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

通过芳香双加氧酶基因的DNA改组构建新的代谢途径

• 批准号: 11460048
• 负责人: FURUKAWA Kensuke
• 金额: $ 7.68万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11460048/
• 关键词: oxygenase; hybrid enzyme; DNA shuffling; substrate specificity; degradative genes; alkyl benzene; molecular evolutionary engineering; ダイオキシン; ジオキシゲナーゼ; Pseudomonas; PCB; 遺伝子シャフリング; 芳香族炭化水素代謝; ジベンゾフラン

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.

加氧酶参与将分子氧直接引入底物，其由末端双加氧酶（由大小亚基组成）和来自NADH的电子传递系统组成。我们首次发现，大亚基是至关重要的参与底物特异性的加氧酶。然后，我们进行了DNA改组之间的两个大亚基的联苯双加氧酶来自假产碱假单胞菌KF 707和洋葱伯克霍尔德菌LB 400。进化的bphA 1基因在大肠杆菌中与其他bph基因一起沿着表达。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，表现出对各种多氯联苯的增强转化能力，这取决于PCB同系物。其他一些进化的酶获得了新的和新颖的苯，甲苯和一些烷基苯的降解能力。利用随机六核苷酸寡聚体进行突变，获得了一种既能通过侧向双加氧作用又能通过角向双加氧作用降解二苯并呋喃和二苯并二恶英的突变型加氧酶，并测定了其氨基酸序列，讨论了其结构与功能的关系。

项目成果

Suenaga, H., Nishi, A., Watanabe, T., Sakai, M. and Furukawa, K.: "Engineering a hybrid pseudomonad aquired 3,4-dioxygenase activity for polychlorinated biphenyls"J. Biosci. Bioeng.. 87. 430-435 (1999)

Suenaga, H.、Nishi, A.、Watanabe, T.、Sakai, M. 和 Furukawa, K.：“设计具有多氯联苯 3,4-双加氧酶活性的杂合假单胞菌”J。

西 哲人, 古川謙介: "IGEシリーズ29 遺伝情報のダイナミズムとその分子機構 ビフェニル/サリチル酸代謝遺伝子群をコードする自己可動化因子bph-salエレメント"東北大学遺伝生態研究センター出版. 39-50 (2001)

Tetsuto Nishi、Kensuke Furukawa：“IGE Series 29 遗传信息的动态及其分子机制：Bph-sal 元件，编码联苯/水杨酸代谢基因组的自动员因子”东北大学遗传与生态学研究中心出版 39-。 50 (2001)

Suenaga, H., Goto, M., Furukawa, K.: "Emergence of multifunctional oxygenase activities of biphenyl dioxygenase by random -priming recombination"J. Biol. Chem.. 276(25). 22500-22506 (2001)

Suenaga，H.，Goto，M.，Furukawa，K.：“通过随机引发重组产生联苯二加氧酶多功能加氧酶活性”J。

Nishi,A.,Tominaga,K.and Furukawa,K.: "A 90 kilo base conjugative chromosomal element coding for biphenyl and salicylate in Pseudomonas putida KF715"J.Bacteriol.. 182. 1949-1955 (2000)

Nishi,A.、Tominaga,K. 和 Furukawa,K.：“恶臭假单胞菌 KF715 中编码联苯和水杨酸盐的 90 公斤碱基接合染色体元件”J.Bacteriol.. 182. 1949-1955 (2000)

Suenaga, H., Goto, M. and Furukawa, K.: "Emergence of multifunctional oxygenase activities of biphenyl dioxygenase by random-priming recombination"J. Biol. Chem.. 276(25). 22500-22506 (2001)

Suenaga, H.、Goto, M. 和 Furukawa, K.：“通过随机引发重组产生联苯二加氧酶多功能加氧酶活性”J。