We review achievements in the conservation of orchid diversity in China over the last 21 years.We provide updated information on orchid biodiversity and suggestions for orchid conservation in China.We outline national...We review achievements in the conservation of orchid diversity in China over the last 21 years.We provide updated information on orchid biodiversity and suggestions for orchid conservation in China.We outline national policies of biodiversity conservation,especially of orchid conservation,which provide general guidelines for orchid conservation in China.There are now approximately 1708 known species of Orchidaceae in 181 genera in China,including five new genera and 365 new species described over the last 21 years.The assessment of risk of extinction of all 1502 known native orchid species in China in 2013 indicated that 653 species were identified as threatened,132 species were treated as data-deficient,and four species endemic to China were classified as extinct.Approximately 1100 species(ca.65%)are protected in national nature reserves,and another~66 species in provincial nature reserves.About 800 native orchid species have living collections in major botanical gardens.The pollination biology of 74 native orchid species and the genetic diversity and spatial genetic structure of 29 orchid species have been investigated at a local scale and/or across species distributions.The mycorrhizal fungal community composition has been investigated in many genera,such as Bletilla,Coelogyne,Cymbidium,Cypripedium,and Dendrobium.Approximately 292 species will be included in the list of national key protected wild plants this year.Two major tasks for near future include in situ conservation and monitoring population dynamics of endangered species.展开更多
Real-time rapid detection of toxic gases at room temperature is particularly important for public health and environmental monitoring.Gas sensors based on conventional bulk materials often suffer from their poor surfa...Real-time rapid detection of toxic gases at room temperature is particularly important for public health and environmental monitoring.Gas sensors based on conventional bulk materials often suffer from their poor surface-sensitive sites,leading to a very low gas adsorption ability.Moreover,the charge transportation efficiency is usually inhibited by the low defect density of surface-sensitive area than that in the interior.In this work,a gas sensing structure model based on CuS quantum dots/Bi_(2)S_(3) nanosheets(CuS QDs/Bi_(2)S_(3) NSs)inspired by artificial neuron network is constructed.Simulation analysis by density functional calculation revealed that CuS QDs and Bi_(2)S_(3) NSs can be used as the main adsorption sites and charge transport pathways,respectively.Thus,the high-sensitivity sensing of NO_(2) can be realized by designing the artificial neuron-like sensor.The experimental results showed that the CuS QDs with a size of about 8 nm are highly adsorbable,which can enhance the NO_(2) sensitivity due to the rich sensitive sites and quantum size effect.The Bi_(2)S_(3) NSs can be used as a charge transfer network channel to achieve efficient charge collection and transmission.The neuron-like sensor that simulates biological smell shows a significantly enhanced response value(3.4),excellent responsiveness(18 s)and recovery rate(338 s),low theoretical detection limit of 78 ppb,and excellent selectivity for NO_(2).Furthermore,the developed wearable device can also realize the visual detection of NO2 through real-time signal changes.展开更多
Orchidaceae are among the largest plant families of angiosperms,with approximately 750 genera and 28,500 species around the world(Chase et al.,2015).The orchids have been considered a flagship of conservation biology(...Orchidaceae are among the largest plant families of angiosperms,with approximately 750 genera and 28,500 species around the world(Chase et al.,2015).The orchids have been considered a flagship of conservation biology(Zhang et al.,2015;Fay,2018;Liu et al.,2020),and all species have been included in CITES(Convention on International Trade in Endangered Species of Wild Fauna and Flora)Appendix I or II.Over the last two decades,there has been major progress in the classification of Orchidaceae.An updated classification with five subfamilies and 22 tribes(including three new tribes)has been proposed(Chase et al.,2015).Generic classifications in Collabieae,Epidendreae,Malaxideae,Neottieae,Orchideae,Podochileae and Vandeae have been discussed(Xiang et al.,2012a,2012b,2013,2014;Jin et al.,2014;Kocyan and Schuiteman,2014;Zhai et al.,2014;Tang et al.,2015;Raskoti et al.,2016;Simo-Droissart et al.,2018;Yan Peng et al.,2018).The generic delimitation of Orchidaceae in China has been discussed(Jin et al.,2015).展开更多
