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

点击翻译按钮获取中文摘要

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.