Camptothecin,a plant-derived pentacyclic pyrroloquinoline alkaloid,and its derivatives like topotecan and irinotecan have been used as clinical anticancer agents for decades.However,the complete biosynthetic pathway o...Camptothecin,a plant-derived pentacyclic pyrroloquinoline alkaloid,and its derivatives like topotecan and irinotecan have been used as clinical anticancer agents for decades.However,the complete biosynthetic pathway of camptothecin still remains unelucidated due to the unknown complex formation processes and corresponding enzymes for the downstream biosynthetic pathway including the committed hydrolysis of glycosides.Herein,a novel glycoside hydrolase(CaGH1)responsible for the deglycosylation of biosynthetic glycoside intermediates including both quinoline-type alkaloids pumiloside(1),(3S)-deoxypumiloside(2)and indole-type alkaloid strictosamide(3)has been functionally identified.Moreover,CaGH1 exhibits the highly strict stereoselectivity towards the substrates with 3S configuration.Furthermore,a combined strategy for the discovery of the unknown biosynthetic enzyme by employing activity-guided enzyme verification,transcriptome-based gene mining,biochemical assay in vitro,and structurally characterizing the unstable enzymatic products by derivatization,is reported.These findings not only provide a better understanding of the deglycosylation in camptothecin biosynthesis,also lay the foundation for the complete elucidation of camptothecin biosynthetic pathway and biological production of camptothecin.展开更多
Bibenzyls,a kind of important plant polyphenols,have attracted growing attention for their broad and remarkable pharmacological activities.However,due to the low abundance in nature,uncontrollable and environmentally ...Bibenzyls,a kind of important plant polyphenols,have attracted growing attention for their broad and remarkable pharmacological activities.However,due to the low abundance in nature,uncontrollable and environmentally unfriendly chemical synthesis processes,these compounds are not readily accessible.Herein,one high-yield bibenzyl backbone-producing Escherichia coli strain was constructed by using a highly active and substrate-promiscuous bibenzyl synthase identified from Dendrobium officinale in combination with starter and extender biosynthetic enzymes.Three types of efficiently postmodifying modular strains were engineered by employing methyltransferases,prenyltransferase,and glycosyltransferase with high activity and substrate tolerance together with their corresponding donor biosynthetic modules.Structurally different bibenzyl derivatives were tandemly and/or divergently synthesized by co-culture engineering in various combination modes.Especially,a prenylated bibenzyl derivative(12)was found to be an antioxidant that exhibited potent neuroprotective activity in the cellular and rat models of ischemia stroke.RNA-seq,quantitative RT-PCR,and Western-blot analysis demonstrated that 12 could up-regulate the expression level of an apoptosis-inducing factor,mitochondria associated 3(Aifm3),suggesting that Aifm3 might be a new target in ischemic stroke therapy.This study provides a flexible plug-and-play strategy for the easy-to-implement synthesis of structurally diverse bibenzyls through a modular co-culture engineering pipeline for drug discovery.展开更多
基金financially supported by the National Key Research and Development Program of China(No.2020YFA0908000)CAMS Innovation Fund for Medical Sciences(No.CIFMS-2023I2M-2–006)。
文摘Camptothecin,a plant-derived pentacyclic pyrroloquinoline alkaloid,and its derivatives like topotecan and irinotecan have been used as clinical anticancer agents for decades.However,the complete biosynthetic pathway of camptothecin still remains unelucidated due to the unknown complex formation processes and corresponding enzymes for the downstream biosynthetic pathway including the committed hydrolysis of glycosides.Herein,a novel glycoside hydrolase(CaGH1)responsible for the deglycosylation of biosynthetic glycoside intermediates including both quinoline-type alkaloids pumiloside(1),(3S)-deoxypumiloside(2)and indole-type alkaloid strictosamide(3)has been functionally identified.Moreover,CaGH1 exhibits the highly strict stereoselectivity towards the substrates with 3S configuration.Furthermore,a combined strategy for the discovery of the unknown biosynthetic enzyme by employing activity-guided enzyme verification,transcriptome-based gene mining,biochemical assay in vitro,and structurally characterizing the unstable enzymatic products by derivatization,is reported.These findings not only provide a better understanding of the deglycosylation in camptothecin biosynthesis,also lay the foundation for the complete elucidation of camptothecin biosynthetic pathway and biological production of camptothecin.
基金supported by the National Key Research and Development Program of China(2020YFA0908000)CAMS Innovation fund for Medical Sciences(CIFMS-2021-I2M1-028 and CIFMS-2021-I2M-1-029,China)Beijing Key Laboratory of non-Clinical Drug Metabolism and PK/PD Study(Z141102004414062,China)。
文摘Bibenzyls,a kind of important plant polyphenols,have attracted growing attention for their broad and remarkable pharmacological activities.However,due to the low abundance in nature,uncontrollable and environmentally unfriendly chemical synthesis processes,these compounds are not readily accessible.Herein,one high-yield bibenzyl backbone-producing Escherichia coli strain was constructed by using a highly active and substrate-promiscuous bibenzyl synthase identified from Dendrobium officinale in combination with starter and extender biosynthetic enzymes.Three types of efficiently postmodifying modular strains were engineered by employing methyltransferases,prenyltransferase,and glycosyltransferase with high activity and substrate tolerance together with their corresponding donor biosynthetic modules.Structurally different bibenzyl derivatives were tandemly and/or divergently synthesized by co-culture engineering in various combination modes.Especially,a prenylated bibenzyl derivative(12)was found to be an antioxidant that exhibited potent neuroprotective activity in the cellular and rat models of ischemia stroke.RNA-seq,quantitative RT-PCR,and Western-blot analysis demonstrated that 12 could up-regulate the expression level of an apoptosis-inducing factor,mitochondria associated 3(Aifm3),suggesting that Aifm3 might be a new target in ischemic stroke therapy.This study provides a flexible plug-and-play strategy for the easy-to-implement synthesis of structurally diverse bibenzyls through a modular co-culture engineering pipeline for drug discovery.
