红树林土壤微生物卤代酶基因和宏基因组非核糖体肽合酶基因的克隆和表达
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摘要
红树林是分布于热带、亚热带海岸的潮间带的木本植物群落,是海滩上特有的森林类型。红树林生态系统具有高生产率,高归还率,高分解率的“三高”特点。微生物是红树林生态系统中重要的生物组成成份,它在红树林生态系统的能量物质循环中起着关键的作用。由于红树林生态系统处于咸淡水交汇的特殊环境,兼具海洋与陆地的性质,因而其中的微生物形成了独特的生长代谢调控机制和化学防御机制,其独特的代谢产物也将成为我们获取新型天然产物的资源宝库。
首先,用不同的针对卤代酶基因设计的引物对分离自红树林土壤的放线菌进行筛选,发现其中含有丰富的卤代酶基因,且类型多样。由此推测这些放线菌具有产生类型多样的卤代化合物的潜力。并对其中的几个FADH_2依赖型卤代酶进行表达,试图阐明其催化机制,但一直无法实现可溶性表达。
在对分离自红树林土壤沉积物的放线菌进行卤代酶基因筛选时,发现MGR072菌株中的卤代酶基因与Actinosynnema pretiosum subsp.Auranticum中卤代酶基因asm12的相似性为64%,另外用针对安莎类抗生素起始合成酶AHBA合酶基因设计的引物对其进行扩增,发现AHBA合酶基因扩增结果为阳性。综合以上结果,推测其中含有合成安莎类抗生素的生物合成基因簇,构建了其基因组文库,发掘出其中与安莎类抗生素生物合成相关的约70Kb的基因簇。并对其培养条件进行了初步的研究。
我们还构建了含有至少260Mb红树林沉积物环境样品DNA的沉积物宏基因组文库,并利用序列同源性驱动筛选方法对其中所含有的NRPS基因加以系统的发掘和整理,发现了克隆4F11可能包含一段完整的NRPS基因簇。
Mangroves are unique woody plant communities growing in intertidal zones of tropical and subtropical coast. The mangrove ecosystem has the characteristics of high productivity, high return rates and high rates of decomposition which are called "three-high" feature. Microorganisms are an important biological composition in mangrove ecosystem, and play a key role in its energy and substances cycle. Because the mangrove ecosystem is in the intersection of freshwater and seawater and have both marine and terrestrial nature, the microorganisms form particular mechanisms of metabolism regulation and chemical defense and their secondary metaboites will also become a new source of natural products.
First, we investigated the diversity of halogenase genes of the actinomycetes which were isolated from mangrove sediments by using two pairs of specific degenerate primers and found it was abundant in halogenase genes. Based on these results it was believed that these actinomycetes had potential ability to produce kinds of organohalogen compounds. And then three FADH_2-dependent halogenases were expressed in E.coli in order to explain the mechanism of halogenation. But they couldn't express as soluble protein in our experiments.
when investigating the diversity of halogenase genes of the actinomycetes, we found a halogenase gene fragment of strain MGR072 exhibited 64% similarity with the halogenase gene asm12 of Actinosynnema pretiosum subsp. Auranticum. Then we used the primers which could amplify the AHBA synthetase gene to test the strain MGR072, and the result was positive. Based on the results above we presumed the strain MGR072 could produce a new ansamycin compound. After construction the genomic library of it, we got a 70Kb gene cluster associated with the biosynthesis of the new ansamycin compound, and preliminary studied on the cultural condition.
At last, a metagenomic cosmid library was constructed with the large DNA fragments obtained from mangrove sediments, and was screened the NRPS gene by sequence-driven screening. A clone 4F11 was obtained and it maybe contains a whole NRPS gene cluster.
首先,用不同的针对卤代酶基因设计的引物对分离自红树林土壤的放线菌进行筛选,发现其中含有丰富的卤代酶基因,且类型多样。由此推测这些放线菌具有产生类型多样的卤代化合物的潜力。并对其中的几个FADH_2依赖型卤代酶进行表达,试图阐明其催化机制,但一直无法实现可溶性表达。
在对分离自红树林土壤沉积物的放线菌进行卤代酶基因筛选时,发现MGR072菌株中的卤代酶基因与Actinosynnema pretiosum subsp.Auranticum中卤代酶基因asm12的相似性为64%,另外用针对安莎类抗生素起始合成酶AHBA合酶基因设计的引物对其进行扩增,发现AHBA合酶基因扩增结果为阳性。综合以上结果,推测其中含有合成安莎类抗生素的生物合成基因簇,构建了其基因组文库,发掘出其中与安莎类抗生素生物合成相关的约70Kb的基因簇。并对其培养条件进行了初步的研究。
我们还构建了含有至少260Mb红树林沉积物环境样品DNA的沉积物宏基因组文库,并利用序列同源性驱动筛选方法对其中所含有的NRPS基因加以系统的发掘和整理,发现了克隆4F11可能包含一段完整的NRPS基因簇。
Mangroves are unique woody plant communities growing in intertidal zones of tropical and subtropical coast. The mangrove ecosystem has the characteristics of high productivity, high return rates and high rates of decomposition which are called "three-high" feature. Microorganisms are an important biological composition in mangrove ecosystem, and play a key role in its energy and substances cycle. Because the mangrove ecosystem is in the intersection of freshwater and seawater and have both marine and terrestrial nature, the microorganisms form particular mechanisms of metabolism regulation and chemical defense and their secondary metaboites will also become a new source of natural products.