Inorganic solar cells, as durable photovoltaic devices for harvesting electric energy from sun light,have received tremendous attention due to the fear of exhausting the earth's energy resources and damaging the l...Inorganic solar cells, as durable photovoltaic devices for harvesting electric energy from sun light,have received tremendous attention due to the fear of exhausting the earth's energy resources and damaging the living environment due to greenhouse gases. Some recent developments in nanotechnology have opened up new avenues for more relevant inorganic solar cells produced by new photovoltaic conversion concepts and effective solar energy harvesting nanostructures. In this review, the multiple exciton generation effect solar cells, hot carrier solar cells, one dimensional material constructed asymmetrical schottky barrier arrays, noble nanoparticle induced plasmonic enhancement, and light trapping nanostructured semiconductor solar cells are highlighted.展开更多
Bioactive glass is well known for its ability of bone regeneration, and sol-gel bioactive glass has many advantages compared with melt-derived bioactive glass. 3-D scaffold prepared by the sol-gel method is a promisin...Bioactive glass is well known for its ability of bone regeneration, and sol-gel bioactive glass has many advantages compared with melt-derived bioactive glass. 3-D scaffold prepared by the sol-gel method is a promising substrate material for bone tissue engineering and large-scale bone repair. Porous sol-gel glass in the CaO-SiO2-P205 system with macropores larger than 100 μm was prepared by the addition of stearic acid as a pore former. The diameter of the pore created by the pore former varied from 100 to 300 μm. The formation of a hydroxyapatite layer on the glass was analyzed by studying the surface of the porous glass by scanning electron microscopy, energy dispersive spectroscopy, X-ray diffraction, and Raman spectra after they had been immersed in simulated body fluid （SBF） for some time, and the porous glass shows good bioactivity.展开更多
Camptothecin is one of the most commonly used anticancer drugs worldwide,yet the downstream biosynthetic route from strictosidine to camptothecin has remained unclear for more than half a century.Here,we searched for ...Camptothecin is one of the most commonly used anticancer drugs worldwide,yet the downstream biosynthetic route from strictosidine to camptothecin has remained unclear for more than half a century.Here,we searched for proteins involved in camptothecin biosynthesis from the camptothecin-producing plant Ophiorrhiza pumila by chemoproteomics and identified Op CYP716E111.展开更多
Plants produce diverse flavonoids for defense and stress resistance,most of which have health benefits and are widely used as food additives and medicines.Methylation of the free hydroxyl groups of flavonoids,catalyze...Plants produce diverse flavonoids for defense and stress resistance,most of which have health benefits and are widely used as food additives and medicines.Methylation of the free hydroxyl groups of flavonoids,catalyzed by S-adenosyl-l-methionine-dependent O-methyltransferases(OMTs),significantly affects their physicochemical properties and bioactivities.Soybeans(Glycine max)contain a rich pool of O-methylated flavonoids.However,the OMTs responsible for flavonoid methylation in G.max remain largely unknown.We screened the G.max genome and obtained 22 putative OMT-encoding genes that share a broad spectrum of amino acid identities(25–96%);among them,19 OMTs were successfully cloned and heterologously expressed in Escherichia coli.We used the flavonoids containing the free 3,5,7,8,3′,4′hydroxyl group,such as flavones(luteolin and 7,8-dihydroxyflavone),flavonols(kaempferol and quercetin),flavanones(naringenin and eriodictyol),isoflavonoids(daidzein and glycetein),and caffeic acid as substrates,and 15 OMTs were proven to catalyze at least one substrate.The methylation activities of these GmOMTs covered the 3,7,8,3′,4′-hydroxyl of flavonoids and 7,4′-hydroxyl of isoflavonoids.The systematic characterization of G.max flavonoid OMTs provides insights into the biosynthesis of methylated flavonoids in soybeans and OMT bioparts for the production of methylated flavonoids via synthetic biology.展开更多
Ginsenosides, the main pharmacologically active natural compounds in ginseng （Panax ginseng）, are mostly the glycosylated products of protopanaxadiol （PPD） and protopanaxatriol （PPT）. No uridine diphosphate glyc...Ginsenosides, the main pharmacologically active natural compounds in ginseng （Panax ginseng）, are mostly the glycosylated products of protopanaxadiol （PPD） and protopanaxatriol （PPT）. No uridine diphosphate glycosyltransferase （UGT）, which catalyzes PPT to produce PPT-type ginsenosides, has yet been reported. Here, we show that UGTPgl, which has been demonstrated to regio-specifically glycosylate the C20-OH of PPD, also specifically glycosylates the C20-OH of PPT to produce bioactive ginsenoside FI. We report the characterization of four novel UGT genes isolated from P. ginseng, sharing high deduced amino acid identity （〉84%） with