Construction of novel metabolic pathway by DNA shuffling of aromatic dioxygenase genes

加氧酶参与将分子氧直接引入底物，它由末端双加氧酶（由一个大亚基和一个小亚基组成）和NADH的电子传递系统组成。我们首先揭示了大亚基在加氧酶的底物特异性中至关重要。然后，我们对来自假碱性假单胞菌KF707和洋葱伯克氏菌LB400的联苯双加氧酶的两个大亚基进行了DNA洗牌。进化的bphA1基因与其他bph基因在大肠杆菌中表达。一些进化的联苯双加氧酶通过在2,3-和/或3,4位引入分子氧，显示出对各种多氯联苯的转化能力增强，这取决于PCB同系物。其他一些进化的酶获得了对苯、甲苯和某些烷基苯的新的降解能力。利用随机六核苷酸低聚体的突变使我们得到了另一种有趣的突变加氧酶，它可以通过侧向和角向双氧作用降解二苯并呋喃和二苯并对二恶英。测定了上述双加氧酶突变体的氨基酸序列，并讨论了结构与功能的关系。

Oxygenases are involved in the direct introduction of molecular oxygen into the substrates, which consisted with terminal dioxygenase (comprised with a large and a small subunits) and electron transfer system from NADH. We first revealed that the large subunit is crucially involved in the substrate specificity of oxygenase. Then we performed DNA shuffling between the two large subunits of biphenyl dioxygenases derived from Pseudomonas pseudoalcaligenes KF707 and Burkhorderia cepacia LB400. The evolved bphA1 genes were expressed in Escherichia coli along with other bph genes. Some evolved biphenyl dioxygenases exhibited enhanced transformation abilities for various polychlorinated biphenyls by introducing molecular oxygen at the 2,3- and/or 3,4 positions depending on PCB congeners. Some other evolved enzymes acquired new and novel degradation capacities for benzene, toluene and some alkylbenzenes. The mutation using random hexanucleotide oligomers allow us to get another interesting mutant oxygenase which can degrade dibenzofuran and dibenzo-p-dioxin by both lateral dioxygenation and angular dioxygenation.The amino acid sequences of above mutant dioxygenases were determined and the relationships between structure and function were discussed.