Dihydrochalcones(DHCs)are highly accumulated in tender leaves of Lithocarpus litseifolius but their biosynthetic pathway and accumulation mechanism remain unclear.In this study,candidate genes including one cinnamoyl-...Dihydrochalcones(DHCs)are highly accumulated in tender leaves of Lithocarpus litseifolius but their biosynthetic pathway and accumulation mechanism remain unclear.In this study,candidate genes including one cinnamoyl-CoA reductase(LlccR),two double bond reductases(LIDBR1~2),three aldehyde hydrogenases(LlALDH1~3),two 4-coumaroyl:CoA ligases(Ll4CL1~2)and four phloretin glycosyltransferases(LlP4'GT,LlP2'GT1~3)were comprehensively investigated.The substrate specificities and catalytic kinetics of these gene-encoded enzymes were achieved.Through successive catalysis of LlALDH1,Ll4CL2,and chalcone synthase 1(LlCHS1)or combined action of LlCCR and LlCHS1,phloretin was biosynthesized from direct precursor dihydro-p-coumaraldehyde,which had been converted from initial precursor p-coumaroyl-CoA by LlCcR-mediated carboxylic acid reduction and LlDBR1-catalyzedα,β-double bond saturation.High accumulation of the DHCs in tender leaves of L.litseifolius was mainly driven by efficient catalysis of LlccR toward p-coumaroyl-CoA and highly expressed genes in the pathway,especially the LlP4'GT and LlP2'GT1 which contributed to biosynthesis of trilobatin and phlorizin,respectively.Antisense oligodeoxyribonucleotide treatments against the LICCR,LIDBR1,LlALDH1,Ll4CL2,LIP4'GT,and LIP2'GT1 significantly reduced transcripts of the target genes and content of DHCs,confirming these genes might be involved in the pathway.This finding provides insight into the biosynthesis and accumulation mechanism of DHCs in planta.展开更多
Objective Flavonoids are clinically significant natural products,yet their oxygen-glycosylation in aqueous environments relies heavily on expensive nucleotide-activated sugar donors such as UDP-glucose.Liquid-liquid p...Objective Flavonoids are clinically significant natural products,yet their oxygen-glycosylation in aqueous environments relies heavily on expensive nucleotide-activated sugar donors such as UDP-glucose.Liquid-liquid phase separation(LLPS)creates specialized,membraneless physicochemical microenvironments capable of modulating enzymatic functions and overcoming mass transfer limitations.This study aims to investigate whether the gut microbiota-derived DgpB/C complex-a multienzyme system traditionally recognized for cleaving stable C-glycosidic bonds and facilitating isomerization-can undergo functional remodeling within phase-separated condensates.Our core objective is to elucidate the role of phase separation in expanding enzymatic catalytic plasticity and to provide a non-canonical,highly cost-effective biocatalytic mechanism for the direct utilization of free sugars in the synthesis of O-glycosylated natural products.Methods An artificial phase-separation platform was constructed utilizing the multivalent arginine-glycine-glycine motif(RGG)-repeat domain derived from the Caenorhabditis elegans LAF-1 protein.To ensure precise spatial compartmentalization,the DgpB/C complex was specifically recruited into the RGG condensates via a high-affinity SZ1/SZ2 heterodimerization tag system.Condensate formation and substrate partitioning were visualized using light and confocal fluorescence microscopy.The chemical structures and regioselectivity of the reaction products were rigorously characterized using high-performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry(LC-MS)/MS systems.Furthermore,molecular docking and 20-ns molecular dynamics(MD)simulations were performed via the Hermite platform and Uni-GBSA pipeline to elucidate the structural and thermodynamic basis underlying the phase-transitioninduced functional shift.Results We observed that the condensates formed by RGG proteins selectively recruited and significantly enriched hydrophobic flavonoid substrates.Strikingly,within the LLPS microenvironment,the DgpB/C complex-which typically exhibits only degradative or isomerase activities-underwent