Strategy for efficient cloning of biosynthetic gene clusters from fungi
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摘要
Filamentous fungi are excellent sources for the production of a group of bioactive small molecules which are often called secondary metabolites(SMs). The advanced genome sequencing technology combined with bioinformatics analysis reveals a large number of unexplored biosynthetic gene clusters(BGCs) in the fungal genomes. To unlock this fungal SM treasure, many approaches including heterologous expression are being developed and efficient cloning of the BGCs is a crucial step to do this.Here, we present an efficient strategy for the direct cloning of fungal BGCs. This strategy consisted of Splicing by Overlapping Extension(SOE)-PCR and yeast assembly in vivo. By testing 14 BGCs DNA fragments ranging from 7 kb to 52 kb, the average positive rate was over 80%. The maximal insertion size for fungal BGC assembly was 52 kb. Those constructs could be used conveniently for the heterologous expression leading to the discovery of novel natural products. Thus, our results provide an efficient and quick method for the low cost direct cloning of fungal BGCs.
Filamentous fungi are excellent sources for the production of a group of bioactive small molecules which are often called secondary metabolites(SMs). The advanced genome sequencing technology combined with bioinformatics analysis reveals a large number of unexplored biosynthetic gene clusters(BGCs) in the fungal genomes. To unlock this fungal SM treasure, many approaches including heterologous expression are being developed and efficient cloning of the BGCs is a crucial step to do this.Here, we present an efficient strategy for the direct cloning of fungal BGCs. This strategy consisted of Splicing by Overlapping Extension(SOE)-PCR and yeast assembly in vivo. By testing 14 BGCs DNA fragments ranging from 7 kb to 52 kb, the average positive rate was over 80%. The maximal insertion size for fungal BGC assembly was 52 kb. Those constructs could be used conveniently for the heterologous expression leading to the discovery of novel natural products. Thus, our results provide an efficient and quick method for the low cost direct cloning of fungal BGCs.
Filamentous fungi are excellent sources for the production of a group of bioactive small molecules which are often called secondary metabolites(SMs). The advanced genome sequencing technology combined with bioinformatics analysis reveals a large number of unexplored biosynthetic gene clusters(BGCs) in the fungal genomes. To unlock this fungal SM treasure, many approaches including heterologous expression are being developed and efficient cloning of the BGCs is a crucial step to do this.Here, we present an efficient strategy for the direct cloning of fungal BGCs. This strategy consisted of Splicing by Overlapping Extension(SOE)-PCR and yeast assembly in vivo. By testing 14 BGCs DNA fragments ranging from 7 kb to 52 kb, the average positive rate was over 80%. The maximal insertion size for fungal BGC assembly was 52 kb. Those constructs could be used conveniently for the heterologous expression leading to the discovery of novel natural products. Thus, our results provide an efficient and quick method for the low cost direct cloning of fungal BGCs.
引文
Bilyk,O.,Sekurova,O.N.,Zotchev,S.B.,and Luzhetskyy,A.(2016).Cloning and heterologous expression of the grecocycline biosynthetic gene cluster.PLoS ONE 11,e0158682.
Blackwell,M.(2011).The Fungi:1,2,3…5.1 million species?Am J Bot98,426-438.
Brakhage,A.A.,and Schroeckh,V.(2011).Fungal secondary metabolitesStrategies to activate silent gene clusters.Fungal Genets Biol 48,15-22.
Chaffin,D.O.,and Rubens,C.E.(1998).Blue/white screening of recombinant plasmids in Gram-positive bacteria by interruption of alkaline phosphatase gene(phoZ)expression.Gene 219,91-99.
Clevenger,K.D.,Bok,J.W.,Ye,R.,Miley,G.P.,Verdan,M.H.,Velk,T.,Chen,C.,Yang,K.H.,Robey,M.T.,Gao,P.,et al.(2017).A scalable platform to identify fungal secondary metabolites and their gene clusters.Nat Chem Biol 13,895-901.
Clutterbuck,P.W.,Lovell,R.,and Raistrick,H.(1932).Studies in the biochemistry of micro-organisms.Biochem J 26,1907-1918.
Cohen,S.N.,Chang,A.C.Y.,Boyer,H.W.,and Helling,R.B.(1973).Construction of biologically functional bacterial plasmids in vitro.Proc Natl Acad Sci USA 70,3240-3244.
