培养调控激活卡伍尔氏链霉菌NA4产frontalamides类物质的研究
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摘要
微生物(尤其是链霉菌)是农用抗生素的主要来源之一。前期基因筛选发现卡沃尔氏链霉菌NA4具有产生多种PKS和NRPS类型化合物的潜能,但只分离到PKS I型的抗植物病原真菌的物质bafilomycins,为了挖掘更多的抗真菌先导化合物,采用Hiseq2500高通量测序技术获得其基因组扫描图,经生物信息学分析和文献检索发现,其除了能产生bafilomycins,还具有产生polycyclic tetramate macrolactam(PTM)、alkylresorcinol和valinomycin类型抗真菌活性物质的潜力。为了激活这些基因簇,采用基于OSMAC策略中的培养调控为手段,通过改变培养基碳氮源组分,添加氨基酸前体及不同金属离子,使用活性追踪和HPLC-UV发现用麸皮替代NM2培养基中甘油的培养条件能激活PTM类型frontalamides合成基因簇,经LC-MS分析有7个主要的化合物是PTM类化合物,其中的3个可能为新化合物。通过培养调控激活了PTM基因簇,为新型PTM类化合物进一步的分离鉴定提供参考。
Microorganisms(especially Streptomyces) are one of the main sources of agricultural antibiotics. Gene screening in early stage has found that Streptomyces cavourensis NA4 to have the potential to produce a variety of PKS and NRPS type compounds,however,only bafilomycins of PKS I type against plant pathogenic fungi were isolated. In order to burrow more antifungal guiding compounds,a genome scanning draft of S. cavourensis NA4 was obtained using Hiseq2500 high throughput sequencing technology,it was found through bioinformatic analysis and literature retrieval,strain NA4 also possessing the potential production of polycyclic tetramate macrolactam(PTM),alkylresorcinol,and valinomycin type antifungal active matter,except for producing bafilomycins. In order to activate these gene clusters,through changing the components of carbon and nitrogen sources in the medium,and adding amino acid precursors and different metal ions,and applying activity tracing as well as HPLC-UV,it was found that using pollard to replace glycerol in NM2 medium cultural conditions could activate frontalamides synthetic gene cluster of PTM. There were seven main compounds being PTM type compound,among them three possibly were new compounds. In this study,the PTM gene cluster was activated adopting culture regulation,and it provided a guidance for further separation and identification of novel PTM compounds.
Microorganisms(especially Streptomyces) are one of the main sources of agricultural antibiotics. Gene screening in early stage has found that Streptomyces cavourensis NA4 to have the potential to produce a variety of PKS and NRPS type compounds,however,only bafilomycins of PKS I type against plant pathogenic fungi were isolated. In order to burrow more antifungal guiding compounds,a genome scanning draft of S. cavourensis NA4 was obtained using Hiseq2500 high throughput sequencing technology,it was found through bioinformatic analysis and literature retrieval,strain NA4 also possessing the potential production of polycyclic tetramate macrolactam(PTM),alkylresorcinol,and valinomycin type antifungal active matter,except for producing bafilomycins. In order to activate these gene clusters,through changing the components of carbon and nitrogen sources in the medium,and adding amino acid precursors and different metal ions,and applying activity tracing as well as HPLC-UV,it was found that using pollard to replace glycerol in NM2 medium cultural conditions could activate frontalamides synthetic gene cluster of PTM. There were seven main compounds being PTM type compound,among them three possibly were new compounds. In this study,the PTM gene cluster was activated adopting culture regulation,and it provided a guidance for further separation and identification of novel PTM compounds.
引文
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[2]李扬,王耀雯,王育荣,等.水稻稻瘟病菌研究进展[J].广西农业科学,2010,41(8):789-792.
[3]张洁,伊艳杰,王金水,等.小麦赤霉病的防治技术研究进展[J].中国植保导刊,2014,34(1):24-28.
[4]Thaden JT,Lewis SS,Hazen KC,et al.Rising rates of carbapenem-resistant enterobacteriaceae in community hospitals:a mixed-methods review of epidemiology and microbiology practices in a network of community hospitals in the southeastern United States[J].Infection Control&Hospital Epidemiology,2014,35(8):978-983.
[5]武英,王辂.开发微生物来源天然产物的新技术[J].国外医药抗生素,2010,31(1):1-6.
[6]Katz L,Baltz RH.Natural product discovery:past,present,and future[J].Journal of Industrial Microbiology&Biotechnology,2016,43(2):155-176.
[7]Udaany DW,Zeigler L,Asolkar RN,et al.Genome sequencing reveals complex secondary metabolome in the marine actinomycete Salinispora tropica[J].Proceedings of the National Academy of Sciences,2007,104(25):10376-10381.
[8]Omura S,Ikeda H,Ishikawa J,et al.Genome sequence of an industrial microorganism Streptomyces avermitilis:Deducing the ability of producing secondary metabolites[J].Proceedings of the National Academy of Sciences,2001,98(21):12215-12220.
