春雷霉素低产和高产菌株的比较基因组分析
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摘要
氨基糖苷类抗生素春雷霉素由春雷链霉菌和小金色链霉菌产生,广泛应用于农业病害的防治。本研究利用二代和三代测序技术相结合的策略对低产的春雷链霉菌BCRC12349(Streptomyces kasugaensis BCRC12349,LY)和高产的小金色链霉菌XM301(S.microaureus XM301,HY)进行了全基因组测序,发现高产菌株染色体比低产菌株小了将近200 kb。比较基因组分析发现,与低产菌株相比,高产菌株中存在41个插入缺失(In Del)和164个单碱基突变位点(SNV)。基于RNA-seq的比较转录组分析发现,高产菌株中的一些初级代谢关键基因、春雷霉素前体合成相关基因、春雷霉素生物合成基因簇内部基因的转录发生了上调。同时发现基因组中调控基因有44个显著上调、16个显著下调,转运蛋白基因有32个显著上调、11个显著下调。高产菌株和低产菌株基因组和转录组的比较分析为春雷霉素产生菌的进一步高产改造提供了理论基础。
Aminoglycoside antibiotics Kasugamycin, produced by Streptomyces kasugaensis and S. microaureus,is widely used in the prevention and control of agricultural diseases. The whole genomes of the low-yielding S.kasugaensis BCRC12349(LY) and high-yielding S. microaureus XM301(HY) were sequenced by combining the second and third generation sequencing technologies, which indictaed that the chromosomes of high-yielding strains were nearly 200 kb smaller than those of low-yielding strains. Through comparative genomic analysis, it revealed that there were 41 In Dels and 164 single nucleotide variations(SNVs) in S. microaureus XM301(HY).Comparative genome analysis based on RNA-seq indicated that the expression of genes in high-yielding strains involved in primary metabolic pathway, kasugamycin precursor synthesis and most kasugamycin biosynthetic genes was up-regulated. Moreover, in the genome, 42 genes were significantly up-regulated and 16 down-regulated of regulatory genes while 32 genes were significantly up-regulated and 11 down-regulated of transporter genes.The comparative genome and transcriptome analysis between the low-yielding and high-yielding strains would provide theoretical basis for further transformation towards high-yielding of Kasugamycin strains.
Aminoglycoside antibiotics Kasugamycin, produced by Streptomyces kasugaensis and S. microaureus,is widely used in the prevention and control of agricultural diseases. The whole genomes of the low-yielding S.kasugaensis BCRC12349(LY) and high-yielding S. microaureus XM301(HY) were sequenced by combining the second and third generation sequencing technologies, which indictaed that the chromosomes of high-yielding strains were nearly 200 kb smaller than those of low-yielding strains. Through comparative genomic analysis, it revealed that there were 41 In Dels and 164 single nucleotide variations(SNVs) in S. microaureus XM301(HY).Comparative genome analysis based on RNA-seq indicated that the expression of genes in high-yielding strains involved in primary metabolic pathway, kasugamycin precursor synthesis and most kasugamycin biosynthetic genes was up-regulated. Moreover, in the genome, 42 genes were significantly up-regulated and 16 down-regulated of regulatory genes while 32 genes were significantly up-regulated and 11 down-regulated of transporter genes.The comparative genome and transcriptome analysis between the low-yielding and high-yielding strains would provide theoretical basis for further transformation towards high-yielding of Kasugamycin strains.