a profound functional remodeling,transforming into an efficient O-glycosyltransferase.Diverging from canonical pathways that require high-energy donors,the sequestered DgpB/C complex directly utilized unactivated free glucose to catalyze the formation of new O-glycosidic bonds.This remodeled activity was found to be evolutionarily conserved across DgpB/C homologs from diverse gut microbiota strains,such as P581a and W974-1.LC-MS/MS analysis further revealed that the phase-separated environment reduced the regioselectivity constraints of the enzyme,enabling catalytic action on multiple hydroxyl positions of the flavonoid scaffold.MD simulations further indicated that the low-water-activity microenvironment of the condensates reconfigured the conformational dynamics of the catalytic pocket,favoring a spatial orientation highly conducive to dehydration condensation.Conclusion This study demonstrates that LLPS drives the functional remodeling of the gut microbiota enzyme DgpB/C through the reconfiguration of the physicochemical microenvironment.These findings challenge traditional perceptions regarding the functional boundaries of metabolic enzymes and reveal a novel biocatalytic pathway that bypasses the requirement for nucleotide-activated sugars.Consequently,this provides a highly promising artificial compartmentalization strategy for the green manufacturing of complex,high-value-added natural products.展开更多
B S W集团是英国最大的林业和锯木综合业务公司,现已被私募股权公司Endless LLP收购。作为英国的林业产业领导者,B S W集团拥有1200多名员工,每年营业额超过3.5亿英镑,之前通过收购Tilhill Forestry、Alvic Plastics和最近的Maelor Fore...B S W集团是英国最大的林业和锯木综合业务公司,现已被私募股权公司Endless LLP收购。作为英国的林业产业领导者,B S W集团拥有1200多名员工,每年营业额超过3.5亿英镑,之前通过收购Tilhill Forestry、Alvic Plastics和最近的Maelor Forest Nurseries扩大了业务并整合了其供应链。BSW于1848年在伯威克郡(Ber wickshire)成立,是英国最大的锯木集团,位列欧洲木材供应商前二十强。收购之后,原有管理层不会发生大的调整,BSW首席执行官Tony Hackney和首席财务官Alan Milne将继续管理公司业务,并将继续协助推动集团的未来发展。展开更多
Against the backdrop of the issuance of Chinese Auditing Standard for Certified Public Accountants No.1504—Communicating Key Audit Matters in the Independent Auditor's Report,both the inclusion of Key Audit Matte...Against the backdrop of the issuance of Chinese Auditing Standard for Certified Public Accountants No.1504—Communicating Key Audit Matters in the Independent Auditor's Report,both the inclusion of Key Audit Matters(KAMs)in audit reports by accounting firms and the issuance of regulatory inquiry letters by stock exchanges are intended to enhance the vitality of China's capital market.Using data from companies audited by Pan-China Certified Public Accountants LLP between 2020 and 2024,this paper examines the disclosure of key audit matters.The findings indicate that the disclosure of KAMs exhibits a serious degree of homogenization and is often disconnected from the material issues actually faced by audited companies.In light of these findings,this paper suggests that stock exchanges should strengthen regulatory penalties and establish channels for public feedback,while accounting firms should develop rigorous procedures for identifying key audit matters and enhance the entity-specific nature of KAM disclosures.展开更多
为了解国外电力公司的雷电定位系统建设与技术研发情况,结合近年来日本雷电定位系统的发展动态,对日本雷电定位系统的技术更新、主要雷电定位方法、雷电流观测等方面的最新研究进展进行了总结与分析。主要内容有:(1)总结了雷电定位与防...为了解国外电力公司的雷电定位系统建设与技术研发情况,结合近年来日本雷电定位系统的发展动态,对日本雷电定位系统的技术更新、主要雷电定位方法、雷电流观测等方面的最新研究进展进行了总结与分析。主要内容有:(1)总结了雷电定位与防护(lightning location protection,LLP)系统、雷电定位和跟踪(lightning position and tracking system,LPATS)与SAFIR系统(system de surveillance et d’alerte foudre par interferometrie radioelectrique)的定位原理及技术发展,及其在日本各大电力公司与一般商用服务中的应用现状及探测站分布情况;并对3种雷电定位系统或雷电信息采集系统特性进行了对比分析。(2)调查分析了日本电力中央研究所关于Tokyo Skytree的雷击电流观测、雷击电磁波观测等方面的最新研究进展。由调研结果可知:雷击电流、电磁波以及上行先导的联合试验观测体系可以获取大量雷电活动规律与特征参数,非常有助于揭示雷电发展过程中的基本物理现象,探索并完善上行先导的机理与模型等;随着观测设备及技术的提高,可以获取更准确的数据、更精细的雷电发展过程参数,非常有助于进一步提高雷电定位系统的性能与定位精度,有效促进电网主动防雷技术的发展。展开更多
基金financially supported by the National Natural Science Foundation of China(grant 32272763)China Agriculture Research System of MOF and MARA.and the Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding-Tea Plant(grant 2021C02067-6).
