Small, cysteine-rich, highly stable antifungal proteins secreted by filamentous Ascomycetes have great po- tential for the development of novel antifungal strate- gies. However, their practical application is still li...Small, cysteine-rich, highly stable antifungal proteins secreted by filamentous Ascomycetes have great po- tential for the development of novel antifungal strate- gies. However, their practical application is still limited due to their not fully clarified mode of action. The aim of this work was to provide a deep insight into the anti-fungal mechanism of Neosartorya fischeri antifungal protein （NFAP）, a novel representative of this protein group. Within a short exposure time to NFAP, reduced cellular metabolism, apoptosis induction, changes in the actin distribution and chitin deposition at the hyphal tip were observed in NFAP-sensitive Aspergillus nidulans. NFAP did show neither a direct membrane disrupting- effect nor uptake by endocytosis. Investigation of A. nidulans signalling mutants revealed that NFAP acti- vates the cAMP/protein kinase A pathway via G-protein signalling which leads to apoptosis and inhibition of polar growth. In contrast, NFAP does not have any in- fluence on the cell wall integrity pathway, but an un- known cell wall integrity pathway-independent mitogen activated protein kinase A-activated target is assumed to be involved in the cell death induction. Taken to- gether, it was concluded that NFAP shows similarities, but also differences in its mode of antifungal action compared to two most investigated NFAP-related pro-teins from Aspergillus giganteus and Penicillium chrysogenum.展开更多
Laccases, multicopper oxidoreductases, are mainly produced in white-rot fungi and are considered as ideal green catalysts in industrial and biotechnological applications. However, the development of laccases is limite...Laccases, multicopper oxidoreductases, are mainly produced in white-rot fungi and are considered as ideal green catalysts in industrial and biotechnological applications. However, the development of laccases is limited due to the slow growth of natural laccase producing strains and the low expression levels of laccases. In this study, we designed three regulation strategies for laccase gene expression in the model fungus Aspergillus nidulans. By introducing various promoters in front of the laccase gene pslcc from the white-rot fungus Pycnoporus sanguineus, we found that the laccase gene with the original promoter had effective expression in A. nidulans. Using the previously identified transcription factor RsmA regulatory mechanism, the aflR promoter was inserted into the pslcc expression vectors, and the laccase production was 15-fold higher in the strain overexpressing of RsmA compared to the control strain. To improve the laccase yield, the dipeptidyl-peptidase DppV, aspartic protease PepA and mannosyltransferase Mnn9 were successfully deleted in the A. nidulans host. The laccase activities were increased approximately 8-fold and 13-fold in the double deletions strains of Δmnn9ΔpepA and ΔdppVΔpepA over the control strains, respectively.Taken together, these results not only demonstrate an efficient system for heterologous protein production in the model fungus A.nidulans but also provide a general approach to applying regulatory methods to control gene expression.展开更多
A new acyclic peroxide derivative asperoxide A(1),along with 13 known compounds,namely,microperfuranone(2),9-hydroxymicroperfuranone(3),gibellulin A(4),lecanoric acid(5),terrequinone A(6),sterigmatocystin(7),isosecost...A new acyclic peroxide derivative asperoxide A(1),along with 13 known compounds,namely,microperfuranone(2),9-hydroxymicroperfuranone(3),gibellulin A(4),lecanoric acid(5),terrequinone A(6),sterigmatocystin(7),isosecosterigmatocystin(8),arugosin C(9),curvularin(10),3,3'-diindolylmethane(11),austinol(12),austin(13),and dehydroaustin(14),were isolated and identified from the culture extract of Aspergillus nidulans SD-531,a fungus obtained from the deep-sea sediment of cold spring in the South China Sea.Their structures were determined based on detailed interpretation of nuclear magnetic resonance(NMR)spectroscopic and mass spectrometry data analysis.All the isolated compounds were evaluated for antimicrobial activities against human and aquatic bacteria as well as plant pathogenic fungi.Compounds 1–8,10,and 11 exhibited antimicrobial activities against some of the tested strains with minimum inhibitory concentration(MIC)values ranging from^2 to 64μg/mL.Compounds 4 and 6 displayed strongest activities among the tested samples and might be used as promising molecules for the development of natural antimicrobial agents.展开更多