引文
Cho H.,Choi D.B.,and Lim C.K.,2008,Mutagenesis of Streptomyces kasugaensis for kasugamycin production,Journal of Environmental and Sanitary Engineering,23(4):23-27
Fu G.J.,Liu J.F.,Wang H.H.,Huang X.G.,Cao F.J.,and Zhang Y.X.,2013,Mutation and fermentation of kasugamycin high-yield strain by injecting nitrogen ion,Shenyang Gongye Xuebao(Journal of Shenyang Pharmaceutical University),30(12):977-981(付桂杰,刘俊芳,王海慧,黄秀果,曹凤娟,张怡轩,2013,氮离子注入法选育春雷霉素高产菌株和生产发酵,沈阳工业学报,30(12):977-981)
Fukagawa Y.,Sawa T.,Homma I.,Takeuchi T.,and Umezawa H.,1968,Studies on biosynthesis of kasugamycin.V.Biosynthesis of the amidine group,Journal of Antibiotics(Tokyo),21(6):410-412
Fukagawa Y.,Sawa T.,Homma I.,Takeuchi T.,and Umezawa H.,1968,Studies on biosynthesis of kasugamycin.IV.Biosynthesis of the kasugamine moiety from[1-14C]-glucosamine and[1,2 or 6-14C]-glucose,Journal of Antibiotics(Tokyo),21(5):358-360
Fukagawa Y.,Sawa T.,Takeuchi T.,and Umezawa H.,1968,Studies on biosynthesis of kasugamycin.I Biosynthesis of kasugamycin and the kasugamine moiety,Journal of Antibiotics(Tokyo),21(1):50-54
Fukagawa Y.,Sawa T.,Takeuchi T.,and Umezawa H.,1968,Studies on biosynthesis of kasugamycin.ⅢBiosynthesis of the D-inositol moiety,Journal of Antibiotics(Tokyo),21(3):185-188
Ikeno S.,Aoki D.,Hamada M.,Hori M.,and Tsuchiya K.S.,2006,DNA sequencing and transcriptional analysis of the kasugamycin biosynthetic gene cluster from Streptomyces kasugaensis M338-M1,Journal of Antibiotics,59(1):18-28
Ikeno S.,Tsuji T.,Higashide K.,Kinoshita N.,Hamada M.,and Hori M.,1998,A7.6 kb DNA region from Streptomyces kasugaensis M338-M1 includes some genes responsible for kasugamycin biosynthesis,Journal of Antibiotics(Tokyo),51(3):341-352
Jin C.X.,Piao S.A.,Cui Y.N.,Jing X.,and Guo X.W.,2004,Screening and fermentation processing control of US95#-a high yielding Chunlei Mycin bacterial strain,Shengwuxue Zazhi(Journal of Biology),21(5):34-35(金昌燮,朴顺爱,崔英兰,金星,郭小威,2004,春雷霉素高产菌株US95#的选育及大罐发酵工艺控制,生物学杂志,21(5):34-35)
Kasuga K.,Sasaki A.,Matsuo T.,Yamamoto C.,Minato Y.,Kuwahara N.,Fujii C.,Kobayashi M.,Agematu H.,Tamura T.,Komatsu M.,Ishikawa J.,Ikeda H.,and Kojima I.,2017,Heterologous production of kasugamycin,an aminoglycoside antibiotic from Streptomyces kasugaensis,in Streptomyces lividans and Rhodococcus erythropolis L-88 by constitutive expression of the biosynthetic gene cluster,Applied Microbiology and Biotechnology,101(10):4259-4268
Kim C.J.,Chang Y.K.,Chun G.T.,Jeong Y.H.,and Lee S.J.,2001,Continuous culture of immobilized Streptomyces cells for kasugamycin production,Biotechnology Progress,17(3):453-461
Kim C.J.,Chang Y.K.,and Chun G.T.,2000,Enhancement of kasugamycin production by p H shock in batch cultures of Streptomyces kasugaensis,Biotechnology Progress,16(4):548-552
