一个源于皮革肾岛衣地衣型真菌中的HR-PKS基因克隆与鉴定
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摘要
本研究通过对皮革肾岛衣(Nephrmopsis pallescens)地衣型真菌转录组数据的分析,首次克隆得到一种高度还原型聚酮合酶(Highly reducing PKS)基因全长,并对该基因进行生物信息学分析,并检测该基因在不同培养基上的表达量。该基因cDNA全长7 491 bp (命名为NpPKS1),可编码2 496个氨基酸,是一种稳定存在于细胞质基质中的非分泌蛋白;结构域与其他真菌的洛伐他汀九酮合成酶(LNKS)结构域极为相似,且聚类分析与其他真菌的洛伐他汀九酮合成酶(LNKS)聚为一类;在以葡萄糖和麦芽糖作为碳源的情况下,番茄浸粉、牛肉浸粉、酪蛋白胨等作为氮源可强烈刺激NpPKS1基因的表达,而胰蛋白胨对该基因的表达存在一定程度的抑制效应。本研究为皮革肾岛衣地衣型真菌中的基因资源利用、聚酮化合物异源表达和其合成机制的研究奠定了基础。
The present study cloned the full length of a highly reducing PKS gene through analyzing the transcriptome data of Lichenized-fungi of Nephrmopsis pallescens, simultaneously took a bioinformatics analysis to this gene, and tested the gene expression level in different medium. The cDNA of this gene had a total length of 7 491 bp(named NpPKS1), could encode 2 496 amino acids, was a stable non-secretory protein locating in cytoplasmic matrix. The domain was very similar to that of lovastatin nonaketide synthase(LNKS) of other fungi, and cluster analysis was the same as that of LNKS of other fungi. With glucose and maltose as carbon sources, tomato extract,beef extract and casein peptone as nitrogen sources could strongly stimulate the expression of NpPKS1 gene.Inversely, tryptone had certain inhibitory effect to the gene expression. The present study could lay a foundation for the study of gene resource utilization, heterologous expression and the biosynthesis mechanism of polyketide in Lichenized-fungi of Nephrmopsis pallescens.
The present study cloned the full length of a highly reducing PKS gene through analyzing the transcriptome data of Lichenized-fungi of Nephrmopsis pallescens, simultaneously took a bioinformatics analysis to this gene, and tested the gene expression level in different medium. The cDNA of this gene had a total length of 7 491 bp(named NpPKS1), could encode 2 496 amino acids, was a stable non-secretory protein locating in cytoplasmic matrix. The domain was very similar to that of lovastatin nonaketide synthase(LNKS) of other fungi, and cluster analysis was the same as that of LNKS of other fungi. With glucose and maltose as carbon sources, tomato extract,beef extract and casein peptone as nitrogen sources could strongly stimulate the expression of NpPKS1 gene.Inversely, tryptone had certain inhibitory effect to the gene expression. The present study could lay a foundation for the study of gene resource utilization, heterologous expression and the biosynthesis mechanism of polyketide in Lichenized-fungi of Nephrmopsis pallescens.
