to the development of the more sensitive regulatory technology.

Taxol is a widely-applied anticancer drug that inhibits microtubule dynamics in actively replicating cells. Although a minimum 19-step biosynthetic pathway has been proposed and 16 enzymes likely involved have been characterized, stepwise biosynthetic reactions from the well-characterized di-oxygenated taxoids to Taxol tetracyclic core skeleton are yet to be elucidated. Here, we uncover the biosynthetic pathways for a few tri-oxygenated taxoids via confirming the critical reaction order of the second and third hydroxylation steps, unearth a taxoid 9 α -hydroxylase catalyzing the fourth hydroxylation, and identify CYP725A55 catalyzing the oxetane ester formation via a cascade oxidation-concerted acyl rearrangement mechanism. After identifying a acetyltransferase catalyzing the formation of C7-OAc, the pathway producing the highly-oxygenated 1 β -dehydroxybaccatin VI with the Taxol tetracyclic core skeleton is elucidated and its complete biosynthesis from taxa-4(20),11(12)-diene-5 α -ol is achieved in an engineered yeast. These systematic studies lay the foundation for the complete elucidation of the biosynthetic pathway of Taxol.

The downstream biosynthetic route of camptothecin has remained unclear for more than half a century. Here, we discovered a P450 enzyme involved in camptothecin biosynthesis from plant by chemoproteomics, which lays the foundation for synthesis of camptothecin and highlights the effectiveness of chemoproteomic profiling for discovering unknown enzymes.

BACKGROUND:Alzheimer's disease (AD) is the most prevalent form of dementia, but no effective therapeutic strategy is available to date. Rhythmic magnetic stimulation is an attractive means of neuron modulation that could be beneficial for restoring learning and memory abilities. OBJECTIVE:To assess the effect of a compound pulsed rhythmic magnetic field (cPMF) on cognition during AD progression and to explore the appropriate cPMF intervention period. METHODS:Female 5xFAD mice aged 10 weeks and 18 weeks were exposed to cPMF with a carrier frequency of 40 Hz, repeated at 5 Hz for 1 h/d for 8 consecutive weeks. The Morris water maze (MWM) test was used for cognitive behavioral assessment. Furthermore, changes in molecular pathology within the brain were detected using immunofluorescence staining and real-time PCR. RESULTS:10-week-old AD mice treated with cPMF explored the target quadrant more frequently than sham-exposed AD mice in MWM test, exhibiting improved learning and memory abilities. Additionally, cPMF exposure alleviated Aβ plaque deposition and astrogliosis in the AD brain. Moreover, neurotrophic factor fibroblast growth factor 1 (FGF1) in the AD brain was upregulated by cPMF treatment. However, in 18-week-old AD mice treated with cPMF, cognitive performance and Fgf1 gene expression were not significantly improved, although Aβ plaque deposition and astrogliosis were alleviated. CONCLUSION:Early intervention via long-term rhythmic cPMF stimulation may alleviate the histopathological features and enhance neuroprotective gene Fgf1 expression, thereby improving the cognitive performance of 5xFAD mice, which should provide promising insight for the clinical treatment of patients with AD.

Protein methylation is a commonly posttranslational modification of transcriptional regulators to fine-tune protein function, however, whether this regulation strategy participates in the regulation of lignocellulase synthesis and secretion in Trichoderma reesei remains unexplored. Here, a putative protein methyltransferase (TrSAM) is screened from a T. reesei mutant with the ability to express heterologous β-glucosidase efficiently even under glucose repression. The deletion of its encoding gene trsam causes a significant increase of cellulase activities in all tested T. reesei strains, including transformants of expressing heterologous genes using cbh1 promotor. Further investigation confirms that TrSAM interacts with the cellulase negative regulator ACE1 via its amino acid residue Arg383, which causes a decrease in the ACE1-DNA binding affinity. The enzyme activity of a T. reesei strain harboring ACE1R383Q increases by 85.8%, whereas that of the strains with trsam or ace1 deletion increases by more than 100%. By contrast, the strain with ACE1R383K shows no difference to the parent strain. Taken together, our results demonstrate that TrSAM plays an important role in regulating the expression of cellulase and heterologous proteins initiated by cbh1 promotor through interacting with ACE1R383. Elimination and mutation of TrSAM and its downstream ACE1 alleviate the carbon catabolite repression (CCR) in expressing cellulase and heterologous protein in varying degrees. This provides a new solution for the exquisite modification of T. reesei chassis.

Magnetotactic bacteria (MTB), possessing chains of iron oxide nanoparticles, are potential tools for controlled therapy due to their responsiveness to weak magnetic fields. This study aims to evaluate the targeting and aggregation capabilities of axial AMB-1 and polar MO -1 MTB for therapeutic applications. A three-dimensional focusing magnetic field (fMF)-producing device was designed and established with a gradient magnetic field of 0.08 mT/mm at the central region for the guidance of MTB in vitro and in vivo . Under a unidirectional magnetic field, polar Magneto -ovoid MO -1 formed a single line in the culture dish, whereas axial Magnetospirillum magneticum AMB-1 displayed a small portion aggregated on the centerline. With simultaneous X and Y magnetic field application, MO -1 cells aggregated at a point, while AMB-1 bacteria gathered incompletely. Further, magnetic resonance imaging of breast cancer -bearing nude mice injected subcutaneously with MTB peritumorally and navigated by a fMF revealed more substantial aggregation of MO -1 cells within the tumor than AMB-1 bacteria. Prussian blue staining corroborated the penetration of MO -1 cells into the tumor tissue. These findings suggest that polar MO -1 bacteria exhibit superior targeting and aggregation properties compared to axial AMB-1, indicating their potential suitability for targeted therapeutic applications.