UGTPgl. We demonstrate that UGTPgl00 specifically glycosylates the C6-OH of PPT to produce bioactive ginsenoside Rhl, and UGTPgl01 catalyzes PPT to produce F1, followed by the generation of ginsenoside Rgl from FI. However, UGTPgl02 and UGTPgl03 were found to have no detectable activity on PPT. Through structural modeling and site-directed mutagenesis, we identified several key amino acids of these UGTs that may play important roles in determining their activities and substrate regio-specificities. Moreover, we constructed yeast recombinants to biosynthesize F1 and Rhl by introducing the genetically engineered PPT-producing pathway and UGTPgl or UGTPgl00. Our study reveals the possible biosynthetic pathways of PPT-type ginsenosides in Panax plants, and provides a sound manufacturing approach for bioactive PPT-type ginsenosides in yeast via synthetic biology strategies.展开更多
Icaritin is a prenylflavonoid present in the Chinese herbal medicinal plant Epimedium spp. and is under investigation in a phase Ⅲ clinical trial for advanced hepatocellular carcinoma. Here, we report the biosynthesi...Icaritin is a prenylflavonoid present in the Chinese herbal medicinal plant Epimedium spp. and is under investigation in a phase Ⅲ clinical trial for advanced hepatocellular carcinoma. Here, we report the biosynthesis of icaritin from glucose by engineered microbial strains. We initially designed an artificial icaritin biosynthetic pathway by identifying a novel prenyltransferase from the Berberidaceae-family species Epimedium sagittatum(EsPT2) that catalyzes the C8 prenylation of kaempferol to yield 8-prenlykaempferol and a novel methyltransferase GmOMT2 from soybean to transfer a methyl to C4’-OH of 8-prenlykaempferol to produce icaritin. We next introduced 11 heterologous genes and modified 12 native yeast genes to construct a yeast strain capable of producing 8-prenylkaempferol with high efficiency. GmOMT2 was sensitive to low pH and lost its activity when expressed in the yeast cytoplasm. By relocating GmOMT2 into mitochondria(higher pH than cytoplasm) of the 8-prenylkaempferol–producing yeast strain or co-culturing the 8-prenylkaempferol–producing yeast with an Escherichia coli strain expressing GmOMT2, we obtained icaritin yields of 7.2 and 19.7 mg/L, respectively. Beyond the characterizing two previously unknown plant enzymes and conducting the first biosynthesis of icaritin from glucose, we describe two strategies of overcoming the widespread issue of incompatible pH conditions encountered in basic and applied bioproduction research. Our findings will facilitate industrial-scale production of icaritin and other prenylflavonoids.展开更多
Ginsenoside Compound K(CK)has been recognized as a major functional component that is absorbed into the systemic circulation after oral administration of ginseng.CK demonstrates diverse bioactivities.A phase I clinica...Ginsenoside Compound K(CK)has been recognized as a major functional component that is absorbed into the systemic circulation after oral administration of ginseng.CK demonstrates diverse bioactivities.A phase I clinical study indicated that CK was a potential candidate for arthritis therapy.However,a phase II clinical study was suspended because of the high cost associated with the present CK manufacturing approach,which is based on the traditional planting-extracting-biotransforming process.We previously elucidated the complete CK biosynthetic pathway and realized for the first time de novo biosynthesis of CK from glucose by engineered yeast.However,CK production was not sufficient for industrial application.Here,we systematically engineered Saccharomyces cerevisiae to achieve high titer production of CK from glucose using a previously constructed protopanaxadiol(PPD)-producing chassis,optimizing UGTPg1 expression,improving UDP-glucose biosynthesis,and tuning down UDP-glucose consumption.Our final engineered yeast strain produced CK with a titer of 5.74 g/L in fed-batch fermentation,which represents the highest CK production in microbes reported to date.Once scaled-up,this high titer de novo microbial biosynthesis platform will enable a robust and stable supply of CK,thus facilitating study and medical application of CK.展开更多