Fan,A.,Mi,W.,Liu,Z.,Zeng,G.,Zhang,P.,Hu,Y.,Fang,W.,and Yin,W.B.(2017).Deletion of a Histone Acetyltransferase Leads to the Pleiotropic Activation of Natural Products in Metarhizium robertsii.Org Lett 19,1686-1689.
Fekete,E.,Karaffa,L.,Seiboth,B.,Fekete,é.,P.Kubicek,C.,and Flipphi,M.(2012).Identification of a permease gene involved in lactose utilisation in Aspergillus nidulans.Fungal Genets Biol 49,415-425.
Fu,J.,Bian,X.,Hu,S.,Wang,H.,Huang,F.,Seibert,P.M.,Plaza,A.,Xia,L.,Müller,R.,Stewart,A.F.,et al.(2012).Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting.Nat Biotechnol 30,440-446.
Gibson,D.G.,Young,L.,Chuang,R.Y.,Venter,J.C.,Hutchison,C.A.,and Smith,H.O.(2009).Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat Methods 6,343-345.
Han,X.,Chakrabortti,A.,Zhu,J.,Liang,Z.X.,and Li,J.(2016).Sequencing and functional annotation of the whole genome of the filamentous fungus Aspergillus westerdijkiae.BMC Genomics 17,633.
Inglis,D.O.,Binkley,J.,Skrzypek,M.S.,Arnaud,M.B.,Cerqueira,G.C.,Shah,P.,Wymore,F.,Wortman,J.R.,and Sherlock,G.(2013).Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans,A.fumigatus,A.niger and A.oryzae.BMC Microbiol 13,91.
Khaldi,N.,Seifuddin,F.T.,Turner,G.,Haft,D.,Nierman,W.C.,Wolfe,K.H.,and Fedorova,N.D.(2010).SMURF:Genomic mapping of fungal secondary metabolite clusters.Fungal Genets Biol 47,736-741.
Kouprina,N.,and Larionov,V.(2008).Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae.Nat Protoc 3,371-377.
Kupiec,M.(2000).Damage-induced recombination in the yeast Saccharomyces cerevisiae.Mutat Res-Fund Mol M 451,91-105.
Larionov,V.,Kouprina,N.,Graves,J.,Chen,X.N.,Korenberg,J.R.,and Resnick,M.A.(1996).Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination..Proc Natl Acad Sci USA 93,491-496.
Li,W.,Fan,A.,Wang,L.,Zhang,P.,Liu,Z.,An,Z.,and Yin,W.B.(2018).Asperphenamate biosynthesis reveals a novel two-module NRPSsystem to synthesize amino acid esters in fungi.Chem Sci 9,2589-2594.
Lin,H.,Lyu,H.,Zhou,S.,Yu,J.,Keller,N.P.,Chen,L.,and Yin,W.B.(2018).Deletion of a global regulator LaeB leads to the discovery of novel polyketides in Aspergillus nidulans.Org Biomol Chem 16,4973-4976.
Ma,H.,Kunes,S.,Schatz,P.J.,and Botstein,D.(1987).Plasmid construction by homologous recombination in yeast.Gene 58,201-216.
Ma,Z.,Li,W.,Zhang,P.,Lyu,H.,Hu,Y.,and Yin,W.B.(2018).Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans.Appl Microbiol Biotechnol 102,297-304.
Mayorga,M.E.,and Timberlake,W.E.(1990).Isolation and molecular characterization of the Aspergillus nidulans wA gene.Genetics 126,73-79.
Muller,H.,Annaluru,N.,Schwerzmann,J.W.,Richardson,S.M.,Dymond,J.S.,Cooper,E.M.,Bader,J.S.,Boeke,J.D.,and Chandra-segaran,S.(2012).Assembling large DNA segments in yeast.In Gene Synthesis:Methods and Protocols,J.Peccoud,ed.(New Jersey:Humana Press),pp.133-150.
Niu,G.,Zheng,J.,and Tan,H.(2017).Biosynthesis and combinatorial biosynthesis of antifungal nucleoside antibiotics.Sci China Life Sci 60,939-947.
Paques,F.,and Haber,J.E.(1999).Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae.Microbiol Mol Biol Rev 63,349-404.
