Ergot alkaloids (EAs) are secondary metabolites produced by ergot fungi (e.g., Claviceps purpurea), which are parasites of Gramineae grasses. EAs and their analogs are used to treat migraine, postpartum uterine bleedi...Ergot alkaloids (EAs) are secondary metabolites produced by ergot fungi (e.g., Claviceps purpurea), which are parasites of Gramineae grasses. EAs and their analogs are used to treat migraine, postpartum uterine bleeding, and Parkinson's syndrome. Recent studies have reported additional new bioactive activities of EAs and their analogs, making them essential compounds for drug development, drug repositioning, and clinical applications. EAs are produced industrially by field cultivation of ergot or liquid fermentation in the mycelial phase, but there are few published studies of the production of EAs by cereal culture and thus this approach is poorly understood. This study searched for Claviceps strains that produce EAs cultured artificially in the mycelial phase, then the selected strains were cultured on cereal media (white rice, brown rice, and rye) to examine their ability to produce EAs on each medium. C. purpurea var. agropyri produced the Clavine-type EAs pyroclavine (1), festuclavine (2), and agroclavine (3) in the mycelial phase. When cultured with white rice, brown rice, or rye, C. purpurea var. agropyri produced 1 - 3 on all cereal media. The total amount of 1 - 3 in each cereal medium (150 g of cereal per Roux flask) was 2220.5 ± 564.1 μg for white rice, 920.0 ± 463.6 μg for brown rice, and 595.4 ± 52.1 μg for rye. The white rice medium supported the highest production of 1 - 3, with the total amount of EAs (150 g of white rice per Roux flask) being about 34 times higher than that in the T25 liquid medium (190 mL per 1 L Erlenmeyer flask) (equivalent amount per flask).展开更多
Endophyte infected tall fescue (E+) is the base diet for nearly all beef cattle in the southern USA. It has been linked to a variety of toxicological conditions due to the presence of large numbers of ergot alkaloids....Endophyte infected tall fescue (E+) is the base diet for nearly all beef cattle in the southern USA. It has been linked to a variety of toxicological conditions due to the presence of large numbers of ergot alkaloids. This study was designed to investigate the effects of E+ seed extract and selected ergot alkaloids on the detoxification pathway by cytochrome P450 (CYP3A4) enzyme system. Tests were performed using the P450- Glo CYP3A4 enzyme activity kit (Promega, WI), according to the manufacturer’s manual. Luminescence was measured using a single tube TD20/20 luminometer. Endophyte infected tall fescue seed was extracted with 50/50 methanol/25 mM ammonium carbonate, cleaned and concentrated on Strata-X reversed phase column (Phenomenex). The extracts were evaluated on an HPLC, and then tested using a serial dilution method. Commercially available ergonovine (EN), ergocorine (ER), bromocryptine (BC) and ergocryptine (EC) were tested individually using 0 to 44 nM concentrations. Seed extract of E+ produced a significant (P P < 0.05) dose dependent manner with EC being most potent, followed by ER, BC, and then EN (70%, 40%, 30% and 10% at 44 nM concentration). The similarity of the inhibition curves of seed extract to that of the commercially available ergot alkaloids suggests a related mode of action and that the use of such ergot alkaloids and CYP3A4 assay is a good model to study the toxicity of tall fescue. Furthermore, it provides the foundation to identify the individual toxic components of purified endophyte infected tall fescue extract.展开更多
Microbial cell factories(MCFs)and cell-free systems(CFSs)are generally considered as two unrelated approaches for the biosynthesis of biomolecules.In the current study,two systems were combined together for the overpr...Microbial cell factories(MCFs)and cell-free systems(CFSs)are generally considered as two unrelated approaches for the biosynthesis of biomolecules.In the current study,two systems were combined together for the overproduction of agroclavine(AC),a structurally complex ergot alkaloid.The whole biosynthetic pathway for AC was split into the early pathway and the late pathway at the point of the FAD-linked oxidoreductase EasE,which was reconstituted in an MCF(Aspergillus nidulans)and a four-enzyme CFS,respectively.The final titer of AC of this combined system is 1209 mg/L,which is the highest one that has been reported so far,to the best of our knowledge.The development of such a combined route could potentially avoid the limitations of both MCF and CFS systems,and boost the production of complex ergot alkaloids with polycyclic ring systems.展开更多
Mycotoxins are toxic secondary metabolites produced by fungus kingdom. Fungi (molds) under aerobic and optimum conditions of humidity and temperature consume nutrients for proliferation and mycotoxin production (secre...Mycotoxins are toxic secondary metabolites produced by fungus kingdom. Fungi (molds) under aerobic and optimum conditions of humidity and temperature consume nutrients for proliferation and mycotoxin production (secretion). There are seven major groups of mycotoxins produced by different species of toxigenic fungal genus. Mycotoxins production from these toxigenic fungi depends on the surrounding intrinsic and extrinsic environments. These seven mycotoxins groups that contaminate grains, foods and animal feeds are: Aflatoxins, Trichothecene, Ochratoxins, Ergot alkaloid (Ergolin), Fumonisins, Patulin, and Zearalenone. These mycotoxins are capable of causing health hazards and death for both human and animals by effecting mammalian cells, causing a number of problems in normal cell function and a wide variety of clinical symptoms of diseases. These mycotoxins are varied in their toxicity depending on the infected host (human or animal) and the host