Claviceps purpurea produces many pharmacologically important ergot alkaloids(EAS),which are widely used to treat migraine and hypertension and to aid childbirth.Although an EAS biosynthetic cluster of C.purpurea has b...Claviceps purpurea produces many pharmacologically important ergot alkaloids(EAS),which are widely used to treat migraine and hypertension and to aid childbirth.Although an EAS biosynthetic cluster of C.purpurea has been discovered more than 20 years ago,the complete biosynthetic pathway of EAS has not been fully characterized until now.The main obstacle to elucidating this pathway and strain modification is the lack of efficient genome-editing tools for C.purpurea.The conventional gene manipulation method for C.purpurea relies on homologous recombination(HR),although the efficiency of HR in C.purpurea is very low(~1-5%).Consequently,the disruption of target genes is laborious and time-consuming.Although CRISPR/Cas9 genome-editing methods based on in vivo Cas9 expression and gRNA transcription have been reported recently,their gene-disruption efficiency is still very low.Here,we developed an efficient genome-editing system in C.purpurea based on in vitro assembled CRISPR/Cas9 gRNA ribonucleoprotein complexes.As proof of principle,three target genes were efficiently knocked out using this CRISPR/Cas9 ribonucleoprotein complex-mediated HR system,with editing efficiencies ranging from 50%to 100%.Inactivation of the three genes,which are closely related to uridine biosynthesis(ura5),hypha morphology(rac),and EAS production(easA),resulted in a uridine auxotrophic mutant,a mutant with a drastically different phenotype in axenic culture,and a mutant that did not produce EAS,respectively.Our ribonucleoprotein-based genome-editing system has a great advantage over conventional and in vivo CRISPR/Cas9 methods for genome editing in C.purpurea,which will greatly facilitate elucidation of the EAS biosynthetic pathway and other future basic and applied research on C.purpurea.展开更多
基金supported by Grants from National Forestry and Grassland Administration,China(No.2019073018,2019073019)National Natural Science Foundation of China(No.31870195,31670194)。
文摘We review achievements in the conservation of orchid diversity in China over the last 21 years.We provide updated information on orchid biodiversity and suggestions for orchid conservation in China.We outline national policies of biodiversity conservation,especially of orchid conservation,which provide general guidelines for orchid conservation in China.There are now approximately 1708 known species of Orchidaceae in 181 genera in China,including five new genera and 365 new species described over the last 21 years.The assessment of risk of extinction of all 1502 known native orchid species in China in 2013 indicated that 653 species were identified as threatened,132 species were treated as data-deficient,and four species endemic to China were classified as extinct.Approximately 1100 species(ca.65%)are protected in national nature reserves,and another~66 species in provincial nature reserves.About 800 native orchid species have living collections in major botanical gardens.The pollination biology of 74 native orchid species and the genetic diversity and spatial genetic structure of 29 orchid species have been investigated at a local scale and/or across species distributions.The mycorrhizal fungal community composition has been investigated in many genera,such as Bletilla,Coelogyne,Cymbidium,Cypripedium,and Dendrobium.Approximately 292 species will be included in the list of national key protected wild plants this year.Two major tasks for near future include in situ conservation and monitoring population dynamics of endangered species.
基金supported by the National Natural Science Foundation of China(61971284)the Oceanic Interdisciplinary Program of Shanghai Jiao Tong University(SL2020ZD203 and SL2020MS031)+2 种基金Scientific Research Fund of Second Institute of Oceanography,Ministry of Natural Resources of P.R.China(SL2003)Shanghai Sailing Program(21YF1421400)Startup Fund for Youngman Research at Shanghai Jiao Tong University.
文摘Real-time rapid detection of toxic gases at room temperature is particularly important for public health and environmental monitoring.Gas sensors based on conventional bulk materials often suffer from their poor surface-sensitive sites,leading to a very low gas adsorption ability.Moreover,the charge transportation efficiency is usually inhibited by the low defect density of surface-sensitive area than that in the interior.In this work,a gas sensing structure model based on CuS quantum dots/Bi_(2)S_(3) nanosheets(CuS QDs/Bi_(2)S_(3) NSs)inspired by artificial neuron network is constructed.Simulation analysis by density functional calculation revealed that CuS QDs and Bi_(2)S_(3) NSs can be used as the main adsorption sites and charge transport pathways,respectively.Thus,the high-sensitivity sensing of NO_(2) can be realized by designing the artificial neuron-like sensor.The experimental results showed that the CuS QDs with a size of about 8 nm are highly adsorbable,which can enhance the NO_(2) sensitivity due to the rich sensitive sites and quantum size effect.The Bi_(2)S_(3) NSs can be used as a charge transfer network channel to achieve efficient charge collection and transmission.The neuron-like sensor that simulates biological smell shows a significantly enhanced response value(3.4),excellent responsiveness(18 s)and recovery rate(338 s),low theoretical detection limit of 78 ppb,and excellent selectivity for NO_(2).Furthermore,the developed wearable device can also realize the visual detection of NO2 through real-time signal changes.
