绿僵菌破坏素合成缺失株中分离鉴定pseurotin A
|
摘要
真菌次级代谢产物是医药活性成分的重要来源之一。就真菌基因组编码的次级代谢基因簇数量而言,由于普遍存在的基因沉默现象,常规培养中能够分离鉴定的化合物种类一般很有限。广谱杀虫的罗伯茨绿僵菌在液体培养基中的主要代谢产物为非核糖体环肽类的破坏素,本研究在对破坏素合成缺失突变株ΔdtxS1进行液体培养时获得了新的产物峰,对其中一个产物峰进行分离、纯化及结构鉴定,确定该化合物为螺环类的pseurotin A。结合在烟曲霉菌中解析的pseurotin A合成途径,推测获得了罗伯茨绿僵菌中的潜在合成基因簇,并推测了pseurotin A在绿僵菌中的合成途径。本研究首次在绿僵菌中鉴定获得pseurotin A,并揭示了在真菌中通过对主要次级代谢产物缺失的方法可以鉴定获得新型的化合物。
The secondary metabolites produced by different fungi are the rich sources for pharmaceutical explorations. In terms of the number of the secondary metabolism gene clusters encoded in certain fungal species, only handful of metabolites have been identified due to the effect of gene silencing. In the generalist species of the insect pathogenic fungi such as Metarhizium robertsii, the non-ribosomal cyclopeptide destruxins are the major compounds produced by the fungus grown in artificial liquid media. In this study, we found that the fermentation of the core non-ribosomal peptide synthase gene deletion mutant ΔdtxS1 obtained extra compounds when compared to the wild type(WT) strain of M. robertsii. Purification and structure identification of a chromatographic peak indicated that pseurotin A, a spirocyclic compound, was produced by ΔdtxS1 but not by the WT strain. Based on the elucidation of pseurotin A production in Aspergillus fumigatus, the conserved gene cluster is identified and the putative biosynthetic pathway is proposed in M. robertsii. In conclusion, we first reported the production of pseurotin A by Metarhizium species, and demonstrated that it is an effective way to identify new compounds from fungi by impairment of the production of certain major metabolites.
The secondary metabolites produced by different fungi are the rich sources for pharmaceutical explorations. In terms of the number of the secondary metabolism gene clusters encoded in certain fungal species, only handful of metabolites have been identified due to the effect of gene silencing. In the generalist species of the insect pathogenic fungi such as Metarhizium robertsii, the non-ribosomal cyclopeptide destruxins are the major compounds produced by the fungus grown in artificial liquid media. In this study, we found that the fermentation of the core non-ribosomal peptide synthase gene deletion mutant ΔdtxS1 obtained extra compounds when compared to the wild type(WT) strain of M. robertsii. Purification and structure identification of a chromatographic peak indicated that pseurotin A, a spirocyclic compound, was produced by ΔdtxS1 but not by the WT strain. Based on the elucidation of pseurotin A production in Aspergillus fumigatus, the conserved gene cluster is identified and the putative biosynthetic pathway is proposed in M. robertsii. In conclusion, we first reported the production of pseurotin A by Metarhizium species, and demonstrated that it is an effective way to identify new compounds from fungi by impairment of the production of certain major metabolites.