Niduenes A-F(1-6),six novel sesterterpenoids with unprecedented 5/5/5/5/6 pentacyclic ring skeleton were isolated from endophytic fungus Aspergillus nidulans.Compounds 1 and 2 represent the first examples of aromatic ...Niduenes A-F(1-6),six novel sesterterpenoids with unprecedented 5/5/5/5/6 pentacyclic ring skeleton were isolated from endophytic fungus Aspergillus nidulans.Compounds 1 and 2 represent the first examples of aromatic pentacyclic sesterterpenoids.Their structures and configurations were elucidated by spectroscopic data and single-crystal X-ray diffraction analyses.Compound 4 demonstrated potent resensitization of SW620/AD300 cells to paclitaxel(PTX).Rhodamine 123 accumulation assay and docking analysis further support that 4 inhibitory the efflux function of P-glycoprotein(P-gp).展开更多
Introduction:Secondary metabolites of fungi are receiving an increasing amount of interest due to their prolific bioactivities and the fact that fungal biosynthesis of secondary metabolites often occurs from co-regula...Introduction:Secondary metabolites of fungi are receiving an increasing amount of interest due to their prolific bioactivities and the fact that fungal biosynthesis of secondary metabolites often occurs from co-regulated and co-located gene clusters.This makes the gene clusters attractive for synthetic biology and industrial biotechnology applications.We have previously published a method for accurate prediction of clusters from genome and transcriptome data,which could also suggest cross-chemistry,however,this method was limited both in the number of parameters which could be adjusted as well as in userfriendliness.Furthermore,sensitivity to the transcriptome data required manual curation of the predictions.In the present work,we have aimed at improving these features.Results:FunGeneClusterS is an improved implementation of our previous method with a graphical user interface for off-and on-line use.The new method adds options to adjust the size of the gene cluster(s)being sought as well as an option for the algorithm to be flexible with genes in the cluster which may not seem to be co-regulated with the remainder of the cluster.We have benchmarked the method using data from the well-studied Aspergillus nidulans and found that the method is an improvement over the previous one.In particular,it makes it possible to predict clusters with more than 10 genes more accurately,and allows identification of co-regulated gene clusters irrespective of the function of the genes.It also greatly reduces the need for manual curation of the prediction results.We furthermore applied the method to transcriptome data from A.niger.Using the identified best set of parameters,we were able to identify clusters for 31 out of 76 previously predicted secondary metabolite synthases/synthetases.Furthermore,we identified additional putative secondary metabolite gene clusters.In total,we predicted 432 co-transcribed gene clusters in A.niger(spanning 1.323 genes,12%of the genome).Some of these had functions related to primary metabolism,e.g.we展开更多
Understanding the growth regulatory mechanisms in filamentous fungi is very important for the production of medicines for antifungal therapies. It is well established that Ca2+ gradient is essential for hyphal growth ...Understanding the growth regulatory mechanisms in filamentous fungi is very important for the production of medicines for antifungal therapies. It is well established that Ca2+ gradient is essential for hyphal growth and that one mechanism responsible for the Ca2+ cellular concentration starts with the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by receptor-regulated forms of phosphoinositide-specific phospholipase C (PI-PLC). In the present study the levels of calcium in Aspergillus nidulans wild type (A26) and plcA-deficient mutant (AP27) growing in a carbon source readily assimilated, as glucose or pectin a non-readily assimilated carbon source was investigated. Intracellular calcium levels in A26 were higher in the presence of glucose than in pectin, but lower in AP27 