文摘Orchidaceae are among the largest plant families of angiosperms,with approximately 750 genera and 28,500 species around the world(Chase et al.,2015).The orchids have been considered a flagship of conservation biology(Zhang et al.,2015;Fay,2018;Liu et al.,2020),and all species have been included in CITES(Convention on International Trade in Endangered Species of Wild Fauna and Flora)Appendix I or II.Over the last two decades,there has been major progress in the classification of Orchidaceae.An updated classification with five subfamilies and 22 tribes(including three new tribes)has been proposed(Chase et al.,2015).Generic classifications in Collabieae,Epidendreae,Malaxideae,Neottieae,Orchideae,Podochileae and Vandeae have been discussed(Xiang et al.,2012a,2012b,2013,2014;Jin et al.,2014;Kocyan and Schuiteman,2014;Zhai et al.,2014;Tang et al.,2015;Raskoti et al.,2016;Simo-Droissart et al.,2018;Yan Peng et al.,2018).The generic delimitation of Orchidaceae in China has been discussed(Jin et al.,2015).
基金financial support from National High-Tech R & D Program of China (863, No. 2011AA050504)Natural Science Foundation of shanghai (No. 10ZR1416300), Shanghai Pujiang Program (No. 11PJD011)+2 种基金the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learningthe Foundation for SMC Excellent Young Teacher, "PCSIRT"the Analytical and Testing Center in Shanghai Jiaotong University
文摘Inorganic solar cells, as durable photovoltaic devices for harvesting electric energy from sun light,have received tremendous attention due to the fear of exhausting the earth's energy resources and damaging the living environment due to greenhouse gases. Some recent developments in nanotechnology have opened up new avenues for more relevant inorganic solar cells produced by new photovoltaic conversion concepts and effective solar energy harvesting nanostructures. In this review, the multiple exciton generation effect solar cells, hot carrier solar cells, one dimensional material constructed asymmetrical schottky barrier arrays, noble nanoparticle induced plasmonic enhancement, and light trapping nanostructured semiconductor solar cells are highlighted.
基金the National Natural Science Foundation of China(No.50174059)
文摘Bioactive glass is well known for its ability of bone regeneration, and sol-gel bioactive glass has many advantages compared with melt-derived bioactive glass. 3-D scaffold prepared by the sol-gel method is a promising substrate material for bone tissue engineering and large-scale bone repair. Porous sol-gel glass in the CaO-SiO2-P205 system with macropores larger than 100 μm was prepared by the addition of stearic acid as a pore former. The diameter of the pore created by the pore former varied from 100 to 300 μm. The formation of a hydroxyapatite layer on the glass was analyzed by studying the surface of the porous glass by scanning electron microscopy, energy dispersive spectroscopy, X-ray diffraction, and Raman spectra after they had been immersed in simulated body fluid （SBF） for some time, and the porous glass shows good bioactivity.
基金supported financially by the National Key Research and Development Program of China(Grant No.2018YFA0900700)the National Natural Science Foundation of China(Nos.31901021+1 种基金31921006)the Strategic Biological Resources Service Network Plan of the Chinese Academy of Sciences(Grant No.KFJ-BRP009)。
文摘Camptothecin is one of the most commonly used anticancer drugs worldwide,yet the downstream biosynthetic route from strictosidine to camptothecin has remained unclear for more than half a century.Here,we searched for proteins involved in camptothecin biosynthesis from the camptothecin-producing plant Ophiorrhiza pumila by chemoproteomics and identified Op CYP716E111.
