Phenolic acids are the main bioactive compounds in Salvia miltiorrhiza,which can be increased by salicylic acid(SA)elicitation.However,the specific molecular mechanism remains unclear.The nonexpresser of PR genes 1(NP...Phenolic acids are the main bioactive compounds in Salvia miltiorrhiza,which can be increased by salicylic acid(SA)elicitation.However,the specific molecular mechanism remains unclear.The nonexpresser of PR genes 1(NPR1)and its family members are essential components of the SA signaling pathway.Here,we report an NPR protein,SmNPR4,that showed strong expression in hairy root after SA treatment,acting as a negative moderator of SA-induced phenolic acid biosynthesis in S.miltiorrhiza(S.miltiorrhiza).Moreover,a basic leucine zipper family transcription factor SmTGA5 was identified and was found to interact with SmNPR4.SmTGA5 activates the expression of phenolic acid biosynthesis gene SmTAT1 through binding to the as-1 element.Finally,a series of biochemical assays and dual gene overexpression analysis demonstrated that the SmNPR4 significantly inhibited the function of SmTGA5,and SA can alleviate the inhibitory effect of SmNPR4 on SmTGA5.Overall,our results reveal the molecular mechanism of salicylic acid regulating phenolic acid biosynthesis in S.miltiorrhiza and provide new insights for SA signaling to regulate secondary metabolic biosynthesis.展开更多
Objective Plant-derived terpenoid ginkgolides exhibit significant pharmacological efficacy,however,their extraction remains costly.Given that levopimaradiene is a key biosynthetic precursor to ginkgolides,its high-yie...Objective Plant-derived terpenoid ginkgolides exhibit significant pharmacological efficacy,however,their extraction remains costly.Given that levopimaradiene is a key biosynthetic precursor to ginkgolides,its high-yield production via heterologous gene expression therefore establishes a critical foundation for scaling up their manufacture.This study primarily aims to enhance the yield of levopimaradiene in Escherichia coli(LB medium)by remodeling the unnatural isopentenol utilization pathway(IUP).Methods Plasmid construction was driven by the mechanism of homologous recombination,which utilizes recombinase to facilitate the ligation process.The expression of kinase was carried out using type 7(T7)promoter and isopropylβ-D-1-thiogalactopyranoside(IPTG)as the inducer.The quantification of levopimaradiene produced by E.coli was determined by comparison with a standard curve that we constructed.Results Combining the selection of kinases,ribosome-binding site(RBS)screening,protein directed evolution and optimization of fermentation parameters,the production of levopimaradiene in E.coli was ultimately enhanced to 2691.3 mg/L,surpassing the highest reported titers of levopimaradiene with 6-fold in E.coli to date.Additionally,the engineered E.coli was designed to collaborate with farnesyl pyrophosphate(FPP)synthase and geranylfarnesyl pyrophosphate(GFPP)synthase to efficiently produce FPP and GFPP for sesquiterpene and sesterterpene synthesis.Conclusion Our work showcases a combinatorial engineering strategy that employs an IUP-enhanced E.coli chassis for the microbial production of levopimaradiene,as well as other natural terpenoids.展开更多
In this paper,we have developed a decarboxylative amination of carboxylic acids with nitroarenes for the synthesis of secondary amines.The protocol is performed at mild conditions without the use of noble metals as ca...In this paper,we have developed a decarboxylative amination of carboxylic acids with nitroarenes for the synthesis of secondary amines.The protocol is performed at mild conditions without the use of noble metals as catalysts.A wide range of structurally diverse secondary amines could be obtained in good yields(up to 94%)with good functional group tolerance.This transformation shows good to excellent selectivity,avoiding the generation of over alkylated byproducts.展开更多
基金The researchwas financially supported by the National Natural Science Foundation of China(31670301,32270278)the Natural Science Foundation of Shaanxi Province(2022JM-099)the Innovation Training Program for College Students(202210712222).
