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首页> 外文期刊>Applied Microbiology >Heterologous Expression of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes from a Novel Pyrene-Degrading Betaproteobacterium
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Heterologous Expression of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes from a Novel Pyrene-Degrading Betaproteobacterium

机译：新型降解P的β-变形杆菌的多环芳香烃环加氢双加氧酶基因的异源表达。

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A betaproteobacterium within the family Rhodocyclaceae previously identified as a pyrene degrader via stable-isotope probing (SIP) of contaminated soil (designated pyrene group 1 or PG1) was cultivated as the dominant member of a mixed bacterial culture. A metagenomic library was constructed, and the largest contigs were analyzed for genes associated with polycyclic aromatic hydrocarbon (PAH) metabolism. Eight pairs of genes with similarity to the α- and β-subunits of ring-hydroxylating dioxygenases (RHDs) associated with aerobic bacterial PAH degradation were identified and linked to PG1 through PCR analyses of a simplified enrichment culture. In tandem with a ferredoxin and reductase found in close proximity to one pair of RHD genes, six of the RHDs were cloned and expressed in Escherichia coli . Each cloned RHD was tested for activity against nine PAHs ranging in size from two to five rings. Despite differences in their predicted protein sequences, each of the six RHDs was capable of transforming phenanthrene and pyrene. Three RHDs could additionally transform naphthalene and fluorene, and these genotypes were also associated with the ability of the E. coli constructs to convert indole to indigo. Only one of the six cloned RHDs was capable of transforming anthracene and benz[ a ]anthracene. None of the tested RHDs were capable of significantly transforming fluoranthene, chrysene, or benzo[ a ]pyrene.
机译：培养了以前通过污染的土壤（指定为（族1或PG1）通过稳定同位素探测（SIP）鉴定为as降解物的红景天科内的β变形杆菌，作为混合细菌培养的主要成分。构建了宏基因组文库，并分析了最大重叠群与多环芳烃（PAH）代谢相关的基因。鉴定了八对与需氧细菌PAH降解相关的环羟基化双加氧酶（RHDs）的α-和β-亚基相似的基因，并通过简化的富集培养的PCR分析将其与PG1连接。与铁氧还蛋白和还原酶紧密靠近一对RHD基因相结合，克隆了六个RHD，并在大肠杆菌中表达。测试了每个克隆的RHD对九种PAH的活性，PAH的大小从2个到5个环不等。尽管其预测的蛋白质序列不同，但六个RHD均能够转化菲和pyr。三种RHD可以另外转化萘和芴，这些基因型还与大肠杆菌构建体将吲哚转化为靛蓝的能力有关。六个克隆的RHD中只有一个能够转化蒽和苯并[a]蒽。所测试的RHD均未能够显着转化荧蒽，or或苯并[a] re。

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