Lee J.,Hwang Y.,Kim S.,Kim E.,and Choi C.,2000,Effect of a global regulatory gene,afs R2,from Streptomyces lividans on avermectin production in Streptomyces avermitilis,Journal of Bioscience and Bioengineering,89(6):606-608
Marnix H.M.,Kai B.,Peter C.,Victor D.J.,Piotr Z.,Michael A.F.,Tilmann W.,Eriko T.,and Rainer B.,2011,Anti SMASH:rapid identification,annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences,Nucleic Acids Research,39(Web Server issue):W339-W346
Medini D.,Serruto D.,Parkhill J.,Relman D.A.,Donati C.,Moxon R.,Falkow S.,and Rappuoli R.,2008,Microbiology in the post-genomic era,Nature Reviews Microbiology,6(6):419-430
Mortazavi A.,Williams B.A.,Mc Cue K.,Schaeffer L.,and Wold B.,2008,Mapping and quantifying mmaalian transcriptomes by RNA-Seq,Nature Methods,5(7):621-628
Qi Y.X.,Liu Y.B.,and Rong W.H.,2011,RNA-Seq and its applications:a new technology for transcriptomics,Yichuan(Hereditas),33(11):1191-1202(祁云霞,刘永斌,荣威恒,2011,转录组研究新技术:RNA-Seq及其应用,遗传,33(11):1191-1202)
Tan G.Y.,Peng Y.,Lu C.,Bai L.,and Zhong J.J.,2015,Engineering validamycin production by tandem deletion of gamma-butyrolactone receptor genes in Streptomyces hygroscopicus 5008,Metabolic Engineering,28:74-81
Tilmann W.,Kai B.,Srikanth D.,Daniel K.,Hyun U.K.,Robert B.,Sang Y.L.,Michael A.F.,Rolf M.,Wolfgang W.,Rainer B.,Eriko T.,and Marnix H.M.,2015,Anti SMASH3.0-acomprehensive resource for the genome mining of biosynthetic gene clusters,Nucleic Acids Research,43(Web Server issue):W237
Umezawa H.,Hamada M.,Suhara Y.,Hashimoto T.,and Ikekawa T.,1965,Kasugamycin,a new antibiotic,Antimicrob Agents Chemother(Bethesda),5:753-757
Wang G.,Wu Y.,Yuan Z.Y.,Deng Z.X.,Su E.Z.,and Chen W.Q.,2016,Current research status and perspectives on kasugamycin,Shengwu Jiagong Guocheng(Journal of Bioprocess Engineering),14(4):70-75(汪桂,吴蕴,袁子雨,邓子新,苏二正,陈文青,2016,春雷霉素的研究现状及展望,生物加工过程,14(4):70-75)
Wang Z.,Gerstein M.,and Snyder M.,2009,RNA-seq:a revolutionary tool for transcriptomics,Nature Reviews Genetics.,10(1):57-63
Wu H.,2012,Comparative functional genomics for validamycin overproduction,Dissertation for Ph.D.,Shanghai Jiao Tong University,Supervisor:Zhou X.F.,pp.9-27(吴杭,2012,井冈霉素高产的比较功能基因组研究,博士学位论文,上海交通大学,导师:周秀芬,pp.9-27)
Wu P.,Pan H.,Zhang C.,Wu H.,Yuan L.,Huang X.,Zhou Y.,Ye B.C.,Weaver D.T.,and Zhang L.,2014,SACE_3986,a Tet R family transcriptional regulator,negatively controls erythromycin biosynthesis in Saccharopolyspora erythraea,Journal of Industrial Microbiology and Biotechnology,41(7):1159-1167
Yagi Y.,Kitamura I.,Okamura K.,Ozaki A.,Hamada M.,and Umezawa H.,1971,Production of kasugamycin by Streptomyces kasugaensis:(Ⅰ)oils and fatty acid as carbon sources,Journal of Fermentation Technology,49(2):117-121
Zhu C.,Kang Q.,Bai L.,Cheng L.,and Deng Z.,2016,Identification and engineering of regulation-related genes toward improved kasugamycin production,Applied Microbiology and Biotechnology,100(4):1811-1821
