This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The use of plant essential oils for a variety of medicinal application such as the prevention and treatment of infectious diseases and physiological disorders has been shown to be effective. Careful analyses of these plant essential oils has revealed that one of their active moieties seems to be related to sesquiterpenes and their derivatives, and research and discovery of these compounds for therapeutic applications are quickly becoming more attractive. It is, however, a well known fact that sesquiterpenes and their metabolites are usually poorly produced in a mixture of various isomers which are difficult to separate. In addition, chemical synthesis of these compounds is difficult due to their structural complexity and tends to result in a mixture of isomers. Enzyme dependent biosynthesis from a sesquiterpene precursor, farnesyl pyrophosphate, in genetically engineered E.coli may prove feasible as an alternative method of terpene production. Although it usually produces a single specific compound, the type of terpenes and the amount produced are limited by the number of genes cloned and the properties of those enzymes: expression, stability, and activity. It is usually difficult and time consuming to clone a desired gene responsible for particular sesquiterpene production. The technique called directed evolution has been applied for a decade to improve activity, stability and solubility of target enzymes, and alter their product specificity. We have shown in our previous work that (+)-delta- cadinene synthase was among the sesquiterpene synthase expressed well in E.coli. Therefore (+)-delta-cadinene synthase has been subjected to rounds of random muitagenesis and following saturation mutagenesis, and was successfully converted to germacrene-D-ol synthase with over 95% selectivity to it. Our project seeks to build structural models of both (+)-delta-cadinene synthase and germacrene-D-ol synthase, and thereby clarify the significant structual change within the enzyme that results in alteration of product specificity. This should also provide important information for the rational design of enzyme modifications to improve and diverse the enzyme function.
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