Reen,F.J.,Romano,S.,Dobson,A.D.W.,and O'Gara,F.(2015).The sound of silence:activating silent biosynthetic gene clusters in marine microorganisms.Mar Drugs 13,4754-4783.
Shimizu,K.,and Keller,N.P.(2001).Genetic involvement of a cAMP-dependent protein kinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans.Genetics 157,591-600.
Shinohara,A.,and Ogawa,T.(1995).Homologous recombination and the roles of double-strand breaks.Trends Biochem Sci 20,387-391.
Wiemann,P.,and Keller,N.P.(2014).Strategies for mining fungal natural products.J Ind Microbiol Biotechnol 41,301-313.
Wu,G.,Zhou,H.,Zhang,P.,Wang,X.,Li,W.,Zhang,W.,Liu,X.,Liu,H.W.,Keller,N.P.,An,Z.,et al.(2016).Polyketide production of pestaloficiols and macrodiolide ficiolides revealed by manipulations of epigenetic regulators in an endophytic fungus.Org Lett 18,1832-1835.
Xu,X.,Liu,L.,Zhang,F.,Wang,W.,Li,J.,Guo,L.,Che,Y.,and Liu,G.(2014).Identification of the first diphenyl ether gene cluster for pestheic acid biosynthesis in plant endophyte Pestalotiopsis fici.ChemBioChem15,284-292.
Yin,W.B.,Chooi,Y.H.,Smith,A.R.,Cacho,R.A.,Hu,Y.,White,T.C.,and Tang,Y.(2013).Discovery of Cryptic Polyketide Metabolites from Dermatophytes Using Heterologous Expression in Aspergillus nidulans.ACS Synth Biol 2,629-634.
Zhang,P.,Wang,X.,Fan,A.,Zheng,Y.,Liu,X.,Wang,S.,Zou,H.,Oakley,B.R.,Keller,N.P.,and Yin,W.B.(2017).A cryptic pigment biosynthetic pathway uncovered by heterologous expression is essential for conidial development in Pestalotiopsis fici.Mol Microbiol 105,469-483.
Zhang,A.,P.Lu,A.M.Dahl-Roshak,P.S.Paress,S.Kennedy,J.S.Tkacz,and An,Z.Q.(2003).Efficient disruption of a polyketide synthase gene(pks1)required for melanin synthesis through Ag-robacterium-mediated transformation of Glarea lozoyeasis.Mol Genet Genomics 268,645-655.
Zheng,Y.,Ma,K.,Lyu,H.,Huang,Y.,Liu,H.,Liu,L.,Che,Y.,Liu,X.,Zou,H.,and Yin,W.B.(2017a).Genetic manipulation of the COP9signalosome subunit PfCsnE leads to the discovery of pestaloficins in Pestalotiopsis fici.Org Lett 19,4700-4703.
Zheng,Y.,Wang,X.,Zhang,X.,Li,W.,Liu,G.,Wang,S.,Yan,X.,Zou,H.,and Yin,W.B.(2017b).Cop9 signalosome subunit pfcsne regulates secondary metabolism and conidial formation in pestalotiopsis fici.Sci China Life Sci 60,656-664.
Zhou,S.,Zhang,P.,Zhou,H.,Liu,X.,Li,S.M.,Guo,L.,Li,K.,and Yin,W.B.(2019).A new regulator RsdA mediating fungal secondary metabolism has a detrimental impact on asexual development in Pestalotiopsis fici.Environ Microbiol 21,4163-426.
Zhuo,J.,Ma,B.,Xu,J.,Hu,W.,Zhang,J.,Tan,H.,and Tian,Y.(2017).Reconstruction of a hybrid nucleoside antibiotic gene cluster based on scarless modification of large DNA fragments.Sci China Life Sci 60,968-979.
Blackwell,M.(2011).The Fungi:1,2,3…5.1 million species?Am J Bot98,426-438.
Brakhage,A.A.,and Schroeckh,V.(2011).Fungal secondary metabolitesStrategies to activate silent gene clusters.Fungal Genets Biol 48,15-22.
Chaffin,D.O.,and Rubens,C.E.(1998).Blue/white screening of recombinant plasmids in Gram-positive bacteria by interruption of alkaline phosphatase gene(phoZ)expression.Gene 219,91-99.
