基于转座子策略提高链霉菌中landomycin E产量的研究
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摘要
【目的】对影响放线菌链霉菌Streptomyces globisporus产landomycin E(laE)的代谢网络进行研究,以提高次生代谢物的产量。【方法】通过构建含强启动子和抗性标记的转座子Tn7为基础的转座子,整合至S.globisporus的染色体产生突变库,筛选高产量的突变株并对其代谢网络进行研究分析。【结果】利用构建好的Tn7-转座子连续转化链霉菌S.globisporus,经过数轮的突变和筛选,得到6株产量有较大改变的突变株,对整合位点的亚克隆和测序结果表明,该位点整合导致编码类似细菌的某些调节因子如TetR和GntR家族的蛋白的基因失活。【结论】所构建的经过修饰的微型Tn7-转座子不仅带有抗性标记且有启动子,可插入链霉菌染色体产生突变,进而提高次生代谢物laE的产量,同时也证明,转座子基载体可应用于非模式菌链霉菌。
【Objective】Studies were conducted on the metabolic network affecting the localomycin E(laE)production of S treptomycesglobisporuscan increase the y ield of secondary metabolites.【Methods】By constructing a transposon based on a transposon Tn7 containing a strong promoter and a resistance marker,a chromosome-generated mutation library was integrated into S.globisporus,a high-yielding mutant strain was screened and its metabolic network was studied and analyzed.【Results】Using the constructed Tn7 transposon for continuous transformation of S.globisporus,several rounds of mutations and screening were used to obtain six mutants with large changes in yield.Subcloning and sequencing of the integration site showed that the site integration resulted in the inactivation of genes encoding certain regulatory factors such as the TetR and GntR family of proteins.【Conclusion】The constructed and modified micro Tn7 transposon contains not only antibiotic resistance marker and but also strong promoter which can be inserted into the chromosome of Streptomyces to produce mutations,thereby increasing the yield of secondary metabolite LaE.It also demonstrates that the transposon-based vector can be applied to non-model S treptomyces sp.
【Objective】Studies were conducted on the metabolic network affecting the localomycin E(laE)production of S treptomycesglobisporuscan increase the y ield of secondary metabolites.【Methods】By constructing a transposon based on a transposon Tn7 containing a strong promoter and a resistance marker,a chromosome-generated mutation library was integrated into S.globisporus,a high-yielding mutant strain was screened and its metabolic network was studied and analyzed.【Results】Using the constructed Tn7 transposon for continuous transformation of S.globisporus,several rounds of mutations and screening were used to obtain six mutants with large changes in yield.Subcloning and sequencing of the integration site showed that the site integration resulted in the inactivation of genes encoding certain regulatory factors such as the TetR and GntR family of proteins.【Conclusion】The constructed and modified micro Tn7 transposon contains not only antibiotic resistance marker and but also strong promoter which can be inserted into the chromosome of Streptomyces to produce mutations,thereby increasing the yield of secondary metabolite LaE.It also demonstrates that the transposon-based vector can be applied to non-model S treptomyces sp.
引文
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[13]BOHDAN B,STEPHEN W,MAKSYM M,et al.In vivo random mutagenesis of streptomycetes using mariner-based transposon Himar1[J].Appl Microbiol Biotechnol,2013,97:351-359.
[14]DAMASCENO J D,BEVERLEY S M,TOSI L R.Atransposon toolkit for gene transfer and mutagenesis in protozoan parasites[J].Genetica,2010,38:301-311.
[15]PETZKE L,LUZHETSKYY A.In vivo Tn5-based transposon mutagenesis of Streptomycetes[J].Appl Microbiol Biotechnol,2010,86:1593.
[16]WEADEN J,DYSON P.Transposon mutagenesis with IS6100 in the avermectin-producer Streptomyces avermitilis[J].Microbiology,1998,144:1963-1970.
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[18]RIGALI S,TITGEMEYER F,BARENDS S,et al.Feast or famine:The global regulator DasR links nutrient stress to antibiotic production by Streptomyces[J].EMBO Rep,2008,9:670-675.
[19]RAMOS J L,MARTINEZ-BUENO M,MOLINA-HENARES A J,et al.The TetR family of transcriptional repressors[J].Microbiol Mol Biol Rev,2005,69:326-356.
