Engineering hybrid polyketide synthase systems using high affinity DNA binding do

描述（由申请人提供）：I型模块化PKSs负责生成具有制药，兽医和农业应用的各种聚酮产品的大环内酯核心。I型pks的模块化特性使它们成为酶生物工程研究的特别有吸引力的目标，为发现和开发天然产物药物建立了一种新的令人兴奋的方法。人们非常乐观地认为，非自然杂交pks的创建可以使这些天然产物的结构修饰和发展成为治疗剂，并且已经使用了几种策略。了解多组分聚酮合成酶（PKS）中的单个蛋白质如何相互作用以创建一条功能装配线，对于创建工程生物合成途径是不可或缺的。蛋白质-蛋白质界面被认为在很大程度上是由被称为对接域的卷曲卷曲的末端基序介导的，它使特定的成对相互作用具有有效的催化活性。人们可以设想一种合成生物学方法，利用各种同源码头结构域对来构建新的PKS通路。类似的偶联发生在非核糖体多肽合成酶（NRPSs）的相互作用模块之间。潜在地，新的PKS-NRPS杂交途径可以通过特异性偶联介导蛋白质相互作用来设计。一个关键的问题仍然是，来自系统发育上不同来源的模块化PKS和NPRS蛋白（例如放线菌，海洋蓝藻，黏菌）是否可以通过强结合界面更有效地相互作用。这个问题为探索合成高亲和力DNA结合域（DBDs）介导PKS模块相互作用的能力提供了一个令人感兴趣的动机，从而有效地组装新型聚酮天然产物分子。dbd普遍存在于细菌、真菌、哺乳动物和病毒等生物系统中。DBD是指一个独立折叠的蛋白质结构域，它包含至少一个识别双链或单链DNA的基序。dbd具有许多特性，使它们在蛋白质工程中极具吸引力。值得注意的是它们与序列特异性双链DNA的高亲和力（KD < 50 nM）。因此，dbd是替代亲和力相对较低的PKS对接结构域（KD ~ 50 5M）的理想靶点。本提案的目的是探索使用dbd作为在保持高效催化活性的同时在PKS模块之间建立高亲和相互作用的手段。虽然人们对dbd了解很多，但它们作为人工对接域的应用还没有被探索。DBD的许多特性可能需要优化，包括DBD的选择、域的大小以及用于将两个不同的DBD连接在一起的DNA序列。将测试DBD融合蛋白的功能，并严格控制相互作用的能力将被检查。最后，将测试一些混合PKS系统产生新型聚酮分子的潜力。拟议的工作为解锁PKS和NRPS系统的模块化潜力提供了巨大的潜力，以产生新的生物活性天然产物。

DESCRIPTION (provided by applicant): Type I modular PKSs are responsible for generating the macrolide core of a diverse range of polyketide products with pharmaceutical, veterinary, and agricultural applications. The modular nature of type I PKSs have made them particularly attractive targets for enzyme bioengineering efforts, establishing a new and exciting approach to discovery and development of natural product pharmaceuticals. There is significant optimism that the creation of unnatural hybrid PKSs can enable structural modification and development of these natural products into therapeutic agents, and several strategies have been used. Understanding how individual proteins within a multi-component polyketide synthase (PKS) interact with one another to create a functional assembly line has been integral to creating engineered biosynthetic pathways. The protein-protein interfaces are thought to be largely mediated by the coiled-coil termini motifs called docking domains that enable specific pair-wise interactions for effective catalytic activity. One can envision a synthetic biology approach in utilizing a diverse range of cognate dock domain pairs for the construction of new PKS pathways. A similar type of coupling occurs between interacting modules of non-ribosomal polypeptide synthases (NRPSs). Potentially, novel PKS-NRPS hybrid pathways could be engineered by mediating protein-protein interactions through specific couplings. A key question remains whether modular PKS and NPRS proteins from phylogenetically divergent sources (e.g. actinomycetes, marine cyanobacteria, myxobacteria) can interact more productively if engaged through a strong binding interface. This question provides a compelling motivation to explore the ability of synthetic high affinity DNA binding domains (DBDs) to mediate PKS modular interactions for efficient assembly of novel polyketide natural product molecules. DBDs are ubiquitously found in biological systems including bacteria, fungi, mammals and viruses. DBD refers to an independently folded protein domain, which contains at least one motif that recognizes double- or single-stranded DNA. DBDs have a number of characteristics that make them extremely attractive for protein engineering. The noteworthy feature is their high affinity (KD < 50 nM) to bind to sequence-specific double stranded DNA. Therefore, DBDs are an attractive target to replace the relatively low affinity PKS docking domains (KD ~ 50 5M). The aim of this proposal is to explore the use of DBDs as a means to establish high affinity interactions between PKS modules while maintaining efficient catalytic activity. Although much is known about DBDs, their application as artificial docking domains has not been explored. A number of features of the DBDs will likely need optimizing including the choice of DBD, the size of the domain, and the DNA sequence used to bring two distinct DBDs together. DBD fusion proteins will be tested for functionality and the ability to control interactions strictly will be examined. Finally, a number of hybrid PKS systems will be tested for their potential to generate novel polyketide molecules. The proposed work provides significant potential to unlock the modular potential of PKS and NRPS systems for the generation of new biologically active natural products. PUBLIC HEALTH RELEVANCE: The rapid rise of antibiotic resistant microbes has made the discovery and development of novel antibacterial agents a priority for national health. Polyketide natural products, such as erythromycin, have proven to be a rich source of antimicrobial bioactivity; however, due to their structural complexity, the synthesis of novel polyketides is a challenging, costly, and time consuming endeavor. The modular nature of PKS systems is an attractive feature for discovery and development of new macrolide antibiotics. The synthesis of biologically active polyketide natural product molecules is mediated by a multi-component complex comprised of proteins linked to each other in an analogous fashion to that of a passenger train, with each car representing a protein whose sequential order is dictated by the unique coupling mechanism between proteins. Each protein 'car' performs a specified modification to the polyketide molecule as it transits head-to-tail through the protein 'train'. The order of the protein 'cars' dictates the final size and shape of the polyketide molecule. By re-engineering the coupling mechanism between proteins we plan to rearrange the order of the assembly proteins within the protein 'train' resulting in new, polyketide molecules with diverse biological activities.