引文
Beemelmanns C,Guo H,Rischer M,Poulsen M,2016.Natural products from microbes associated with insects.Beilstein Journal of Organic Chemistry,12:314-327
Bertinetti BV,Pena NI,Cabrera GM,2009.An antifungal tetrapeptide from the culture of Penicillium canescens.Chemistry&Biodiversity,6:1178-1184
Bloch P,Tamm C,Bollinger P,Petcher TJ,Weber HP,1976.Pseurotin,a new metabolite of Pseudeurotium ovalis Stolk having an unusual hetero-spirocyclic system.Helvetica Chimica Acta,59:133-137
Brakhage AA,2013.Regulation of fungal secondary metabolism.Nature Reviews Microbiology,11:21-32
Breitenstein W,Chexal KK,Mohr P,Tamm C,1981.Pseurotin B,C,D,and E.further new metabolites of Pseudeurotium ovalis STOLK.Helvetica Chimica Acta,64:379-388
Cook D,Donzelli BGG,Creamer R,Baucom DL,Gardner DR,Pan J,Moore N,Krasnoff SB,Jaromczyk JW,Schardl CL,2017.Swainsonine biosynthesis genes in diverse symbiotic and pathogenic fungi.G3(Bethesda),7:1791-1797
Donzelli BG,Krasnoff SB,2016.Molecular genetics of secondary chemistry in Metarhizium fungi.Advances in Genetics,94:365-436
Donzelli BG,Krasnoff SB,Churchill AC,Vandenberg JD,Gibson DM,2010.Identification of a hybrid PKS-NRPSrequired for the biosynthesis of NG-391 in Metarhizium robertsii.Current Genetics,56:151-162
Giuliano Garisto Donzelli B,Gibson DM,Krasnoff SB,2015.Intracellular siderophore but not extracellular siderophore is required for full virulence in Metarhizium robertsii.Fungal Genetics and Biology,82:56-68
Hou CX,Yin Y,Qi QY,Song HS,Wang CS,2018.Identification and antibacterial activity detection of compounds from Cordyceps militaris.Mycosystema,37(12):1688-1694(in Chinese)
Hu X,Xiao G,Zheng P,Shang Y,Su Y,Zhang X,Liu X,Zhan S,St Leger RJ,Wang C,2014.Trajectory and genomic determinants of fungal-pathogen speciation and host adaptation.Proceedings of the National Academy of Sciences of the United States of America,111:16796-16801
Ishikawa M,Ninomiya T,Akabane H,Kushida N,Tsujiuchi G,Ohyama M,Gomi S,Shito K,Murata T,2009.Pseurotin Aand its analogues as inhibitors of immunoglobulin E[correction of immunoglobuline E]production.Bioorganic and Medicinal Chemistry Letters,19:1457-1460
Kato H,Tsunematsu Y,Yamamoto T,Namiki T,Kishimoto S,Noguchi H,Watanabe K,2016.New natural products isolated from Metarhizium robertsii ARSEF 23 by chemical screening and identification of the gene cluster through engineered biosynthesis in Aspergillus nidulans A1145.Journal of Antibiotics,69:561-566
Keller NP,2019.Fungal secondary metabolism:regulation,function and drug discovery.Nature Reviews:Microbiology,17:167-180
Lind AL,Wisecaver JH,Lameiras C,Wiemann P,Palmer JM,Keller NP,Rodrigues F,Goldman GH,Rokas A,2017.Drivers of genetic diversity in secondary metabolic gene clusters within a fungal species.PLoS Biology,15:e2003583
Mioso R,Toledo Marante FJ,Herrera Bravo de Laguna I,2015.The chemical diversity of the ascomycete fungus Paecilomyces variotii.Applied Biochemistry and Biotechnology,177:781-791
Moon YS,Donzelli BG,Krasnoff SB,McLane H,Griggs MH,Cooke P,Vandenberg JD,Gibson DM,Churchill AC,2008.Agrobacterium-mediated disruption of a nonribosomal peptide synthetase gene in the invertebrate pathogen Metarhizium anisopliae reveals a peptide spore factor.Applied and Environmental Microbiology,74:4366-4380
Sanchez JF,Entwistle R,Hung JH,Yaegashi J,Jain S,Chiang YM,Wang CC,Oakley BR,2011.Genome-based deletion analysis reveals the prenyl xanthone biosynthesis pathway in Aspergillus nidulans.Journal of the American Chemical Society,133:4010-4017
Sbaraini N,Guedes RL,Andreis FC,Junges A,de Morais GL,Vainstein MH,de Vasconcelos AT,Schrank A,2016.Secondary metabolite gene clusters in the entomopathogen fungus Metarhizium anisopliae:genome identification and patterns of expression in a cuticle infection model.BMC Genomics,17:736