susceptibility (immunity). The major concern of food and feed industries is the contamination of food products and animal feed supplies by these mycotoxins. Worldwide Health Organization (WHO), and Food and Agriculture Organization (FAO) are responsible to regulate the acceptable (tolerable) levels of these mycotoxins in grains, food and feed supplies to ensure the safety and health for both human and animals. Understanding fungal ecology and factors that affect fungal proliferation and mycotoxins production by these toxigenic fungi in agriculture crops as raw materials for both human food and animal feed products, plus understanding the chemistry and property of these mycotoxins, methods of detection, illness symptoms, and comply with regulatory guidance established by World Health Organization (WHO)/Food and Agriculture Organization (FAO) are key factors to prevent or minimize foods/feeds contamination and the toxicity of these mycotoxins for both human and animals health, plus reducing economical loss.展开更多
The present study was designed to isolate and purify chemical constituents from solid culture of endophyte Aspergillus terreus LQ, using silica gel column chromatography, gel filtration with Sephadex LH-20, and HPLC. ...The present study was designed to isolate and purify chemical constituents from solid culture of endophyte Aspergillus terreus LQ, using silica gel column chromatography, gel filtration with Sephadex LH-20, and HPLC. Fumigaclavine I(1), a new alkaloid, was obtained, along with seven known compounds, including fumigaclavine C(2), rhizoctonic acid(3), monomethylsulochrin(4), chaetominine(5), spirotryprostatin A(6), asperfumoid(7), and lumichrome(8). The structure of compound 1 was elucidated by various spectroscopic analyses(UV, MS, 1D and 2D NMR). The in vitro cytotoxicity of compound 1 was determined by MTT assay in human hepatocarcinoma cell line SMMC-7721, showing weaker cytotoxicity, compared with cisplatin, a clinically used cancer chemotherapeutic agent.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
In the present study, we developed a novel approach for the synthesis of the tetracyclic core of fumigaclavines A–D. A palladium-catalyzed intramolecular Larock indole synthesis was utilized to assemble the B/C rings...In the present study, we developed a novel approach for the synthesis of the tetracyclic core of fumigaclavines A–D. A palladium-catalyzed intramolecular Larock indole synthesis was utilized to assemble the B/C rings of the tetracyclic core in one step. Although all attempts to convert compound 18 to fumigaclavine B failed, this study provided useful information for the total synthesis of fumigaclavines A–D.展开更多
文摘Ergot alkaloids (EAs) are secondary metabolites produced by ergot fungi (e.g., Claviceps purpurea), which are parasites of Gramineae grasses. EAs and their analogs are used to treat migraine, postpartum uterine bleeding, and Parkinson's syndrome. Recent studies have reported additional new bioactive activities of EAs and their analogs, making them essential compounds for drug development, drug repositioning, and clinical applications. EAs are produced industrially by field cultivation of ergot or liquid fermentation in the mycelial phase, but there are few published studies of the production of EAs by cereal culture and thus this approach is poorly understood. This study searched for Claviceps strains that produce EAs cultured artificially in the mycelial phase, then the selected strains were cultured on cereal media (white rice, brown rice, and rye) to examine their ability to produce EAs on each medium. C. purpurea var. agropyri produced the Clavine-type EAs pyroclavine (1), festuclavine (2), and agroclavine (3) in the mycelial phase. When cultured with white rice, brown rice, or rye, C. purpurea var. agropyri produced 1 - 3 on all cereal media. The total amount of 1 - 3 in each cereal medium (150 g of cereal per Roux flask) was 2220.5 ± 564.1 μg for white rice, 920.0 ± 463.6 μg for brown rice, and 595.4 ± 52.1 μg for rye. The white rice medium supported the highest production of 1 - 3, with the total amount of EAs (150 g of white rice per Roux flask) being about 34 times higher than that in the T25 liquid medium (190 mL per 1 L Erlenmeyer flask) (equivalent amount per flask).
文摘Endophyte infected tall fescue (E+) is the base diet for nearly all beef cattle in the southern USA. It has been linked to a variety of toxicological conditions due to the presence of large numbers of ergot alkaloids. This study was designed to investigate the effects of E+ seed extract and selected ergot alkaloids on the detoxification pathway by cytochrome P450 (CYP3A4) enzyme system. Tests were performed using the P450- Glo CYP3A4 enzyme activity kit (Promega, WI), according to the manufacturer’s manual. Luminescence was measured using a single tube TD20/20 luminometer. Endophyte infected tall fescue seed was extracted with 50/50 methanol/25 mM ammonium carbonate, cleaned and concentrated on Strata-X reversed phase column (Phenomenex). The extracts were evaluated on an HPLC, and then tested using a serial dilution method. Commercially available ergonovine (EN), ergocorine (ER), bromocryptine (BC) and ergocryptine (EC) were tested individually using 0 to 44 nM concentrations. Seed extract of E+ produced a significant (P P < 0.05) dose dependent manner with EC being most potent, followed by ER, BC, and then EN (70%, 40%, 30% and 10% at 44 nM concentration). The similarity of the inhibition curves of seed extract to that of the commercially available ergot alkaloids suggests a related mode of action and that the use of such ergot alkaloids and CYP3A4 assay is a good model to study the toxicity of tall fescue. Furthermore, it provides the foundation to identify the individual toxic components of purified endophyte infected tall fescue extract.
