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首页> 外文期刊>Journal of bacteriology >A Novel Phenanthrene Dioxygenase fromNocardioides sp. Strain KP7: Expression inEscherichia coli
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A Novel Phenanthrene Dioxygenase fromNocardioides sp. Strain KP7: Expression inEscherichia coli

机译：一种来自诺卡氏菌的新型菲双加氧酶。菌株KP7：在大肠杆菌中的表达

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Nocardioides sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene too-phthalate (phd) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight phd genes was cloned and sequenced. ThephdA, phdB, phdC, andphdD genes, which encode the α and β subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster, phdAB, was located 8.3 kb downstream of the previously characterized phdK gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The phdCD gene cluster was located 2.9 kb downstream of the phdB gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the α and β subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The phdABCD genes were expressed poorly inEscherichia coli, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the phdABCD genes improved their expression in E. coli. E. colicells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activity, and those carrying phdABDor phdABC exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying thephdABCD genes. It was thus concluded that all of thephdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of thephd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in Nocardioides sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases.
机译： Nocardioides sp。 KP7菌株生长在菲上，而不生长在萘上。该生物通过1-羟基-2-萘甲酸，邻苯二甲酸邻苯二甲酸酯和原儿茶酸酯降解菲。通过Southern杂交发现负责将菲降解为邻苯二甲酸邻苯二甲酸盐的基因（em> phd ）位于染色体上。克隆并测序了一个含有8个 phd 基因的10.6kb DNA片段。 phdA ， phdB ， phdC 和 phdD 基因，它们编码加氧酶成分的α和β亚基分别鉴定出菲双氧合酶的铁氧还蛋白和铁氧还蛋白还原酶。 phdAB 基因簇位于先前表征的 phdK 基因下游8.3 kb，该基因编码2-羧基苯甲醛脱氢酶。 phdCD 基因簇位于 phdB 基因下游2.9 kb。 PhdA和PhdB与其他环羟基化双加氧酶的α和β亚基表现出中等（小于60％）的序列同一性。 PhdC序列显示了[3Fe-4S]或[4Fe-4S]型铁氧还蛋白的特征，而不是大多数已报道的环羟基化双加氧酶中发现的[2Fe-2S]型铁氧还蛋白的特征。 PhdD还显示出与已知还原酶的中等（少于40％）序列同一性。即使在强启动子的控制下， phdABCD 基因在大肠杆菌中的表达也很差。在 phdABCD 基因每个起始密码子上游引入Shine-Dalgarno序列可改善其在 E中的表达。大肠杆菌。携带 phdBCD 或 phdACD 的大肠杆菌细胞没有菲降解活性，而携带 phdABD 或 phdABC < / em>的菲降解活性明显低于携带 phdABCD 基因的细胞。因此得出结论，所有 phdABCD 基因对于有效表达菲降解活性是必需的。 phd 基因的遗传组成，这些基因的系统发育差异以及铁氧还蛋白成分的异常类型表明 Nocardioides sp中的菲二加氧酶。菌株KP7是一类新的芳香环-羟化双加氧酶。

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