independently of the carbon source and in AP27 the vesicular calcium distribution occurred mainly at the apex of the hyphae. Delay in nuclear division was also observed if A26 and AP27 were grown in pectin presence when compared with growth in glucose. For the first time, it is demonstrated that the levels of intracellular Ca2+ were higher when A. nidulans was growing in glucose than in a non readily assimilated carbon source as pectin. Further, it also showed that the plcA gene, although not essential, may be responsible for high-molecular weight carbon source recongnation, for the intracellular Ca2+ levels maintenance and consequently by the nuclear division in A. nidulans.展开更多
This study investigates the potential of native biocontrol agents(BCAs)as controls against Ganoderma lucidum causing root rot mortality in Indian mesquite.The disease is prevalent in sandy soils where trees grow under...This study investigates the potential of native biocontrol agents(BCAs)as controls against Ganoderma lucidum causing root rot mortality in Indian mesquite.The disease is prevalent in sandy soils where trees grow under rainfed conditions.In addition,a beetle namely A canthophorus serraticornis damages the roots,resulting in increasing vulnerability of the host thereby allowing easy of the pathogen.In dual culture tests,Ganoderma infected cowpea root bit experiment and compatibility with insecticides revealed that the three BCAs(Trichoderma longibrachiatum,T.harzianum,and Aspergillus nidulans)significantly inhibited G.lucidum mycelial growth.The highest mycelial growth inhibition(47.6%)was recorded after 96 h followed by 39.8%and 29.3%at 72 and 48 h,respectively,by T.longibrachium.Cell free filtrates of T.longibrachiatum,T.harzianum,and A.nidulans were superior in inhibiting mycelium growth.A low concentration(3 ml)of T.longibrachiatum was more effective in inhibiting mycelium growth compared to other BCAs.Both P rosopis julifl ora compost and onion residue compost amendments as food substrates favored the growth of these BCAs,which ultimately reduced the viability of Ganoderma-colonized root bits of cowpea.Studies on compatibility between insecticides and BCAs suggests that T.longibrachiatum,harzianum and A.nidulans can be combined with phorate or chloropyriphos(both organophosphates)at variable concentrations if amended together for partially infected trees,or as a prophylactic measure in healthy trees.These studies demonstrate that there is considerable opportunity for using native BCAs against G.lucidum in managing root rot disease.展开更多
Kaurenoic acid (KA), a kaurane-type diterpene extracted from leaves of Mikania hirsutissima, was previously reported as an inhibitor of vascular contractility mainly by blocking extracellular Ca2+ influx. The compound...Kaurenoic acid (KA), a kaurane-type diterpene extracted from leaves of Mikania hirsutissima, was previously reported as an inhibitor of vascular contractility mainly by blocking extracellular Ca2+ influx. The compound is known for several other biological activities such as antiparasitic, antispasmodic and antibacterial activity. The aim of the present study was to investigate the effect of KA on Aspergillus nidulans. KA (0.3 mM) showed fungistatic activity against A. nidulans with visible hyphal elongation and morphology damage. These effects were reverted by CaCl2 addition showing that KA interferes with intracellular Ca2+ gradient in A. nidulans. This is the first report on the mechanism of action of KA involving calcium levels by altering the elongation of fungi hyphae.展开更多
Copper-induced changes in the urea uptake and urease activity have been investigated in the cyanobacteria Anabaena doliolum and Anacystis nidulans. Copper, at and above 5 μmol/L concentration, inhibited urea uptake a...Copper-induced changes in the urea uptake and urease activity have been investigated in the cyanobacteria Anabaena doliolum and Anacystis nidulans. Copper, at and above 5 μmol/L concentration, inhibited urea uptake and urease activity systems in both the cyanobacteria in a concentration dependent manner. However, the urea uptake and urease activity systems in A. nidulans appeared slightly more tolerant to copper than that of A.doliolum. The inhibitory effect of copper on urea uptake and urease activity was mitigated by sulphur containing amino acids (cystine and cysteine), however, methionine could not do so, indicating the involvement of sulfhydryl (-SH) groups in the assimilation of urea in cyanobacteria展开更多