Clevenger,K.D.,Bok,J.W.,Ye,R.,Miley,G.P.,Verdan,M.H.,Velk,T.,Chen,C.,Yang,K.H.,Robey,M.T.,Gao,P.,et al.(2017).A scalable platform to identify fungal secondary metabolites and their gene clusters.Nat Chem Biol 13,895-901.
Clutterbuck,P.W.,Lovell,R.,and Raistrick,H.(1932).Studies in the biochemistry of micro-organisms.Biochem J 26,1907-1918.
Cohen,S.N.,Chang,A.C.Y.,Boyer,H.W.,and Helling,R.B.(1973).Construction of biologically functional bacterial plasmids in vitro.Proc Natl Acad Sci USA 70,3240-3244.
Fan,A.,Mi,W.,Liu,Z.,Zeng,G.,Zhang,P.,Hu,Y.,Fang,W.,and Yin,W.B.(2017).Deletion of a Histone Acetyltransferase Leads to the Pleiotropic Activation of Natural Products in Metarhizium robertsii.Org Lett 19,1686-1689.
Fekete,E.,Karaffa,L.,Seiboth,B.,Fekete,é.,P.Kubicek,C.,and Flipphi,M.(2012).Identification of a permease gene involved in lactose utilisation in Aspergillus nidulans.Fungal Genets Biol 49,415-425.
Fu,J.,Bian,X.,Hu,S.,Wang,H.,Huang,F.,Seibert,P.M.,Plaza,A.,Xia,L.,Müller,R.,Stewart,A.F.,et al.(2012).Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting.Nat Biotechnol 30,440-446.
Gibson,D.G.,Young,L.,Chuang,R.Y.,Venter,J.C.,Hutchison,C.A.,and Smith,H.O.(2009).Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat Methods 6,343-345.
Han,X.,Chakrabortti,A.,Zhu,J.,Liang,Z.X.,and Li,J.(2016).Sequencing and functional annotation of the whole genome of the filamentous fungus Aspergillus westerdijkiae.BMC Genomics 17,633.
Inglis,D.O.,Binkley,J.,Skrzypek,M.S.,Arnaud,M.B.,Cerqueira,G.C.,Shah,P.,Wymore,F.,Wortman,J.R.,and Sherlock,G.(2013).Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans,A.fumigatus,A.niger and A.oryzae.BMC Microbiol 13,91.
Khaldi,N.,Seifuddin,F.T.,Turner,G.,Haft,D.,Nierman,W.C.,Wolfe,K.H.,and Fedorova,N.D.(2010).SMURF:Genomic mapping of fungal secondary metabolite clusters.Fungal Genets Biol 47,736-741.
Kouprina,N.,and Larionov,V.(2008).Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae.Nat Protoc 3,371-377.
Kupiec,M.(2000).Damage-induced recombination in the yeast Saccharomyces cerevisiae.Mutat Res-Fund Mol M 451,91-105.
Larionov,V.,Kouprina,N.,Graves,J.,Chen,X.N.,Korenberg,J.R.,and Resnick,M.A.(1996).Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination..Proc Natl Acad Sci USA 93,491-496.
Li,W.,Fan,A.,Wang,L.,Zhang,P.,Liu,Z.,An,Z.,and Yin,W.B.(2018).Asperphenamate biosynthesis reveals a novel two-module NRPSsystem to synthesize amino acid esters in fungi.Chem Sci 9,2589-2594.
Lin,H.,Lyu,H.,Zhou,S.,Yu,J.,Keller,N.P.,Chen,L.,and Yin,W.B.(2018).Deletion of a global regulator LaeB leads to the discovery of novel polyketides in Aspergillus nidulans.Org Biomol Chem 16,4973-4976.
Ma,H.,Kunes,S.,Schatz,P.J.,and Botstein,D.(1987).Plasmid construction by homologous recombination in yeast.Gene 58,201-216.
Ma,Z.,Li,W.,Zhang,P.,Lyu,H.,Hu,Y.,and Yin,W.B.(2018).Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans.Appl Microbiol Biotechnol 102,297-304.
Mayorga,M.E.,and Timberlake,W.E.(1990).Isolation and molecular characterization of the Aspergillus nidulans wA gene.Genetics 126,73-79.
