NEW APPROACH TO SUSTAINABLE CHEMICAL RESOURCES BASED ON SEARCH, PRECISE ANALYSIS, AND APPLICATION OF BACTERIAL SUGAR-CARBOCYCLIZATION ENZYMES

基于细菌糖碳环化酶的搜寻、精确分析和应用的可持续化学资源新途径

• 批准号: 11356004
• 负责人: KAKINUMA Katsumi
• 金额: $ 24.23万
• 依托单位: TOKYO INSTITUTE OF TECHNOLOGY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11356004/
• 关键词: sugar-carbocyclization enzymes; aminoglycoside antibiotics; metabolic engineering; 2-deoxy-scyllo-inosose; mechanism-based enzyme inhibitor; benzenoid resources; carbaglucose; secondary metabolism enzymes; 糖質還化酵素; カナマイシン; アミノ基転移酵素; 基質認識; 部位特異的

New paradigm for manufacturing sustainable chemical resources based on the CO_2 fixation is highly desirable to conserve fossil resources and to protect our living environments. An approach using carbohydrate-cyclizing enzyme, 2-deoxy-scyllo-inosose (DOI) synthase (DOIS), being originally involved in the biosynthesis of butirosins in Bacillus circulans, has been developed, and detailed analysis and alternative application of DOIS have been exploited. (1) DOIS of B. circulans was purified, and the responsible gene (btrC) was identified and expressed in E. coli. (2) Fermentation conditions of the transformed E. coli and preparative protocol of DOI were established. (3) Conversion of DOI into catechol or 1, 2, 4-triacetoxybenzene was confirmed by simple chemical treatment. (4) Detailed interaction between the enzyme and substrate was analyzed using substrates analogues to elucidate importance of specific hydrogen-bondings. (3) Active site mapping was pursued with mechanism-based inhibitor … More with aids of enzymatic digestion and LC/ESI/MS/MS analysis. (4) For alternative preparation of DOI by metabolic engineering, the biosynthetic gene cluster of butirosin was extensively analyzed. (5) A new transamination enzyme (BtrS) was identified to be responsible for the step next to DOIS, and was over-expressed in E. coli to confirm the function. (6) The btrS gene in B. circulans was disrupted by in frame deletion. Potential of the disruptant for DOI production is currently under investigation. (7) The over-expressed BtrS was utilized to prepare.2-deoxy-scyllo-inosamine as a potential resource for the development of pharmaceuticals. (8) DOI was successfully converted into carbaglucose which is a promising substitute for glucose without dietary load. (8) A new DOIS was identified from a Streptomyces sp. and overexpressed. Its function was confirmed. All these works appear to constitute significant fundamentals for the precise understanding of DOISs and for its application to the preparation of organic resources. Less

以CO_2固定为基础的可持续化学资源制造新范式是保护化石资源和保护人类生存环境的迫切需要。利用环状芽孢杆菌（Bacillus circulans）中的糖环化酶2-脱氧鲨肌醇（DOI）合酶（DOIS）合成布替罗辛，并对其进行了详细的分析和替代应用。(1)B的DOIS。纯化了circulans，鉴定了该基因的功能，并在大肠杆菌中表达。杆菌(2)转化E.大肠杆菌中表达，并建立了DOI的制备方法。(3)通过简单的化学处理证实了DOI转化为邻苯二酚或1，2，4-三乙酰氧基苯。(4)使用底物类似物分析酶和底物之间的详细相互作用，以阐明特定氢键的重要性。(3)活性位点定位采用基于机制的抑制剂 ...更多信息 借助酶消化和LC/ESI/MS/MS分析。(4)为了通过代谢工程替代DOI的制备，对布替罗新的生物合成基因簇进行了广泛的分析。(5)一种新的转氨酶（BtrS）被鉴定为负责DOIS之后的步骤，并在E. coli进行功能鉴定。(6)B中的btrS基因。通过框内缺失破坏环状蛋白。目前正在调查DOI生产的干扰物的潜力。(7)过表达的BtrS被用来取代。2-脱氧-scyllo-氨基葡萄糖作为一个潜在的资源，用于开发药物。(8)DOI被成功地转化为碳葡萄糖，这是一个有前途的替代葡萄糖没有饮食负荷。(8)从链霉菌中鉴定了一个新的DOIS，并进行了过量表达。它的功能得到了证实。所有这些工作似乎构成了重要的基础，为准确理解DOIS和其应用程序的有机资源的准备。少

项目成果

Fumitaka Kudo: "Molecular Cloning of the Gene for the Key Carbocycle-forming Enzyme in the Biosynthesis of 2-Deoxystreptamine-containing Aminocyclitol Antibiotics and Its Comparison with Dehydroquinate Synthase"Journal of Antibiotics. 52[6]. 559-571 (1999

Fumitaka Kudo：“含 2-脱氧链霉胺的氨基环醇抗生素生物合成中关键碳环形成酶基因的分子克隆及其与脱氢奎宁酸合成酶的比较”抗生素杂志。

T. Eguchi, et al: "Importance of Specific Hydrogen-Bonding Donor-Acceptor Interaction for the Key Carbocycle-forming Reaction Catalyzed by 2-Deoxy-scyllo-inosose Synthase in the Biosynthesis of 2-Deoxystreptamine- containing Aminocyclitol Antibiotics"J. O

T. Eguchi 等人：“特定氢键供体-受体相互作用对于 2-脱氧青蟹肌糖合成酶在含 2-脱氧链霉胺的氨基环醇抗生素生物合成中催化的关键碳环形成反应的重要性”J.

Katsumi Kakinuma: "An Expedition Chemo-Enzymetic Route form Glucose to Catechol by the Uose of 2-Deoxy-scyllo-inosose Synthase"Tetrahedron Lett.,. 41・[12]. 1935-1938 (2000)

Katsumi Kakinuma：“利用 2-脱氧青蟹肌糖合成酶从葡萄糖到儿茶酚的远征化学酶路线”Tetrahedron Lett.，41·[12] 1935-1938。

Hideyuki Tamegai: "Identification of L-Glutamine : 2-Deoxy-scyllo-inosose Aminotransferase Required for the Biosynthesis of Butirosin in Bacillus circulans"Journal of Antibiotics. 55[8]. 707-714 (2002)

Hideyuki Tamegai：“L-谷氨酰胺的鉴定：环状芽孢杆菌中 Butirosin 生物合成所需的 2-脱氧青蟹肌糖转氨酶”抗生素杂志。

Hideyuki Tamegai: "Significance of the 20KDa Subunit of Heterodimeric 2-Deoxy-Slyllo-inosose Synihase for the Biosynthesis Butirosin Antibiotics in Bacillus circulans"Bioscience, Biotechnology and Biochemistry. 66(印刷中). (2002)

Hideyuki Tamegai：“异二聚体 2-脱氧-Slyllo-inosose Synihase 的 20KDa 亚基对于环状芽孢杆菌中生物合成 Butirosin 抗生素的意义”生物科学、生物技术和生物化学 66（出版中）。