引文
Abe Y.,Su zuki T.,Ono C.,Iwamoto K.,Hosobuchi M.,and Yoshikawa H.,2002,Molecular cloning and characterization of an ML-236B(compactin)biosynthetic gene cluster in Penicillium citrinum,Mol.Genet.Genomics,267(5):636-646
Alberts A.W.,Chen J.,Kuron G.,Hunt V.,Huff J.,Hoffman C.,Rothrock J.,Lopez M.,Joshua H.,Harris E.,Patchett A.,Monaghan R.,Currie S.,Stapley E.,Albers-Schonberg G.,Hensens O.,Hirshfield J.,Hoogsteen K.,Liesch J.,and Springer J.,1980,Mevinolin:A highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent,Proc.Natl.Acad.Sci.USA,77(7):3957-3961
Behera B.C.,Verma N.,Sonone A.,and Makhija U.,2005,Evaluation of antioxidant potential of the cultured mycobiont of a lichen Usnea ghattensis,Phytother.Res.,19(1):58-64
Chen J.B.,1994,Understanding and thinking of concept and definition of“Lichen”,Shengwu Duoyangxing(Chinese Biodibersity),2(4):228-230(陈健斌,1994,地衣二元性及其概念的双重理解与思考,生物多样性,2(4):228-230)
Cox R.J.,2007,Polyketi des,proteins and genes in fungi:programmed nano-machines begin to reveal their secrets,Org.Biomol.Chem.,5(13):2010-2026
Crittenden P.D.,and Porter N.,1991,Lichen-forming fungi:potential sources of novel metabolites,Trends Biotechnol.,9(12):409-414
Feng G.H.,and Leonard T.J.,1998,Culture conditions control expression of the genes for aflatoxin and sterigmatocystin biosynthesis in Aspergillus parasiticus and A.nidulans,Appl.Environ.Microbiol.,64(6):2275-2277
Halo L.M.,Marshall J.W.,Yakasai A.A.,Song Z.,Butts C.P.,Crump M.P.,Heneghan M.,Bailey A.M.,Simpson T.J.,Lazarus C.M.,and Cox R.J.,2008,Authentic heterologous expression of the tenellin iterative polyketide synthase nonribosomal peptide synthetase requires coexpression with an enoyl reductase,Chem.Biol.Chem.,9(4):585-594
Hendrickson L.,Davis C.R.,Roach C.,Nguyen D.K.,Aldrich T.,Mcada P.C.,and Reeves C.D.,1999,Lovastatin biosynthesis in Aspergillus terreus:characterization of blocked mutants,enzyme activities and a multifunctional polyketide synthase gene,Chem.Biol.,6(7):429-439
Hertweck C.,2009,The biosynthetic logic of polyketide diversity,Angew.Chem.Int.Ed.Engl.,48(26):4688-4716
Honegger R.,1984,Cytological aspects of the mycobiont-phycobiont relationship in lichens.Haustorial types,phycobiont cell wall types,and the ultrastructure of the cell surface layers in some cultured and symbiotic myco-and phycobionts,Lichenologist,16(2):111-127
Kennedy J.,Auclair K.,Kendrew S.G.,Park C.,Vederas J.C.,and Hutchinson C.R.,1999,Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis,Science,284(5418):1368-1372
Kroken S.,Glass N.L.,Taylor J.W.,Yoder O.C.,and Turgeon B.G.,2003,Phylogenomic analysis of typeⅠpolyketide synthase genes in pathogenic and saprobic ascomycetes,Proc.Natl.Acad.Sci.USA,100(26):15670-15675
Liu N.,Hung Y.S.,Gao S.S.,Hang L.,Zou Y.,Chooi Y.H.,and Tang Y.,2017,Identification and heterologous production of a benzoyl-primed tricarboxylic acid polyketide intermediate from the zaragozic acid a biosynthetic pathway,Org.Lett.,19(13):3560-3563
Ma S.M.,Li J.W.,Choi J.W.,Zhou H.,Lee K.K.,Moorthie V.A.,Xie X.,Kealey J.T.,Silva N.A.D.,Vederas J.C.,and Tang Y.,2009,Complete reconstitution of a highly-reducing iterative polyketide synthase,Science,326(5952):589-592
Miao V.,Coffet-leGal M.F.,Brown D.,Sinnemann S.,Donaldson G.,and Davies J.,2001,Genetic approaches to harvesting lichen products,Trends Biotechnol.,19(9):349-355