文摘Small, cysteine-rich, highly stable antifungal proteins secreted by filamentous Ascomycetes have great po- tential for the development of novel antifungal strate- gies. However, their practical application is still limited due to their not fully clarified mode of action. The aim of this work was to provide a deep insight into the anti-fungal mechanism of Neosartorya fischeri antifungal protein （NFAP）, a novel representative of this protein group. Within a short exposure time to NFAP, reduced cellular metabolism, apoptosis induction, changes in the actin distribution and chitin deposition at the hyphal tip were observed in NFAP-sensitive Aspergillus nidulans. NFAP did show neither a direct membrane disrupting- effect nor uptake by endocytosis. Investigation of A. nidulans signalling mutants revealed that NFAP acti- vates the cAMP/protein kinase A pathway via G-protein signalling which leads to apoptosis and inhibition of polar growth. In contrast, NFAP does not have any in- fluence on the cell wall integrity pathway, but an un- known cell wall integrity pathway-independent mitogen activated protein kinase A-activated target is assumed to be involved in the cell death induction. Taken to- gether, it was concluded that NFAP shows similarities, but also differences in its mode of antifungal action compared to two most investigated NFAP-related pro-teins from Aspergillus giganteus and Penicillium chrysogenum.
基金supported by Beijing Natural Science Foundation (5152018)the National Natural Science Foundation of China (31470178)Wen-Bing Yin is a scholar of "the 100 Talents Project" of CAS
文摘Laccases, multicopper oxidoreductases, are mainly produced in white-rot fungi and are considered as ideal green catalysts in industrial and biotechnological applications. However, the development of laccases is limited due to the slow growth of natural laccase producing strains and the low expression levels of laccases. In this study, we designed three regulation strategies for laccase gene expression in the model fungus Aspergillus nidulans. By introducing various promoters in front of the laccase gene pslcc from the white-rot fungus Pycnoporus sanguineus, we found that the laccase gene with the original promoter had effective expression in A. nidulans. Using the previously identified transcription factor RsmA regulatory mechanism, the aflR promoter was inserted into the pslcc expression vectors, and the laccase production was 15-fold higher in the strain overexpressing of RsmA compared to the control strain. To improve the laccase yield, the dipeptidyl-peptidase DppV, aspartic protease PepA and mannosyltransferase Mnn9 were successfully deleted in the A. nidulans host. The laccase activities were increased approximately 8-fold and 13-fold in the double deletions strains of Δmnn9ΔpepA and ΔdppVΔpepA over the control strains, respectively.Taken together, these results not only demonstrate an efficient system for heterologous protein production in the model fungus A.nidulans but also provide a general approach to applying regulatory methods to control gene expression.
基金the National Key R&D Program of China(No.2018YFC0310800)。
文摘A new acyclic peroxide derivative asperoxide A(1),along with 13 known compounds,namely,microperfuranone(2),9-hydroxymicroperfuranone(3),gibellulin A(4),lecanoric acid(5),terrequinone A(6),sterigmatocystin(7),isosecosterigmatocystin(8),arugosin C(9),curvularin(10),3,3'-diindolylmethane(11),austinol(12),austin(13),and dehydroaustin(14),were isolated and identified from the culture extract of Aspergillus nidulans SD-531,a fungus obtained from the deep-sea sediment of cold spring in the South China Sea.Their structures were determined based on detailed interpretation of nuclear magnetic resonance(NMR)spectroscopic and mass spectrometry data analysis.All the isolated compounds were evaluated for antimicrobial activities against human and aquatic bacteria as well as plant pathogenic fungi.Compounds 1–8,10,and 11 exhibited antimicrobial activities against some of the tested strains with minimum inhibitory concentration(MIC)values ranging from^2 to 64μg/mL.Compounds 4 and 6 displayed strongest activities among the tested samples and might be used as promising molecules for the development of natural antimicrobial agents.