摘要Dihydrochalcones(DHCs)are highly accumulated in tender leaves of Lithocarpus litseifolius but their biosynthetic pathway and accumulation mechanism remain unclear.In this study,candidate genes including one cinnamoyl-CoA reductase(LlccR),two double bond reductases(LIDBR1~2),three aldehyde hydrogenases(LlALDH1~3),two 4-coumaroyl:CoA ligases(Ll4CL1~2)and four phloretin glycosyltransferases(LlP4'GT,LlP2'GT1~3)were comprehensively investigated.The substrate specificities and catalytic kinetics of these gene-encoded enzymes were achieved.Through successive catalysis of LlALDH1,Ll4CL2,and chalcone synthase 1(LlCHS1)or combined action of LlCCR and LlCHS1,phloretin was biosynthesized from direct precursor dihydro-p-coumaraldehyde,which had been converted from initial precursor p-coumaroyl-CoA by LlCcR-mediated carboxylic acid reduction and LlDBR1-catalyzedα,β-double bond saturation.High accumulation of the DHCs in tender leaves of L.litseifolius was mainly driven by efficient catalysis of LlccR toward p-coumaroyl-CoA and highly expressed genes in the pathway,especially the LlP4'GT and LlP2'GT1 which contributed to biosynthesis of trilobatin and phlorizin,respectively.Antisense oligodeoxyribonucleotide treatments against the LICCR,LIDBR1,LlALDH1,Ll4CL2,LIP4'GT,and LIP2'GT1 significantly reduced transcripts of the target genes and content of DHCs,confirming these genes might be involved in the pathway.This finding provides insight into the biosynthesis and accumulation mechanism of DHCs in planta.
摘要Objective Flavonoids are clinically significant natural products,yet their oxygen-glycosylation in aqueous environments relies heavily on expensive nucleotide-activated sugar donors such as UDP-glucose.Liquid-liquid phase separation(LLPS)creates specialized,membraneless physicochemical microenvironments capable of modulating enzymatic functions and overcoming mass transfer limitations.This study aims to investigate whether the gut microbiota-derived DgpB/C complex-a multienzyme system traditionally recognized for cleaving stable C-glycosidic bonds and facilitating isomerization-can undergo functional remodeling within phase-separated condensates.Our core objective is to elucidate the role of phase separation in expanding enzymatic catalytic plasticity and to provide a non-canonical,highly cost-effective biocatalytic mechanism for the direct utilization of free sugars in the synthesis of O-glycosylated natural products.Methods An artificial phase-separation platform was constructed utilizing the multivalent arginine-glycine-glycine motif(RGG)-repeat domain derived from the Caenorhabditis elegans LAF-1 protein.To ensure precise spatial compartmentalization,the DgpB/C complex was specifically recruited into the RGG condensates via a high-affinity SZ1/SZ2 heterodimerization tag system.Condensate formation and substrate partitioning were visualized using light and confocal fluorescence microscopy.The chemical structures and regioselectivity of the reaction products were rigorously characterized using high-performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry(LC-MS)/MS systems.Furthermore,molecular docking and 20-ns molecular dynamics(MD)simulations were performed via the Hermite platform and Uni-GBSA pipeline to elucidate the structural and thermodynamic basis underlying the phase-transitioninduced