Oksanen I.,2006,Ecological and biotechnological aspects of lichens,Appl.Microbiol.Biotechnol.,73(4):723-734
Sato M.,Dander J.E.,Sato C.,Hung Y.S.,Gao S.S.,Tang M.C.,Hang L.,Winter J.M.,Garg N.K.,Watanabe K.,and Tang Y.,2017,Collaborative biosynthesis of maleimide-and succinimide-containing natural products by fungal polyketide megasynthases,J.Am.Chem.Soc.,139(15):5317-5320
Scherlach K.,Boettger D.,Remme N.,and Hertweck C.,2010,The chemistry and biology of cytochalasans,Nat.Prod.Res.,27(6):869-886
Shen B.,2003,Polyketide biosynthesis beyond the typeⅠ,ⅡandⅢpolyketide synthase paradigms,Curr.Opin.Chem.Biol.,7(2):285-295
Stocker-Worgotter E.,2008,Metabolic diversity of lichen-forming ascomycetous fungi:culturing,polyketide and shikimate metabolite production,and PKS genes,Nat.Prod.Rep.,25(1):188-200
Wang Y.,Kim J.A.,Cheong Y.H.,Koh Y.J.,and Hur J.S.,2012,Isolation and characterization of a non-reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima,Mycol.Progress,11(1):75-83
Wang Y.,Zhou X.,Hu Z.X.,and Wang J.,2014,Isolation and characterizaion of a polyketide synthase gene cluster from Usnea longissima,Weishengwu Xuebao(Acta Microbilogica Sinica),54(7):770-777(王毅,周旭,许宰铣,王娟,2014,长松萝中一个聚酮合酶基因簇的克隆和鉴定,微生物学报,54(7):770-777)
Yamamoto Y.,2010,Tissue culture of lichens,Plant Biotechnol.,5(1):44-46
Zhang J.N.,Ding W.Y.,and Yang C.,2016,Effects of fermentation conditions of monascus lovastatin production,Nongchanpin Jiagong(Farm Products Processing),(5):50-52(张江宁,丁卫英,杨春,2016,发酵条件对红曲产洛伐他汀的影响,农产品加工,(5):50-52)
Zhang T.,Almagul B.,Zheng J.S.,Han X.F.,Ren H.,and Jiang D.H.,2016,Screening and identification of Aspergillus clavatus strain with high-yielding lovastatin and its optimization of fermentation conditions,Gongye Weishengwu(Induastrial Microbiology),46(6):24-33(章婷,Almagul B.,郑婕施,韩肖飞,任浩,蒋冬花,2016,高产洛伐他汀棒曲霉菌株的筛选,鉴定及发酵条件优化,工业微生物,46(6):24-33)
Alberts A.W.,Chen J.,Kuron G.,Hunt V.,Huff J.,Hoffman C.,Rothrock J.,Lopez M.,Joshua H.,Harris E.,Patchett A.,Monaghan R.,Currie S.,Stapley E.,Albers-Schonberg G.,Hensens O.,Hirshfield J.,Hoogsteen K.,Liesch J.,and Springer J.,1980,Mevinolin:A highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent,Proc.Natl.Acad.Sci.USA,77(7):3957-3961
Behera B.C.,Verma N.,Sonone A.,and Makhija U.,2005,Evaluation of antioxidant potential of the cultured mycobiont of a lichen Usnea ghattensis,Phytother.Res.,19(1):58-64
Chen J.B.,1994,Understanding and thinking of concept and definition of“Lichen”,Shengwu Duoyangxing(Chinese Biodibersity),2(4):228-230(陈健斌,1994,地衣二元性及其概念的双重理解与思考,生物多样性,2(4):228-230)
Cox R.J.,2007,Polyketi des,proteins and genes in fungi:programmed nano-machines begin to reveal their secrets,Org.Biomol.Chem.,5(13):2010-2026
Crittenden P.D.,and Porter N.,1991,Lichen-forming fungi:potential sources of novel metabolites,Trends Biotechnol.,9(12):409-414
Feng G.H.,and Leonard T.J.,1998,Culture conditions control expression of the genes for aflatoxin and sterigmatocystin biosynthesis in Aspergillus parasiticus and A.nidulans,Appl.Environ.Microbiol.,64(6):2275-2277
Halo L.M.,Marshall J.W.,Yakasai A.A.,Song Z.,Butts C.P.,Crump M.P.,Heneghan M.,Bailey A.M.,Simpson T.J.,Lazarus C.M.,and Cox R.J.,2008,Authentic heterologous expression of the tenellin iterative polyketide synthase nonribosomal peptide synthetase requires coexpression with an enoyl reductase,Chem.Biol.Chem.,9(4):585-594