基金This study was supported by the National Key Research and Development Program of China(grant nos.2021YFC2100600,2019YFA0905100 and 2018YFA0901600)the National Natural Science Foundation of China(grant nos.31872614,32022002,21977113)+1 种基金the Youth Scientists Innovation Promotion Association of CAS(2019090)to S.S.G.,Innovative Cross Team project of Chinese Academy of Sciences,CAS(grant no.JCTD-2019-06)Shandong Provincial Natural Science Foundation(Major Basic Research Projects)(grant no.ZR2019ZD18).
文摘Microbial cell factories(MCFs)and cell-free systems(CFSs)are generally considered as two unrelated approaches for the biosynthesis of biomolecules.In the current study,two systems were combined together for the overproduction of agroclavine(AC),a structurally complex ergot alkaloid.The whole biosynthetic pathway for AC was split into the early pathway and the late pathway at the point of the FAD-linked oxidoreductase EasE,which was reconstituted in an MCF(Aspergillus nidulans)and a four-enzyme CFS,respectively.The final titer of AC of this combined system is 1209 mg/L,which is the highest one that has been reported so far,to the best of our knowledge.The development of such a combined route could potentially avoid the limitations of both MCF and CFS systems,and boost the production of complex ergot alkaloids with polycyclic ring systems.
文摘Mycotoxins are toxic secondary metabolites produced by fungus kingdom. Fungi (molds) under aerobic and optimum conditions of humidity and temperature consume nutrients for proliferation and mycotoxin production (secretion). There are seven major groups of mycotoxins produced by different species of toxigenic fungal genus. Mycotoxins production from these toxigenic fungi depends on the surrounding intrinsic and extrinsic environments. These seven mycotoxins groups that contaminate grains, foods and animal feeds are: Aflatoxins, Trichothecene, Ochratoxins, Ergot alkaloid (Ergolin), Fumonisins, Patulin, and Zearalenone. These mycotoxins are capable of causing health hazards and death for both human and animals by effecting mammalian cells, causing a number of problems in normal cell function and a wide variety of clinical symptoms of diseases. These mycotoxins are varied in their toxicity depending on the infected host (human or animal) and the host susceptibility (immunity). The major concern of food and feed industries is the contamination of food products and animal feed supplies by these mycotoxins. Worldwide Health Organization (WHO), and Food and Agriculture Organization (FAO) are responsible to regulate the acceptable (tolerable) levels of these mycotoxins in grains, food and feed supplies to ensure the safety and health for both human and animals. Understanding fungal ecology and factors that affect fungal proliferation and mycotoxins production by these toxigenic fungi in agriculture crops as raw materials for both human food and animal feed products, plus understanding the chemistry and property of these mycotoxins, methods of detection, illness symptoms, and comply with regulatory guidance established by World Health Organization (WHO)/Food and Agriculture Organization (FAO) are key factors to prevent or minimize foods/feeds contamination and the toxicity of these mycotoxins for both human and animals health, plus reducing economical loss.
基金financially supported by National Natural Science Foundation of China(Nos.21372191 and 31370079)Jiangsu Province Natural Science Foundation(No.BK20130437)
文摘The present study was designed to isolate and purify chemical constituents from solid culture of endophyte Aspergillus terreus LQ, using silica gel column chromatography, gel filtration with Sephadex LH-20, and HPLC. Fumigaclavine I(1), a new alkaloid, was obtained, along with seven known compounds, including fumigaclavine C(2), rhizoctonic acid(3), monomethylsulochrin(4), chaetominine(5), spirotryprostatin A(6), asperfumoid(7), and lumichrome(8). The structure of compound 1 was elucidated by various spectroscopic analyses(UV, MS, 1D and 2D NMR). The in vitro cytotoxicity of compound 1 was determined by MTT assay in human hepatocarcinoma cell line SMMC-7721, showing weaker cytotoxicity, compared with cisplatin, a clinically used cancer chemotherapeutic agent.
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.
基金National Natural Science Foundation of China(Grant No.21372017)
文摘In the present study, we developed a novel approach for the synthesis of the tetracyclic core of fumigaclavines A–D. A palladium-catalyzed intramolecular Larock indole synthesis was utilized to assemble the B/C rings of the tetracyclic core in one step. Although all attempts to convert compound 18 to fumigaclavine B failed, this study provided useful information for the total synthesis of fumigaclavines A–D.