基金financially supported by the National Key Research and Development Program of China(No.2021YFA0910500)the National Natural Science Foundation of China(Nos.U22A20380,82104028,82173706 and 82373755)+2 种基金Innovative Research Groups of the National Natural Science Foundation of China(No.81721005)the Science and Technology Major Project of Hubei Province(No.2021ACA012)the Fundamental Research Funds for the Central Universities,HUST(No.2021JYCXJJ058)。
文摘Niduenes A-F(1-6),six novel sesterterpenoids with unprecedented 5/5/5/5/6 pentacyclic ring skeleton were isolated from endophytic fungus Aspergillus nidulans.Compounds 1 and 2 represent the first examples of aromatic pentacyclic sesterterpenoids.Their structures and configurations were elucidated by spectroscopic data and single-crystal X-ray diffraction analyses.Compound 4 demonstrated potent resensitization of SW620/AD300 cells to paclitaxel(PTX).Rhodamine 123 accumulation assay and docking analysis further support that 4 inhibitory the efflux function of P-glycoprotein(P-gp).
文摘Introduction:Secondary metabolites of fungi are receiving an increasing amount of interest due to their prolific bioactivities and the fact that fungal biosynthesis of secondary metabolites often occurs from co-regulated and co-located gene clusters.This makes the gene clusters attractive for synthetic biology and industrial biotechnology applications.We have previously published a method for accurate prediction of clusters from genome and transcriptome data,which could also suggest cross-chemistry,however,this method was limited both in the number of parameters which could be adjusted as well as in userfriendliness.Furthermore,sensitivity to the transcriptome data required manual curation of the predictions.In the present work,we have aimed at improving these features.Results:FunGeneClusterS is an improved implementation of our previous method with a graphical user interface for off-and on-line use.The new method adds options to adjust the size of the gene cluster(s)being sought as well as an option for the algorithm to be flexible with genes in the cluster which may not seem to be co-regulated with the remainder of the cluster.We have benchmarked the method using data from the well-studied Aspergillus nidulans and found that the method is an improvement over the previous one.In particular,it makes it possible to predict clusters with more than 10 genes more accurately,and allows identification of co-regulated gene clusters irrespective of the function of the genes.It also greatly reduces the need for manual curation of the prediction results.We furthermore applied the method to transcriptome data from A.niger.Using the identified best set of parameters,we were able to identify clusters for 31 out of 76 previously predicted secondary metabolite synthases/synthetases.Furthermore,we identified additional putative secondary metabolite gene clusters.In total,we predicted 432 co-transcribed gene clusters in A.niger(spanning 1.323 genes,12%of the genome).Some of these had functions related to primary metabolism,e.g.we
基金J.A.R.received a Doctoral Fellowship from Coordenacao de Apoio de Pessoal de Nível Superior(CAPES).
文摘Understanding the growth regulatory mechanisms in filamentous fungi is very important for the production of medicines for antifungal therapies. It is well established that Ca2+ gradient is essential for hyphal growth and that one mechanism responsible for the Ca2+ cellular concentration starts with the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by receptor-regulated forms of phosphoinositide-specific phospholipase C (PI-PLC). In the present study the levels of calcium in Aspergillus nidulans wild type (A26) and plcA-deficient mutant (AP27) growing in a carbon source readily assimilated, as glucose or pectin a non-readily assimilated carbon source was investigated. Intracellular calcium levels in A26 were higher in the presence of glucose than in pectin, but lower in AP27 independently of the carbon source and in AP27 the vesicular calcium distribution occurred mainly at the apex of the hyphae. Delay in nuclear division was also observed if A26 and AP27 were grown in pectin presence when compared with growth in glucose. For the first time, it is demonstrated that the levels of intracellular Ca2+ were higher when A. nidulans was growing in glucose than in a non readily assimilated carbon source as pectin. Further, it also showed that the plcA gene, although not essential, may be responsible for high-molecular weight carbon source recongnation, for the intracellular Ca2+ levels maintenance and consequently by the nuclear division in A. nidulans.
