Creating natural product diversity via the fabrication of a hybrid fatty acid-nonribosomal peptide synthetase

摘要：天然产物和天然产物的衍生品/仿制品构成了现代 治疗药物，因为其独特的复杂有机支架有助于其广泛的生物活性。 这些天然产物是由非核糖体多肽合成酶脂肪酸合成酶(FAS)合成的 (NRPS)或聚酮合成酶(PKS)。这个项目研究了蛋白质·蛋白质相互作用在 模块合成酶负责形成临床相关的脂肪酸和非核糖体多肽 重新设计和完全控制生物合成流水线的长期目标。一个例子， 临床上治疗的天然产物是抗菌剂达托霉素，它是一种由 一种用于治疗甲氧西林的混合型脂肪酸合成酶(FAS)和非核糖体肽合成酶(NRPS)。 耐药金黄色葡萄球菌。因此，Fas/NRPS混合动力系统是一个有研究和控制价值的系统 可使具有新的或增强的生物活性的天然产品多样化。在过去的三十年里，真正控制了 生物合成装配线仍然难以捉摸；缺乏对巨型... 合成酶的蛋白质·蛋白质相互作用支配着天然产物的生物合成，阻碍了这一有希望的进展。 为了控制和发展Fas/NRPS的杂交生物合成途径，我们首先必须了解 指导相关生物合成机械的基本原理。FASS和NRPS都依赖于 载体蛋白(CP)；CP在多个伙伴蛋白(PP)之间穿梭生长的底物，以 功能化和并入天然产品。脂肪酸和非核糖体的模块化性质 多肽的生物合成在生物中是保守的，然而，CP·PP在每个生物体中的识别机制 途径是明确的。因此，为了重新设计这些生物合成流水线，本项目重点研究了 CP·PP界面的分子细节。我们的目标是1)通过X射线表征NRPS CP·PP相互作用 结晶学，2)建立设计混合NRPS/Fas相互作用的计算方法，以及3) 探索混合NRPS/Fas相互作用的跨途径生产率。这些研究的结果将 是未来这些模块合成酶生物工程不可或缺的一部分。

Abstract: Natural products and derivatives/mimics of natural products make up the majority of modern therapeutic drugs due to their unique complex organic scaffolds that contribute to their wide range of bioactivities. These natural products are synthesized by the fatty acid synthase (FAS), the nonribosomal peptide synthetase (NRPS) or the polyketide synthase (PKS). This program studies the role of protein•protein interactions in the modular synthases responsible for the formation of clinically relevant fatty acids and nonribosomal peptides for the long-term goal of re-designing and completely controlling the biosynthetic assembly lines. An example of a natural product therapeutic in the clinic is the antimicrobial daptomycin, which is a lipopeptide synthesized from a hybrid fatty acid synthase (FAS) and nonribosomal peptide synthetase (NRPS) that is used to treat methicillin- resistant Staphylococcus aureus. Thus, the hybrid FAS/NRPS are valuable systems to study and control so we may diversify natural products with new or enhanced bioactivities. Over the last three decades, true control over the biosynthetic assembly lines has remained elusive; the lack of a fundamental understanding on how the mega- synthase’s protein•protein interactions govern natural product biosynthesis has hindered this promising advance. In order to control and develop a hybrid FAS/NRPS biosynthetic pathway, first we must understand the fundamentals that guides the related biosynthetic machinery. Both FASs and NRPSs are dependent on the carrier protein (CP); the CP shuttles the growing substrate between multiple partner proteins (PP) for functionalization and incorporation into the natural product. The modular nature of fatty acid and nonribosomal peptide biosynthesis is conserved among organisms, however, the mechanism of CP•PP recognition in each pathway is distinct. Thus, to re-design these biosynthetic assembly lines, this project focuses on studying the molecular details of the CP•PP interface. We aim to 1) Characterize NRPS CP•PP interactions via X-ray crystallography, 2) establish a computational methodology to design a hybrid NRPS/FAS interaction, and 3) explore the cross-pathway productivity of the hybrid NRPS/FAS interaction. The results from these studies will be integral to the future bioengineering of these modular synthases.