Hybrid Enzymes for Nonribosomal Peptides and Ketides

描述（由申请人提供）：本提案描述了一种使用来自天然同源生物合成系统的酶亚基的杂合重建来产生非核糖体/聚酮化合物天然产物变体的方法。涉及的天然产物是埃博霉素、耶尔森菌素和绿脓菌螯铁蛋白，所有这些在癌症治疗和抗生素治疗中具有已知的治疗价值。将通过评价跨杂交界面的酰基转移步骤的有效性来评估杂交、非同源酶组装线成功产生所需变体的能力。这些杂合系统的功能特性将与非同源酶亚基之间的蛋白质/蛋白质识别特性相关。遗传蛋白工程如接头/结构域交换或甲基转移酶结构域插入也将用于提高酰基转移的效率或增加杂交水平以产生天然产物的更多样化的变体。治疗上重要的天然产物的“非天然”变体将被用作探针，以帮助发现未实现的药用益处。工程系统的功能和结构方面之间的关系所获得的信息将进一步扩大组合生物合成作为克服癌症和抗生素耐药性的新策略的能力。

DESCRIPTION (provided by applicant): This proposal describes an approach of using hybrid reconstitution of enzyme subunits from native, cognate biosynthetic systems to generate variants of nonribosomal/polyketide natural products. The natural products involved are the epothilones, yersiniabactin, and pyochelin, all of which have known therapeutic value in cancer therapy and antibiotic treatment. The ability of the hybrid, noncognate enzyme assembly lines to successfully produce the desired variants will be assessed by evaluating the effectiveness of the acyl-transfer step across the hybrid interface. The functional properties of these hybrid systems will be correlated to the protein/protein recognition characteristics between the noncognate enzyme subunits. Genetic protein engineering such as linker/domain swapping or methyltransferase domain insertion will also be employed to either improve the efficiency of acyl-transfer or increase the level of hybridization for generation of more diverse variants of the natural products. The "unnatural" variants of therapeutically important natural products will be used as probes to help discover unrealized medicinal benefits. The information gained on the relationship between the functional and structural aspects of the engineered systems will further expand the capabilities of combinatorial biosynthesis as a novel strategy to overcome cancer and antibiotic resistance.