Tsunematsu Y,Fukutomi M,Saruwatari T,Noguchi H,Hotta K,Tang Y,Watanabe K,2014.Elucidation of pseurotin biosynthetic pathway points to trans-acting C-methyltransferase:generation of chemical diversity.Angewandte Chemie International Edition in English,53:8475-8479
Wang B,Kang Q,Lu Y,Bai L,Wang C,2012.Unveiling the biosynthetic puzzle of destruxins in Metarhizium species.Proceedings of the National Academy of Sciences of the United States of America,109:1287-1292
Wang C,Wang S,2017.Insect pathogenic fungi:genomics,molecular interactions,and genetic improvements.Annual Review of Entomology,62:73-90
Wang CS,Feng MG,2014.Advances in fundamental and applied studies in China of fungal biocontrol agents for use against arthropod pests.Biological Control,68:129-135
Wenke J,Anke H,Sterner O,1993.Pseurotin-a and8-O-demethylpseurotin-a from Aspergillus fumigatus and their inhibitory activities on chitin synthase.Bioscience Biotechnology and Biochemistry,57:961-964
Wiemann P,Guo CJ,Palmer JM,Sekonyela R,Wang CC,Keller NP,2013.Prototype of an intertwined secondary-metabolite supercluster.Proceedings of the National Academy of Sciences of the United States of America,110:17065-17070
Xu YJ,Luo F,Li B,Shang Y,Wang C,2016.Metabolic conservation and diversification of Metarhizium species correlate with fungal host-specificity.Frontiers in Microbiology,7:2020
Yamada T,Ohshima M,Yuasa K,Kikuchi T,Tanaka R,2016.Assignment of the CD cotton effect to the chiral center in pseurotins,and the stereochemical revision of pseurotin A(2).Marine Drugs,14:e74
Zeng G,Zhang P,Zhang Q,Zhao H,Li Z,Zhang X,Wang C,Yin WB,Fang W,2018.Duplication of a Pks gene cluster and subsequent functional diversification facilitate environmental adaptation in Metarhizium species.PLoSGenet,14:e1007472
侯春晓,殷颖,齐秋月,宋红生,王成树,2018.蝉虫草(蝉花)来源的化合物鉴定与抗菌活性检测.菌物学报,37(12):1688-1694
Bertinetti BV,Pena NI,Cabrera GM,2009.An antifungal tetrapeptide from the culture of Penicillium canescens.Chemistry&Biodiversity,6:1178-1184
Bloch P,Tamm C,Bollinger P,Petcher TJ,Weber HP,1976.Pseurotin,a new metabolite of Pseudeurotium ovalis Stolk having an unusual hetero-spirocyclic system.Helvetica Chimica Acta,59:133-137
Brakhage AA,2013.Regulation of fungal secondary metabolism.Nature Reviews Microbiology,11:21-32
Breitenstein W,Chexal KK,Mohr P,Tamm C,1981.Pseurotin B,C,D,and E.further new metabolites of Pseudeurotium ovalis STOLK.Helvetica Chimica Acta,64:379-388
Cook D,Donzelli BGG,Creamer R,Baucom DL,Gardner DR,Pan J,Moore N,Krasnoff SB,Jaromczyk JW,Schardl CL,2017.Swainsonine biosynthesis genes in diverse symbiotic and pathogenic fungi.G3(Bethesda),7:1791-1797
Donzelli BG,Krasnoff SB,2016.Molecular genetics of secondary chemistry in Metarhizium fungi.Advances in Genetics,94:365-436
Donzelli BG,Krasnoff SB,Churchill AC,Vandenberg JD,Gibson DM,2010.Identification of a hybrid PKS-NRPSrequired for the biosynthesis of NG-391 in Metarhizium robertsii.Current Genetics,56:151-162
Giuliano Garisto Donzelli B,Gibson DM,Krasnoff SB,2015.Intracellular siderophore but not extracellular siderophore is required for full virulence in Metarhizium robertsii.Fungal Genetics and Biology,82:56-68
Hou CX,Yin Y,Qi QY,Song HS,Wang CS,2018.Identification and antibacterial activity detection of compounds from Cordyceps militaris.Mycosystema,37(12):1688-1694(in Chinese)
Hu X,Xiao G,Zheng P,Shang Y,Su Y,Zhang X,Liu X,Zhan S,St Leger RJ,Wang C,2014.Trajectory and genomic determinants of fungal-pathogen speciation and host adaptation.Proceedings of the National Academy of Sciences of the United States of America,111:16796-16801
