Hybrid Enzymes for Nonribosomal Peptides and Ketides

描述（由申请人提供）：该提案描述了一种使用天然同源生物合成系统中酶亚基的杂交重组来生成非核糖体/聚酮天然产物变体的方法。所涉及的天然产物是埃泊霉素、耶尔希那巴辛和吡咯霉素，它们在癌症治疗和抗生素治疗中都有已知的治疗价值。杂交、非同源酶装配线成功生产所需变体的能力将通过评估跨杂交界面酰基转移步骤的有效性来评估。这些杂交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。基因蛋白工程，如连接子/结构域交换或甲基转移酶结构域插入，也将被用于提高酰基转移的效率或增加杂交水平，以产生更多样化的天然产物变体。具有重要治疗意义的天然产物的“非自然”变体将被用作探针，帮助发现尚未实现的药用价值。关于工程系统的功能和结构方面之间关系的信息将进一步扩展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.