Fu G.J.,Liu J.F.,Wang H.H.,Huang X.G.,Cao F.J.,and Zhang Y.X.,2013,Mutation and fermentation of kasugamycin high-yield strain by injecting nitrogen ion,Shenyang Gongye Xuebao(Journal of Shenyang Pharmaceutical University),30(12):977-981(付桂杰,刘俊芳,王海慧,黄秀果,曹凤娟,张怡轩,2013,氮离子注入法选育春雷霉素高产菌株和生产发酵,沈阳工业学报,30(12):977-981)
Fukagawa Y.,Sawa T.,Homma I.,Takeuchi T.,and Umezawa H.,1968,Studies on biosynthesis of kasugamycin.V.Biosynthesis of the amidine group,Journal of Antibiotics(Tokyo),21(6):410-412
Fukagawa Y.,Sawa T.,Homma I.,Takeuchi T.,and Umezawa H.,1968,Studies on biosynthesis of kasugamycin.IV.Biosynthesis of the kasugamine moiety from[1-14C]-glucosamine and[1,2 or 6-14C]-glucose,Journal of Antibiotics(Tokyo),21(5):358-360
Fukagawa Y.,Sawa T.,Takeuchi T.,and Umezawa H.,1968,Studies on biosynthesis of kasugamycin.I Biosynthesis of kasugamycin and the kasugamine moiety,Journal of Antibiotics(Tokyo),21(1):50-54
Fukagawa Y.,Sawa T.,Takeuchi T.,and Umezawa H.,1968,Studies on biosynthesis of kasugamycin.ⅢBiosynthesis of the D-inositol moiety,Journal of Antibiotics(Tokyo),21(3):185-188
Ikeno S.,Aoki D.,Hamada M.,Hori M.,and Tsuchiya K.S.,2006,DNA sequencing and transcriptional analysis of the kasugamycin biosynthetic gene cluster from Streptomyces kasugaensis M338-M1,Journal of Antibiotics,59(1):18-28
Ikeno S.,Tsuji T.,Higashide K.,Kinoshita N.,Hamada M.,and Hori M.,1998,A7.6 kb DNA region from Streptomyces kasugaensis M338-M1 includes some genes responsible for kasugamycin biosynthesis,Journal of Antibiotics(Tokyo),51(3):341-352
Jin C.X.,Piao S.A.,Cui Y.N.,Jing X.,and Guo X.W.,2004,Screening and fermentation processing control of US95#-a high yielding Chunlei Mycin bacterial strain,Shengwuxue Zazhi(Journal of Biology),21(5):34-35(金昌燮,朴顺爱,崔英兰,金星,郭小威,2004,春雷霉素高产菌株US95#的选育及大罐发酵工艺控制,生物学杂志,21(5):34-35)
Kasuga K.,Sasaki A.,Matsuo T.,Yamamoto C.,Minato Y.,Kuwahara N.,Fujii C.,Kobayashi M.,Agematu H.,Tamura T.,Komatsu M.,Ishikawa J.,Ikeda H.,and Kojima I.,2017,Heterologous production of kasugamycin,an aminoglycoside antibiotic from Streptomyces kasugaensis,in Streptomyces lividans and Rhodococcus erythropolis L-88 by constitutive expression of the biosynthetic gene cluster,Applied Microbiology and Biotechnology,101(10):4259-4268
Kim C.J.,Chang Y.K.,Chun G.T.,Jeong Y.H.,and Lee S.J.,2001,Continuous culture of immobilized Streptomyces cells for kasugamycin production,Biotechnology Progress,17(3):453-461
Kim C.J.,Chang Y.K.,and Chun G.T.,2000,Enhancement of kasugamycin production by p H shock in batch cultures of Streptomyces kasugaensis,Biotechnology Progress,16(4):548-552
Lee J.,Hwang Y.,Kim S.,Kim E.,and Choi C.,2000,Effect of a global regulatory gene,afs R2,from Streptomyces lividans on avermectin production in Streptomyces avermitilis,Journal of Bioscience and Bioengineering,89(6):606-608