基金supported by the Core Research Grant of SERB–DST,New Delhi,Government of India。
文摘This study investigates the potential of native biocontrol agents(BCAs)as controls against Ganoderma lucidum causing root rot mortality in Indian mesquite.The disease is prevalent in sandy soils where trees grow under rainfed conditions.In addition,a beetle namely A canthophorus serraticornis damages the roots,resulting in increasing vulnerability of the host thereby allowing easy of the pathogen.In dual culture tests,Ganoderma infected cowpea root bit experiment and compatibility with insecticides revealed that the three BCAs(Trichoderma longibrachiatum,T.harzianum,and Aspergillus nidulans)significantly inhibited G.lucidum mycelial growth.The highest mycelial growth inhibition(47.6%)was recorded after 96 h followed by 39.8%and 29.3%at 72 and 48 h,respectively,by T.longibrachium.Cell free filtrates of T.longibrachiatum,T.harzianum,and A.nidulans were superior in inhibiting mycelium growth.A low concentration(3 ml)of T.longibrachiatum was more effective in inhibiting mycelium growth compared to other BCAs.Both P rosopis julifl ora compost and onion residue compost amendments as food substrates favored the growth of these BCAs,which ultimately reduced the viability of Ganoderma-colonized root bits of cowpea.Studies on compatibility between insecticides and BCAs suggests that T.longibrachiatum,harzianum and A.nidulans can be combined with phorate or chloropyriphos(both organophosphates)at variable concentrations if amended together for partially infected trees,or as a prophylactic measure in healthy trees.These studies demonstrate that there is considerable opportunity for using native BCAs against G.lucidum in managing root rot disease.
基金J.A.R.received a Doctoral Fellowship from Coorde-nacao de Apoio de Pessoal de Nível Superio(CAPES)N.S.A.would like to thank Universidade Estadual de Londrina and Fundacao Araucária.
文摘Kaurenoic acid (KA), a kaurane-type diterpene extracted from leaves of Mikania hirsutissima, was previously reported as an inhibitor of vascular contractility mainly by blocking extracellular Ca2+ influx. The compound is known for several other biological activities such as antiparasitic, antispasmodic and antibacterial activity. The aim of the present study was to investigate the effect of KA on Aspergillus nidulans. KA (0.3 mM) showed fungistatic activity against A. nidulans with visible hyphal elongation and morphology damage. These effects were reverted by CaCl2 addition showing that KA interferes with intracellular Ca2+ gradient in A. nidulans. This is the first report on the mechanism of action of KA involving calcium levels by altering the elongation of fungi hyphae.
文摘Copper-induced changes in the urea uptake and urease activity have been investigated in the cyanobacteria Anabaena doliolum and Anacystis nidulans. Copper, at and above 5 μmol/L concentration, inhibited urea uptake and urease activity systems in both the cyanobacteria in a concentration dependent manner. However, the urea uptake and urease activity systems in A. nidulans appeared slightly more tolerant to copper than that of A.doliolum. The inhibitory effect of copper on urea uptake and urease activity was mitigated by sulphur containing amino acids (cystine and cysteine), however, methionine could not do so, indicating the involvement of sulfhydryl (-SH) groups in the assimilation of urea in cyanobacteria
文摘采用固体平板培养和液体发酵筛选产生甲壳素脱乙酰酶的真菌,并且研究了产酶条件.结果显示在42株真菌中有26株有甲壳素脱乙酰酶活性,最终确定了构巢曲霉和蓝色犁头霉两株产酶活性较高的菌株,它们产生的酶活性分别是343U·mL-1和289 U·mL-1.不同的菌株的产酶培养基中的碳源均为含乙酰基的碳源,最适氮源分别为酵母粉和蛋白胨,产酶能力都受Mn2+和Co2+促进,受Cu2+、Fe2+、Fe3+的抑制;构巢曲霉的最适温度、最适pH分别为29 ℃,7.0～7.5,蓝色犁头霉的最适温度、最适pH分别为31 ℃,6.5～7.0,最适发酵时间均为96 h.