[2]黄敏红,刘田甜,陆群,等.链霉菌抗生素生物合成的基因调控[J].中国抗生素杂志,2009,34(7):392-398.HUANG M H,LIU T T,LU Q,et al.Genetic regulation of antibiotic biosynthesis in Streptomyces[J].Chinese Journal of Antibiotics,2009,34(7):392-398.
[3]HORBAL L,FEDORENKO V,BECHTHOLD A,et al.A transposon-based strategy to identify the regulatory gene network responsible for landomycin E biosynthesis[J].FEMS Microbiol Lett,2013,342(2):138-146.
[4]KUTAS P,FECKOVA L,REHAKOVA A,et al.Strict control of auricin production in Streptomyces aureofaciensCCM 3239involves a feedback mechanism[J].Appl Microbiol Biotechnol,2013,97(6):2413-2421.
[5]ZOTCHEV S B,SEKUROVA O N,KATZ L.Genomebased bioprospecting of microbes for new therapeutics[J].Curr Opin Biotechnol,2012,23(6):941-947.
[6]REBETS Y,OSTASH B,LUZHETSKYY A,et al.Production of landomycins in Streptomyces globisporus1912and S.cyanogenus S136is regulated by genes encoding putative transcriptional activators[J].FEMS Microbiol Lett,2003,222(1):149-153.
[7]REBETS Y,OSTASH B,LUZHETSKYY A,et al.DNA-binding activity of LndI protein and temporal expression of the gene that upregulates landomycin E production in Streptomyces globisporus 1912[J].Microbiology,2005,151:281-290.
[8]DUTKO L,REBETS Y,OSTASH B,et al.A putative proteinase gene is involved in regulation of landomycin E biosynthesis in Streptomyces globisporus 1912[J].Fems Microbiol Lett,2006,255:280-285.
[9]OSTASH B,REBETS Y,MYRONOVSKYY M.Identification and characterization of the Streptomyces globisporus 1912 regulatory gene lndYR that affects sporulation and antibiotic production[J].Microbiology,2011,157:1240-1249.
[10]萨姆布鲁克J,拉塞尔D W.分子克隆实验指南[M].黄培堂,译.第3版.北京:科学出版社,2002.SAM BROOK J,RUSSELL D W.Molecular cloning-Alaboratory manual[M].HUANG P T(trans).3rd edition.Beijing:Science Press,2002.
[11]WANG Q,CHEN T,ZHAO X,et al.Metabolic engineering of thermophilic Bacillus licheniformis for chiral pure D-2,3-butanediol production[J].Biotechnol Bioeng,2012,109:1610-1621.
[12]QI G,KANG Y,LI L,et al.Deletion of meso-2,3-butanediol dehydrogenase gene budC for enhanced D-2,3-butanediol production in Bacillus licheniformis[J].Biotechnol Biofuels,2014,7:16.doi:10.1186/1754-6834-7-16.
[13]BOHDAN B,STEPHEN W,MAKSYM M,et al.In vivo random mutagenesis of streptomycetes using mariner-based transposon Himar1[J].Appl Microbiol Biotechnol,2013,97:351-359.
[14]DAMASCENO J D,BEVERLEY S M,TOSI L R.Atransposon toolkit for gene transfer and mutagenesis in protozoan parasites[J].Genetica,2010,38:301-311.
[15]PETZKE L,LUZHETSKYY A.In vivo Tn5-based transposon mutagenesis of Streptomycetes[J].Appl Microbiol Biotechnol,2010,86:1593.
[16]WEADEN J,DYSON P.Transposon mutagenesis with IS6100 in the avermectin-producer Streptomyces avermitilis[J].Microbiology,1998,144:1963-1970.
[17]OSTASH B,REBETS Y,MYRONOVSKYY M.Identification and characterization of the Streptomyces globisporus 1912regulatory gene lndYR that affects sporulation and antibiotic production[J].Microbiology,2011,157(4):1240-1249.
[18]RIGALI S,TITGEMEYER F,BARENDS S,et al.Feast or famine:The global regulator DasR links nutrient stress to antibiotic production by Streptomyces[J].EMBO Rep,2008,9:670-675.
[19]RAMOS J L,MARTINEZ-BUENO M,MOLINA-HENARES A J,et al.The TetR family of transcriptional repressors[J].Microbiol Mol Biol Rev,2005,69:326-356.