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

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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