文摘Phenolic acids are the main bioactive compounds in Salvia miltiorrhiza,which can be increased by salicylic acid(SA)elicitation.However,the specific molecular mechanism remains unclear.The nonexpresser of PR genes 1(NPR1)and its family members are essential components of the SA signaling pathway.Here,we report an NPR protein,SmNPR4,that showed strong expression in hairy root after SA treatment,acting as a negative moderator of SA-induced phenolic acid biosynthesis in S.miltiorrhiza(S.miltiorrhiza).Moreover,a basic leucine zipper family transcription factor SmTGA5 was identified and was found to interact with SmNPR4.SmTGA5 activates the expression of phenolic acid biosynthesis gene SmTAT1 through binding to the as-1 element.Finally,a series of biochemical assays and dual gene overexpression analysis demonstrated that the SmNPR4 significantly inhibited the function of SmTGA5,and SA can alleviate the inhibitory effect of SmNPR4 on SmTGA5.Overall,our results reveal the molecular mechanism of salicylic acid regulating phenolic acid biosynthesis in S.miltiorrhiza and provide new insights for SA signaling to regulate secondary metabolic biosynthesis.
基金co-financed by the Young Scientists Fund of the National Natural Science Foundation of China(NSFC)(No.82003608)the Key Project of NSFC(No.81991524)+3 种基金the Innovation Projects of State Key Laboratory on Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture(No.NZYSKL240109)the Interdisciplinary Project of Nanjing University of Chinese Medicine(No.ZYXJC2024-003 and No.JC202402)the Youth Project of Jiangsu Commission of Health(No.QN202405)the Jiangsu Annual Basic Science Institutions(No.25KJB350006).
文摘Objective Plant-derived terpenoid ginkgolides exhibit significant pharmacological efficacy,however,their extraction remains costly.Given that levopimaradiene is a key biosynthetic precursor to ginkgolides,its high-yield production via heterologous gene expression therefore establishes a critical foundation for scaling up their manufacture.This study primarily aims to enhance the yield of levopimaradiene in Escherichia coli(LB medium)by remodeling the unnatural isopentenol utilization pathway(IUP).Methods Plasmid construction was driven by the mechanism of homologous recombination,which utilizes recombinase to facilitate the ligation process.The expression of kinase was carried out using type 7(T7)promoter and isopropylβ-D-1-thiogalactopyranoside(IPTG)as the inducer.The quantification of levopimaradiene produced by E.coli was determined by comparison with a standard curve that we constructed.Results Combining the selection of kinases,ribosome-binding site(RBS)screening,protein directed evolution and optimization of fermentation parameters,the production of levopimaradiene in E.coli was ultimately enhanced to 2691.3 mg/L,surpassing the highest reported titers of levopimaradiene with 6-fold in E.coli to date.Additionally,the engineered E.coli was designed to collaborate with farnesyl pyrophosphate(FPP)synthase and geranylfarnesyl pyrophosphate(GFPP)synthase to efficiently produce FPP and GFPP for sesquiterpene and sesterterpene synthesis.Conclusion Our work showcases a combinatorial engineering strategy that employs an IUP-enhanced E.coli chassis for the microbial production of levopimaradiene,as well as other natural terpenoids.
基金National Key Research and Development Program of China(2022YFA1503200,2021YFC2101901)the National Natural Science Foundation of China(22122103,22101130,22001117,21971108,22271144)Fundamental Research Funds for the Central Universities(020514380304,020514380252,020514380272)for financial support.
文摘In this paper,we have developed a decarboxylative amination of carboxylic acids with nitroarenes for the synthesis of secondary amines.The protocol is performed at mild conditions without the use of noble metals as catalysts.A wide range of structurally diverse secondary amines could be obtained in good yields(up to 94%)with good functional group tolerance.This transformation shows good to excellent selectivity,avoiding the generation of over alkylated byproducts.