Muller,H.,Annaluru,N.,Schwerzmann,J.W.,Richardson,S.M.,Dymond,J.S.,Cooper,E.M.,Bader,J.S.,Boeke,J.D.,and Chandra-segaran,S.(2012).Assembling large DNA segments in yeast.In Gene Synthesis:Methods and Protocols,J.Peccoud,ed.(New Jersey:Humana Press),pp.133-150.
Niu,G.,Zheng,J.,and Tan,H.(2017).Biosynthesis and combinatorial biosynthesis of antifungal nucleoside antibiotics.Sci China Life Sci 60,939-947.
Paques,F.,and Haber,J.E.(1999).Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae.Microbiol Mol Biol Rev 63,349-404.
Reen,F.J.,Romano,S.,Dobson,A.D.W.,and O'Gara,F.(2015).The sound of silence:activating silent biosynthetic gene clusters in marine microorganisms.Mar Drugs 13,4754-4783.
Shimizu,K.,and Keller,N.P.(2001).Genetic involvement of a cAMP-dependent protein kinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans.Genetics 157,591-600.
Shinohara,A.,and Ogawa,T.(1995).Homologous recombination and the roles of double-strand breaks.Trends Biochem Sci 20,387-391.
Wiemann,P.,and Keller,N.P.(2014).Strategies for mining fungal natural products.J Ind Microbiol Biotechnol 41,301-313.
Wu,G.,Zhou,H.,Zhang,P.,Wang,X.,Li,W.,Zhang,W.,Liu,X.,Liu,H.W.,Keller,N.P.,An,Z.,et al.(2016).Polyketide production of pestaloficiols and macrodiolide ficiolides revealed by manipulations of epigenetic regulators in an endophytic fungus.Org Lett 18,1832-1835.
Xu,X.,Liu,L.,Zhang,F.,Wang,W.,Li,J.,Guo,L.,Che,Y.,and Liu,G.(2014).Identification of the first diphenyl ether gene cluster for pestheic acid biosynthesis in plant endophyte Pestalotiopsis fici.ChemBioChem15,284-292.
Yin,W.B.,Chooi,Y.H.,Smith,A.R.,Cacho,R.A.,Hu,Y.,White,T.C.,and Tang,Y.(2013).Discovery of Cryptic Polyketide Metabolites from Dermatophytes Using Heterologous Expression in Aspergillus nidulans.ACS Synth Biol 2,629-634.
Zhang,P.,Wang,X.,Fan,A.,Zheng,Y.,Liu,X.,Wang,S.,Zou,H.,Oakley,B.R.,Keller,N.P.,and Yin,W.B.(2017).A cryptic pigment biosynthetic pathway uncovered by heterologous expression is essential for conidial development in Pestalotiopsis fici.Mol Microbiol 105,469-483.
Zhang,A.,P.Lu,A.M.Dahl-Roshak,P.S.Paress,S.Kennedy,J.S.Tkacz,and An,Z.Q.(2003).Efficient disruption of a polyketide synthase gene(pks1)required for melanin synthesis through Ag-robacterium-mediated transformation of Glarea lozoyeasis.Mol Genet Genomics 268,645-655.
Zheng,Y.,Ma,K.,Lyu,H.,Huang,Y.,Liu,H.,Liu,L.,Che,Y.,Liu,X.,Zou,H.,and Yin,W.B.(2017a).Genetic manipulation of the COP9signalosome subunit PfCsnE leads to the discovery of pestaloficins in Pestalotiopsis fici.Org Lett 19,4700-4703.
Zheng,Y.,Wang,X.,Zhang,X.,Li,W.,Liu,G.,Wang,S.,Yan,X.,Zou,H.,and Yin,W.B.(2017b).Cop9 signalosome subunit pfcsne regulates secondary metabolism and conidial formation in pestalotiopsis fici.Sci China Life Sci 60,656-664.
Zhou,S.,Zhang,P.,Zhou,H.,Liu,X.,Li,S.M.,Guo,L.,Li,K.,and Yin,W.B.(2019).A new regulator RsdA mediating fungal secondary metabolism has a detrimental impact on asexual development in Pestalotiopsis fici.Environ Microbiol 21,4163-426.
Zhuo,J.,Ma,B.,Xu,J.,Hu,W.,Zhang,J.,Tan,H.,and Tian,Y.(2017).Reconstruction of a hybrid nucleoside antibiotic gene cluster based on scarless modification of large DNA fragments.Sci China Life Sci 60,968-979.