[9]Eerikly M,Challis G.Strategies for the discovery of new natural products by genome mining[J].Challis Chem Bio Chem,2009,10(4):625-633.
[10]钟晶晶,赫卫清.链霉菌隐性次级代谢产物生物合成基因簇的激活[J].生命的化学,2016,36(2):231-236.
[11]Bode HB,Bethe B,H¨ofs R.Big effects from small changes:possible ways to explore nature's chemical diversity[J].ChemBio Chem,2002,3:619-627.
[12]Zhang W,Liu Z,Li S,et al.Spiroindimicins A-D:new bisindole alkaloids from a deep-sea-derived actinomycete[J].Organic Letters,2012,14(13):3364-3367.
[13]Saurav K,Zhang W,Saha S,et al.In silico molecular docking,preclinical evaluation of spiroindimicins A-D,lynamicin A and D isolated from deep marine sea derived Streptomyces sp.SCSIO 03032[J].Interdisciplinary Sciences:Computational Life Sciences,2014,6(3):187-196.
[14]Zhang W,Li S,Zhu Y,et al.Heronamides D-F,polyketide macrolactams from the deep-sea-derived Streptomyces sp.SCSIO 03032[J].Journal of Natural Products,2014,77(2):388-391.
[15]Pan HQ,Yu SY,Song CF,et al.Identification and characterization of the cntifungal substances of a novel Streptomyces cavourensis NA4[J].Journal of Microbiology and Biotechnology,2015,25(3):353-357.
[16]Ramadhar TR,Beemelmanns C,Currie CR,et al.Bacterial symbionts in agricultural systems provide a strategic source for antibiotic discovery[J].Journal of Antibiotics,2013,67(1):53-58.
[17]Matter AM,Hoot SB,Anderson PD,et al.Valinomycin biosynthetic gene cluster in Streptomyces:conservation,ecology and evolution[J].Plos One,2009,4(9):761-768.
[18]Kanda N,Ishizaki N,Inoue N,et al.DB-2073,a new alkylresorcinol antibiotic.I.taxonomy,isolation and characterization[J].Journal of Antibiotics,1975,28(12):935-942.
[19]Weber T,Blin K,Duddela S,et al.Anti SMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene cluster[J].Nucleic Acids Research,2015,43(W1):W237-W243.
[20]Walton LJ,Corre C,Challis GL.Mechanisms for incorporation of glycerol-derived precursors into polyketide metabolites[J].Journal of Industrial Microbiology&Biotechnology,2005,33(2):105-120.
[21]Zhang W,Fortman JL,Carlson JC,et al.Characterization of the bafilomycin biosynthetic gene cluster from Streptomyces lohii[J].Chem Bio Chem,2013,14(3):301-306.
[22]陈竹,李万钧,储炬,等.工业羽毛蛋白胨对红霉素基因工程菌ZL1004发酵过程的影响[J].中国抗生素杂志,2011,36(10):751-757.
[23]Dobson LF,O'Shea DG.Antagonistic effect of divalent cations Ca2+and Mg2+on the morphological development of Streptomyces hygroscopicus var.geldanus[J].Applied Microbiology and Biotechnology,2008,81(1):119-126.
[24]Zhang G,Zhang W,Saha S,et al.Recent advances in discovery,biosynthesis and genome mining of medicinally relevant polyayclic totramate macrolactams[J].Current Topics in Medicinal Chemistry,2016,16(15):1727-1739.
[25]Jomon K,Kuroda Y,Ajisaka M,et al.A new antibiotic,ikarugamycin[J].Journal of Antibiotics,1972,25:271-280.
[26]Luo Y,Huang H,Liang J,et al.Activation and characterization of a cryptic polycyclic tetramate macrolactam biosynthetic gene cluster[J].Nature Communications,2013,4(1):1-8.
[27]Blodgett JA,Oh DC,Cao S,et al.Common biosynthetic origins for polycyclic tetramate macrolactams from phylogenetically diverse bacteria[J].Proceedings of the National Academy of Sciences of the United States of America,2010,107(26):11692-11697.
[28]Lou L,Qian G,Xie Y,et al.Biosynthesis of HSAF,a tetramic acid-containing macrolactam from Lysobacter enzymogenes[J].Journal of the American Chemical Society,2011,133(4):643-645.
[29]Zhang G,Zhang W,Zhang QZ,et al.Mechanistic insights into polycycle formation by reductive cyclization in ikarugamycin biosynthesis[J].Angewandte Chemie International Edition,2014,53(19):4840-4844.
[30]Saha S,Zhang W,Zhang G et al.Activation and characterization of a cryptic gene cluster reveals a cyclization cascade for polycyclic tetramate macrolactams[J].Chemical Science,2017,8(2):1607-1612.