APPROACHES FOR OVERPRODUCTION OF POLYKETIDE ANTIMICRO.

传统的菌株改良过程对于使具有重要治疗意义的聚酮具有商业可行性是必要的，但既耗时又繁琐。这项提议的长期目标是开发解决聚酮类药物过量生产的通用方法，并将这些方法应用于新型聚酮类抗菌剂的生产。特别是，我们将针对新的红霉素类似物和节省成本的中间体来制备酮内酯类抗生素，这些抗生素对MLS耐药微生物具有更广泛的作用范围。该方案的具体目的是：i)用一个或多个异源PKS基因以及低产突变基因替换高产菌株中的PKS基因，并分析其对聚酮生产的影响；ii)将高产菌株中的PKS基因克隆到已知的天蓝色链霉菌表达载体中，并与天蓝色链霉菌中野生型基因的表达进行比较。这些概念验证实验将为我们的第二阶段目标铺平道路，该阶段涉及进一步探索聚酮过量生产的分子和生理基础，并最终使用定义明确的菌株过度生产新型抗微生物药物。拟议的商业应用：与现有的传统方法相比，非专利生产链霉菌宿主将能够以显著的成本和时间节省生产具有商业价值的抗菌多酮。

Conventional strain improvement processes are necessary to make therapeutically important polyketides commercially viable but are time consuming and tedious. The long term goals of this proposal are to develop generic approaches to polyketide overproduction and to apply these approaches to the generation of novel polyketide antimicrobials. In particular, we will target novel erythromycin analogs and cost-saving intermediates for the preparation of the ketolide class of antibiotics which have a broader range of action against MLS resistant organisms. The Specific Aims of the proposal are: i) to substitute the PKS genes in an overproducing strain with one or more heterologous PKS genes as well as low producing mutant genes and analyze the effects on polyketide production; and ii) to clone the PKS genes from an overproducer into a known S. coelicolor expression vector and compare expression to the wild-type genes in S. coelicolor. These proof-of-concept experiments will pave the way for our Phase II objectives which involve further probing of the molecular and physiological basis of polyketide overproduction and ultimately the overproduction of novel anti-microbial pharmaceuticals using well defined strains. PROPOSED COMMERCIAL APPLICATION: A generic overproducing Streptomycete host will enable production of commercially valuable antimicrobial polyketides with significant cost and time savings over existing conventional methods.