functional shift.Results We observed that the condensates formed by RGG proteins selectively recruited and significantly enriched hydrophobic flavonoid substrates.Strikingly,within the LLPS microenvironment,the DgpB/C complex-which typically exhibits only degradative or isomerase activities-underwent a profound functional remodeling,transforming into an efficient O-glycosyltransferase.Diverging from canonical pathways that require high-energy donors,the sequestered DgpB/C complex directly utilized unactivated free glucose to catalyze the formation of new O-glycosidic bonds.This remodeled activity was found to be evolutionarily conserved across DgpB/C homologs from diverse gut microbiota strains,such as P581a and W974-1.LC-MS/MS analysis further revealed that the phase-separated environment reduced the regioselectivity constraints of the enzyme,enabling catalytic action on multiple hydroxyl positions of the flavonoid scaffold.MD simulations further indicated that the low-water-activity microenvironment of the condensates reconfigured the conformational dynamics of the catalytic pocket,favoring a spatial orientation highly conducive to dehydration condensation.Conclusion This study demonstrates that LLPS drives the functional remodeling of the gut microbiota enzyme DgpB/C through the reconfiguration of the physicochemical microenvironment.These findings challenge traditional perceptions regarding the functional boundaries of metabolic enzymes and reveal a novel biocatalytic pathway that bypasses the requirement for nucleotide-activated sugars.Consequently,this provides a highly promising artificial compartmentalization strategy for the green manufacturing of complex,high-value-added natural products.
摘要Against the backdrop of the issuance of Chinese Auditing Standard for Certified Public Accountants No.1504—Communicating Key Audit Matters in the Independent Auditor's Report,both the inclusion of Key Audit Matters(KAMs)in audit reports by accounting firms and the issuance of regulatory inquiry letters by stock exchanges are intended to enhance the vitality of China's capital market.Using data from companies audited by Pan-China Certified Public Accountants LLP between 2020 and 2024,this paper examines the disclosure of key audit matters.The findings indicate that the disclosure of KAMs exhibits a serious degree of homogenization and is often disconnected from the material issues actually faced by audited companies.In light of these findings,this paper suggests that stock exchanges should strengthen regulatory penalties and establish channels for public feedback,while accounting firms should develop rigorous procedures for identifying key audit matters and enhance the entity-specific nature of KAM disclosures.
摘要为了解国外电力公司的雷电定位系统建设与技术研发情况,结合近年来日本雷电定位系统的发展动态,对日本雷电定位系统的技术更新、主要雷电定位方法、雷电流观测等方面的最新研究进展进行了总结与分析。主要内容有:(1)总结了雷电定位与防护(lightning location protection,LLP)系统、雷电定位和跟踪(lightning position and tracking system,LPATS)与SAFIR系统(system de surveillance et d’alerte foudre par interferometrie radioelectrique)的定位原理及技术发展,及其在日本各大电力公司与一般商用服务中的应用现状及探测站分布情况;并对3种雷电定位系统或雷电信息采集系统特性进行了对比分析。(2)调查分析了日本电力中央研究所关于Tokyo Skytree的雷击电流观测、雷击电磁波观测等方面的最新研究进展。由调研结果可知:雷击电流、电磁波以及上行先导的联合试验观测体系可以获取大量雷电活动规律与特征参数,非常有助于揭示雷电发展过程中的基本物理现象,探索并完善上行先导的机理与模型等;随着观测设备及技术的提高,可以获取更准确的数据、更精细的雷电发展过程参数,非常有助于进一步提高雷电定位系统的性能与定位精度,有效促进电网主动防雷技术的发展。