Ishikawa M,Ninomiya T,Akabane H,Kushida N,Tsujiuchi G,Ohyama M,Gomi S,Shito K,Murata T,2009.Pseurotin Aand its analogues as inhibitors of immunoglobulin E[correction of immunoglobuline E]production.Bioorganic and Medicinal Chemistry Letters,19:1457-1460
Kato H,Tsunematsu Y,Yamamoto T,Namiki T,Kishimoto S,Noguchi H,Watanabe K,2016.New natural products isolated from Metarhizium robertsii ARSEF 23 by chemical screening and identification of the gene cluster through engineered biosynthesis in Aspergillus nidulans A1145.Journal of Antibiotics,69:561-566
Keller NP,2019.Fungal secondary metabolism:regulation,function and drug discovery.Nature Reviews:Microbiology,17:167-180
Lind AL,Wisecaver JH,Lameiras C,Wiemann P,Palmer JM,Keller NP,Rodrigues F,Goldman GH,Rokas A,2017.Drivers of genetic diversity in secondary metabolic gene clusters within a fungal species.PLoS Biology,15:e2003583
Mioso R,Toledo Marante FJ,Herrera Bravo de Laguna I,2015.The chemical diversity of the ascomycete fungus Paecilomyces variotii.Applied Biochemistry and Biotechnology,177:781-791
Moon YS,Donzelli BG,Krasnoff SB,McLane H,Griggs MH,Cooke P,Vandenberg JD,Gibson DM,Churchill AC,2008.Agrobacterium-mediated disruption of a nonribosomal peptide synthetase gene in the invertebrate pathogen Metarhizium anisopliae reveals a peptide spore factor.Applied and Environmental Microbiology,74:4366-4380
Sanchez JF,Entwistle R,Hung JH,Yaegashi J,Jain S,Chiang YM,Wang CC,Oakley BR,2011.Genome-based deletion analysis reveals the prenyl xanthone biosynthesis pathway in Aspergillus nidulans.Journal of the American Chemical Society,133:4010-4017
Sbaraini N,Guedes RL,Andreis FC,Junges A,de Morais GL,Vainstein MH,de Vasconcelos AT,Schrank A,2016.Secondary metabolite gene clusters in the entomopathogen fungus Metarhizium anisopliae:genome identification and patterns of expression in a cuticle infection model.BMC Genomics,17:736
Tsunematsu Y,Fukutomi M,Saruwatari T,Noguchi H,Hotta K,Tang Y,Watanabe K,2014.Elucidation of pseurotin biosynthetic pathway points to trans-acting C-methyltransferase:generation of chemical diversity.Angewandte Chemie International Edition in English,53:8475-8479
Wang B,Kang Q,Lu Y,Bai L,Wang C,2012.Unveiling the biosynthetic puzzle of destruxins in Metarhizium species.Proceedings of the National Academy of Sciences of the United States of America,109:1287-1292
Wang C,Wang S,2017.Insect pathogenic fungi:genomics,molecular interactions,and genetic improvements.Annual Review of Entomology,62:73-90
Wang CS,Feng MG,2014.Advances in fundamental and applied studies in China of fungal biocontrol agents for use against arthropod pests.Biological Control,68:129-135
Wenke J,Anke H,Sterner O,1993.Pseurotin-a and8-O-demethylpseurotin-a from Aspergillus fumigatus and their inhibitory activities on chitin synthase.Bioscience Biotechnology and Biochemistry,57:961-964
Wiemann P,Guo CJ,Palmer JM,Sekonyela R,Wang CC,Keller NP,2013.Prototype of an intertwined secondary-metabolite supercluster.Proceedings of the National Academy of Sciences of the United States of America,110:17065-17070
Xu YJ,Luo F,Li B,Shang Y,Wang C,2016.Metabolic conservation and diversification of Metarhizium species correlate with fungal host-specificity.Frontiers in Microbiology,7:2020
Yamada T,Ohshima M,Yuasa K,Kikuchi T,Tanaka R,2016.Assignment of the CD cotton effect to the chiral center in pseurotins,and the stereochemical revision of pseurotin A(2).Marine Drugs,14:e74
Zeng G,Zhang P,Zhang Q,Zhao H,Li Z,Zhang X,Wang C,Yin WB,Fang W,2018.Duplication of a Pks gene cluster and subsequent functional diversification facilitate environmental adaptation in Metarhizium species.PLoSGenet,14:e1007472
侯春晓,殷颖,齐秋月,宋红生,王成树,2018.蝉虫草(蝉花)来源的化合物鉴定与抗菌活性检测.菌物学报,37(12):1688-1694