基金This work was financially supported by the National Key Research and Development Program of China(Grant No.2018YFA0900700)the National Natural Science Foundation of China(Nos.31901021+1 种基金31921006)the Strategic Biological Resources Service Network Plan of the Chinese Academy of Sciences(Grant No.KFJ-BRP-009).
文摘Plants produce diverse flavonoids for defense and stress resistance,most of which have health benefits and are widely used as food additives and medicines.Methylation of the free hydroxyl groups of flavonoids,catalyzed by S-adenosyl-l-methionine-dependent O-methyltransferases(OMTs),significantly affects their physicochemical properties and bioactivities.Soybeans(Glycine max)contain a rich pool of O-methylated flavonoids.However,the OMTs responsible for flavonoid methylation in G.max remain largely unknown.We screened the G.max genome and obtained 22 putative OMT-encoding genes that share a broad spectrum of amino acid identities(25–96%);among them,19 OMTs were successfully cloned and heterologously expressed in Escherichia coli.We used the flavonoids containing the free 3,5,7,8,3′,4′hydroxyl group,such as flavones(luteolin and 7,8-dihydroxyflavone),flavonols(kaempferol and quercetin),flavanones(naringenin and eriodictyol),isoflavonoids(daidzein and glycetein),and caffeic acid as substrates,and 15 OMTs were proven to catalyze at least one substrate.The methylation activities of these GmOMTs covered the 3,7,8,3′,4′-hydroxyl of flavonoids and 7,4′-hydroxyl of isoflavonoids.The systematic characterization of G.max flavonoid OMTs provides insights into the biosynthesis of methylated flavonoids in soybeans and OMT bioparts for the production of methylated flavonoids via synthetic biology.
文摘Ginsenosides, the main pharmacologically active natural compounds in ginseng （Panax ginseng）, are mostly the glycosylated products of protopanaxadiol （PPD） and protopanaxatriol （PPT）. No uridine diphosphate glycosyltransferase （UGT）, which catalyzes PPT to produce PPT-type ginsenosides, has yet been reported. Here, we show that UGTPgl, which has been demonstrated to regio-specifically glycosylate the C20-OH of PPD, also specifically glycosylates the C20-OH of PPT to produce bioactive ginsenoside FI. We report the characterization of four novel UGT genes isolated from P. ginseng, sharing high deduced amino acid identity （〉84%） with UGTPgl. We demonstrate that UGTPgl00 specifically glycosylates the C6-OH of PPT to produce bioactive ginsenoside Rhl, and UGTPgl01 catalyzes PPT to produce F1, followed by the generation of ginsenoside Rgl from FI. However, UGTPgl02 and UGTPgl03 were found to have no detectable activity on PPT. Through structural modeling and site-directed mutagenesis, we identified several key amino acids of these UGTs that may play important roles in determining their activities and substrate regio-specificities. Moreover, we constructed yeast recombinants to biosynthesize F1 and Rhl by introducing the genetically engineered PPT-producing pathway and UGTPgl or UGTPgl00. Our study reveals the possible biosynthetic pathways of PPT-type ginsenosides in Panax plants, and provides a sound manufacturing approach for bioactive PPT-type ginsenosides in yeast via synthetic biology strategies.
基金supported by the National Key Research and Development Program of China (2018YFA0900700)the National Natural Science Foundation of China (31901021 and 31921006)+2 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB27020206)the International Partnership Program of Chinese Academy of Sciences (153D31KYSB20170121)the Strategic Biological Resources Service Network Plan of the Chinese Academy of Sciences (KFJ-BRP-009)。
文摘Icaritin is a prenylflavonoid present in the Chinese herbal medicinal plant Epimedium spp. and is under investigation in a phase Ⅲ clinical trial for advanced hepatocellular carcinoma. Here, we report the biosynthesis of icaritin from glucose by engineered microbial strains. We initially designed an artificial icaritin biosynthetic pathway by identifying a novel prenyltransferase from the Berberidaceae-family species Epimedium sagittatum(EsPT2) that catalyzes the C8 prenylation of kaempferol to yield 8-prenlykaempferol and a novel methyltransferase GmOMT2 from soybean to transfer a methyl to C4’-OH of 8-prenlykaempferol to produce icaritin. We next introduced 11 heterologous genes and modified 12 native yeast genes to construct a yeast strain capable of producing 8-prenylkaempferol with high efficiency. GmOMT2 was sensitive to low pH and lost its activity when expressed in the yeast cytoplasm. By relocating GmOMT2 into mitochondria(higher pH than cytoplasm) of the 8-prenylkaempferol–producing yeast strain or co-culturing the 8-prenylkaempferol–producing yeast with an Escherichia coli strain expressing GmOMT2, we obtained icaritin yields of 7.2 and 19.7 mg/L, respectively. Beyond the characterizing two previously unknown plant enzymes and conducting the first biosynthesis of icaritin from glucose, we describe two strategies of overcoming the widespread issue of incompatible pH conditions encountered in basic and applied bioproduction research. Our findings will facilitate industrial-scale production of icaritin and other prenylflavonoids.