First, we investigated the diversity of halogenase genes of the actinomycetes which were isolated from mangrove sediments by using two pairs of specific degenerate primers and found it was abundant in halogenase genes. Based on these results it was believed that these actinomycetes had potential ability to produce kinds of organohalogen compounds. And then three FADH_2-dependent halogenases were expressed in E.coli in order to explain the mechanism of halogenation. But they couldn't express as soluble protein in our experiments.
when investigating the diversity of halogenase genes of the actinomycetes, we found a halogenase gene fragment of strain MGR072 exhibited 64% similarity with the halogenase gene asm12 of Actinosynnema pretiosum subsp. Auranticum. Then we used the primers which could amplify the AHBA synthetase gene to test the strain MGR072, and the result was positive. Based on the results above we presumed the strain MGR072 could produce a new ansamycin compound. After construction the genomic library of it, we got a 70Kb gene cluster associated with the biosynthesis of the new ansamycin compound, and preliminary studied on the cultural condition.
At last, a metagenomic cosmid library was constructed with the large DNA fragments obtained from mangrove sediments, and was screened the NRPS gene by sequence-driven screening. A clone 4F11 was obtained and it maybe contains a whole NRPS gene cluster.
引文
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[2]林鹏.中国红树林研究进展[J].厦门大学学报(自然科学版),2001,40(2):592.
[3]林鹏.中国红树林生态系[M].北京:科学出版社.1997.
[4]Alongi D M.Bacterial productivity and microbial biomass intropical mangrove sediments[J].Microbial Ecology,1988,15(1):59-79.
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[6]Hyde K D,Lee S Y.Ecology of mangrove fungi and their role in nutriert cycling:What gaps occur in our knowledge?.[J].Hydrobiologia,1995,295(1-3):107-188.
[7]Volkmann-Kohlmeyer B,Kohlmeyer J.Biogeographic observation on Pacific marine fungi[J].Mycologia,1993,85(3):337-346.
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[10]Wu R Y.Studies on the microbial ecology of Tansui Estuary[J].Botanical Bulletin of Academin Sinica,1993,34(1):13-30.
[11]江晓路,梁晓婷,曹杰铭.深圳福田红树林生态中放线菌的筛选及其抗菌活性测定[J].中国海洋大学学报,2006,36(4):601-605.
[12]闫莉萍,洪葵,胡申才,et al.海南近海30株抗B16细胞活性放线菌的16SrDNA多样性分析[J].微生物学报,2005,45(2):185-189.
[13]林文翰.红树附生微生物的化学成分研究第三届海洋高技术论坛(下册)[M].2005.
[14]Poch G K,Gloer J B.Auranticins A and B:two new depsidones from a mangrove isolate of the fungus Preussia aurantiaca[J].J Nat Prod,1991,54(1):213-217.
[15]Schlingmann G,Milne L,Williams D R,et al.Cell wall active antifungal compounds produced by the marine fungus Hypoxylon oceanicum LL-15G256.Ⅱ.Isolation and structure determination[J].J Antibiot(Tokyo),1998,51(3):303-316.
[16]李厚金.六种南海海洋微生物的代谢产物及柳珊瑚酸衍生物的生物合成[D].中山大学博士学位论文,2002.
[17]Wu X,Liu X,Jiang G,et al.Xyloketal G,a Novel Metabolite from the Mangrove Fungus Xylaria sp.2508[J].Chemistry of Natural Compounds,2005,41(1):27-29.
[18]Asolkar R N,Maskey R P,Helmke E,et al.Chalcomycin B,a new macrolide antibiotic from the marine isolate Streptomyces sp.B7064[J].J Antibiot(Tokyo),2002,55(10):893-898.
[19]Stritzke K,Schulz S,Laatsch H,et al.Novel caprolactones from a marine streptomycete[J].J Nat Prod,2004,67(3):395-401.
[20]朱九滨,洪葵,庄令等.红树林细胞毒活性放线菌的筛选及其所产活性物质的初步研究[J].华南热带农业大学学报,2005,11(3):5-8.
[21]Gribble G W.The diversity of naturally produced organohalogens[J].Chemosphere,2003,52(2):289-297.
[22]Gribble G W.Naturally occurring organohalogen compounds--a comprehensive survey[J].Fortsehr Chem Org Naturst,1996,68(1-423.
[23]van Pee K H.Biosynthesis of halogenated metabolites by bacteria[J].Annu Rev Mierobiol,1996,50(375-399.
[24]van Pee K H,Ligon J M.Biosynthesis of pyrrolnitrin and other phenylpyrrole derivatives by bacteria[J].Nat Prod Rep,2000,17(2):157-164.
[25]Pereira E R,Belin L,Sancelme M,et al.Structure-activity relationships in a series of substituted indolocarbazoles:topoisomerase I and protein kinase C inhibition and antitumoral and antimicrobial properties[J].J Med Chem,1996,39(22):4471-4477.
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