Marnix H.M.,Kai B.,Peter C.,Victor D.J.,Piotr Z.,Michael A.F.,Tilmann W.,Eriko T.,and Rainer B.,2011,Anti SMASH:rapid identification,annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences,Nucleic Acids Research,39(Web Server issue):W339-W346
Medini D.,Serruto D.,Parkhill J.,Relman D.A.,Donati C.,Moxon R.,Falkow S.,and Rappuoli R.,2008,Microbiology in the post-genomic era,Nature Reviews Microbiology,6(6):419-430
Mortazavi A.,Williams B.A.,Mc Cue K.,Schaeffer L.,and Wold B.,2008,Mapping and quantifying mmaalian transcriptomes by RNA-Seq,Nature Methods,5(7):621-628
Qi Y.X.,Liu Y.B.,and Rong W.H.,2011,RNA-Seq and its applications:a new technology for transcriptomics,Yichuan(Hereditas),33(11):1191-1202(祁云霞,刘永斌,荣威恒,2011,转录组研究新技术:RNA-Seq及其应用,遗传,33(11):1191-1202)
Tan G.Y.,Peng Y.,Lu C.,Bai L.,and Zhong J.J.,2015,Engineering validamycin production by tandem deletion of gamma-butyrolactone receptor genes in Streptomyces hygroscopicus 5008,Metabolic Engineering,28:74-81
Tilmann W.,Kai B.,Srikanth D.,Daniel K.,Hyun U.K.,Robert B.,Sang Y.L.,Michael A.F.,Rolf M.,Wolfgang W.,Rainer B.,Eriko T.,and Marnix H.M.,2015,Anti SMASH3.0-acomprehensive resource for the genome mining of biosynthetic gene clusters,Nucleic Acids Research,43(Web Server issue):W237
Umezawa H.,Hamada M.,Suhara Y.,Hashimoto T.,and Ikekawa T.,1965,Kasugamycin,a new antibiotic,Antimicrob Agents Chemother(Bethesda),5:753-757
Wang G.,Wu Y.,Yuan Z.Y.,Deng Z.X.,Su E.Z.,and Chen W.Q.,2016,Current research status and perspectives on kasugamycin,Shengwu Jiagong Guocheng(Journal of Bioprocess Engineering),14(4):70-75(汪桂,吴蕴,袁子雨,邓子新,苏二正,陈文青,2016,春雷霉素的研究现状及展望,生物加工过程,14(4):70-75)
Wang Z.,Gerstein M.,and Snyder M.,2009,RNA-seq:a revolutionary tool for transcriptomics,Nature Reviews Genetics.,10(1):57-63
Wu H.,2012,Comparative functional genomics for validamycin overproduction,Dissertation for Ph.D.,Shanghai Jiao Tong University,Supervisor:Zhou X.F.,pp.9-27(吴杭,2012,井冈霉素高产的比较功能基因组研究,博士学位论文,上海交通大学,导师:周秀芬,pp.9-27)
Wu P.,Pan H.,Zhang C.,Wu H.,Yuan L.,Huang X.,Zhou Y.,Ye B.C.,Weaver D.T.,and Zhang L.,2014,SACE_3986,a Tet R family transcriptional regulator,negatively controls erythromycin biosynthesis in Saccharopolyspora erythraea,Journal of Industrial Microbiology and Biotechnology,41(7):1159-1167
Yagi Y.,Kitamura I.,Okamura K.,Ozaki A.,Hamada M.,and Umezawa H.,1971,Production of kasugamycin by Streptomyces kasugaensis:(Ⅰ)oils and fatty acid as carbon sources,Journal of Fermentation Technology,49(2):117-121
Zhu C.,Kang Q.,Bai L.,Cheng L.,and Deng Z.,2016,Identification and engineering of regulation-related genes toward improved kasugamycin production,Applied Microbiology and Biotechnology,100(4):1811-1821