基金supported by the National Key Research and Development Program of China(Grant No.2018YFA0900700)the Drug Innovation Major Project(2018ZX09711001-006-002)+3 种基金the National Natural Science Foundation of China(Nos.31901021,31921006,and 32071425)the Strategic Priority Research Program of the Chinese Academy of Sciences(Grant No.XDB27020206)the Strategic Biological Resources Service Network Plan of the Chinese Academy of Sciences(Grant No.KFJ-BRP-009)the National Key Research and Development Program of Yunnan Province(2019ZF011-1).
文摘Ginsenoside Compound K(CK)has been recognized as a major functional component that is absorbed into the systemic circulation after oral administration of ginseng.CK demonstrates diverse bioactivities.A phase I clinical study indicated that CK was a potential candidate for arthritis therapy.However,a phase II clinical study was suspended because of the high cost associated with the present CK manufacturing approach,which is based on the traditional planting-extracting-biotransforming process.We previously elucidated the complete CK biosynthetic pathway and realized for the first time de novo biosynthesis of CK from glucose by engineered yeast.However,CK production was not sufficient for industrial application.Here,we systematically engineered Saccharomyces cerevisiae to achieve high titer production of CK from glucose using a previously constructed protopanaxadiol(PPD)-producing chassis,optimizing UGTPg1 expression,improving UDP-glucose biosynthesis,and tuning down UDP-glucose consumption.Our final engineered yeast strain produced CK with a titer of 5.74 g/L in fed-batch fermentation,which represents the highest CK production in microbes reported to date.Once scaled-up,this high titer de novo microbial biosynthesis platform will enable a robust and stable supply of CK,thus facilitating study and medical application of CK.
基金This work was financially supported by the National Key Research and Development Program of China(Grant No.2018YFA0900500)the National Natural Science Foundation of China(Nos.31921006,31470201,and,31741003)the Strategic Biological Resources Service Network Plan of the Chinese Academy of Sciences(Grant No.KFJ-BRP-009).
文摘Claviceps purpurea produces many pharmacologically important ergot alkaloids(EAS),which are widely used to treat migraine and hypertension and to aid childbirth.Although an EAS biosynthetic cluster of C.purpurea has been discovered more than 20 years ago,the complete biosynthetic pathway of EAS has not been fully characterized until now.The main obstacle to elucidating this pathway and strain modification is the lack of efficient genome-editing tools for C.purpurea.The conventional gene manipulation method for C.purpurea relies on homologous recombination(HR),although the efficiency of HR in C.purpurea is very low(~1-5%).Consequently,the disruption of target genes is laborious and time-consuming.Although CRISPR/Cas9 genome-editing methods based on in vivo Cas9 expression and gRNA transcription have been reported recently,their gene-disruption efficiency is still very low.Here,we developed an efficient genome-editing system in C.purpurea based on in vitro assembled CRISPR/Cas9 gRNA ribonucleoprotein complexes.As proof of principle,three target genes were efficiently knocked out using this CRISPR/Cas9 ribonucleoprotein complex-mediated HR system,with editing efficiencies ranging from 50%to 100%.Inactivation of the three genes,which are closely related to uridine biosynthesis(ura5),hypha morphology(rac),and EAS production(easA),resulted in a uridine auxotrophic mutant,a mutant with a drastically different phenotype in axenic culture,and a mutant that did not produce EAS,respectively.Our ribonucleoprotein-based genome-editing system has a great advantage over conventional and in vivo CRISPR/Cas9 methods for genome editing in C.purpurea,which will greatly facilitate elucidation of the EAS biosynthetic pathway and other future basic and applied research on C.purpurea.