Hendrickson L.,Davis C.R.,Roach C.,Nguyen D.K.,Aldrich T.,Mcada P.C.,and Reeves C.D.,1999,Lovastatin biosynthesis in Aspergillus terreus:characterization of blocked mutants,enzyme activities and a multifunctional polyketide synthase gene,Chem.Biol.,6(7):429-439
Hertweck C.,2009,The biosynthetic logic of polyketide diversity,Angew.Chem.Int.Ed.Engl.,48(26):4688-4716
Honegger R.,1984,Cytological aspects of the mycobiont-phycobiont relationship in lichens.Haustorial types,phycobiont cell wall types,and the ultrastructure of the cell surface layers in some cultured and symbiotic myco-and phycobionts,Lichenologist,16(2):111-127
Kennedy J.,Auclair K.,Kendrew S.G.,Park C.,Vederas J.C.,and Hutchinson C.R.,1999,Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis,Science,284(5418):1368-1372
Kroken S.,Glass N.L.,Taylor J.W.,Yoder O.C.,and Turgeon B.G.,2003,Phylogenomic analysis of typeⅠpolyketide synthase genes in pathogenic and saprobic ascomycetes,Proc.Natl.Acad.Sci.USA,100(26):15670-15675
Liu N.,Hung Y.S.,Gao S.S.,Hang L.,Zou Y.,Chooi Y.H.,and Tang Y.,2017,Identification and heterologous production of a benzoyl-primed tricarboxylic acid polyketide intermediate from the zaragozic acid a biosynthetic pathway,Org.Lett.,19(13):3560-3563
Ma S.M.,Li J.W.,Choi J.W.,Zhou H.,Lee K.K.,Moorthie V.A.,Xie X.,Kealey J.T.,Silva N.A.D.,Vederas J.C.,and Tang Y.,2009,Complete reconstitution of a highly-reducing iterative polyketide synthase,Science,326(5952):589-592
Miao V.,Coffet-leGal M.F.,Brown D.,Sinnemann S.,Donaldson G.,and Davies J.,2001,Genetic approaches to harvesting lichen products,Trends Biotechnol.,19(9):349-355
Oksanen I.,2006,Ecological and biotechnological aspects of lichens,Appl.Microbiol.Biotechnol.,73(4):723-734
Sato M.,Dander J.E.,Sato C.,Hung Y.S.,Gao S.S.,Tang M.C.,Hang L.,Winter J.M.,Garg N.K.,Watanabe K.,and Tang Y.,2017,Collaborative biosynthesis of maleimide-and succinimide-containing natural products by fungal polyketide megasynthases,J.Am.Chem.Soc.,139(15):5317-5320
Scherlach K.,Boettger D.,Remme N.,and Hertweck C.,2010,The chemistry and biology of cytochalasans,Nat.Prod.Res.,27(6):869-886
Shen B.,2003,Polyketide biosynthesis beyond the typeⅠ,ⅡandⅢpolyketide synthase paradigms,Curr.Opin.Chem.Biol.,7(2):285-295
Stocker-Worgotter E.,2008,Metabolic diversity of lichen-forming ascomycetous fungi:culturing,polyketide and shikimate metabolite production,and PKS genes,Nat.Prod.Rep.,25(1):188-200
Wang Y.,Kim J.A.,Cheong Y.H.,Koh Y.J.,and Hur J.S.,2012,Isolation and characterization of a non-reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima,Mycol.Progress,11(1):75-83
Wang Y.,Zhou X.,Hu Z.X.,and Wang J.,2014,Isolation and characterizaion of a polyketide synthase gene cluster from Usnea longissima,Weishengwu Xuebao(Acta Microbilogica Sinica),54(7):770-777(王毅,周旭,许宰铣,王娟,2014,长松萝中一个聚酮合酶基因簇的克隆和鉴定,微生物学报,54(7):770-777)
Yamamoto Y.,2010,Tissue culture of lichens,Plant Biotechnol.,5(1):44-46
Zhang J.N.,Ding W.Y.,and Yang C.,2016,Effects of fermentation conditions of monascus lovastatin production,Nongchanpin Jiagong(Farm Products Processing),(5):50-52(张江宁,丁卫英,杨春,2016,发酵条件对红曲产洛伐他汀的影响,农产品加工,(5):50-52)
Zhang T.,Almagul B.,Zheng J.S.,Han X.F.,Ren H.,and Jiang D.H.,2016,Screening and identification of Aspergillus clavatus strain with high-yielding lovastatin and its optimization of fermentation conditions,Gongye Weishengwu(Induastrial Microbiology),46(6):24-33(章婷,Almagul B.,郑婕施,韩肖飞,任浩,蒋冬花,2016,高产洛伐他汀棒曲霉菌株的筛选,鉴定及发酵条件优化,工业微生物,46(6):24-33)