Engineering hybrid polyketide synthase systems using high affinity DNA binding do

描述(由申请人提供)：第一类模块化PKSS负责产生具有制药、兽医和农业应用的各种聚酮产品的大环内酯核心。I型PKS的模块化性质使它们成为酶生物工程努力的特别吸引人的目标，建立了一种发现和开发天然产物药物的新的令人兴奋的方法。人们非常乐观地认为，创建非天然杂化PKS可以使这些天然产物能够进行结构修饰并开发成治疗剂，并且已经使用了几种策略。了解多组分聚酮合成酶(PKS)中的单个蛋白质是如何相互作用以创建功能装配线的，这是创建工程生物合成途径所不可或缺的。蛋白质-蛋白质界面被认为在很大程度上是由称为对接结构域的卷曲末端基序介导的，这些末端基序使特定的成对相互作用能够实现有效的催化活性。人们可以设想一种合成生物学的方法，利用不同范围的同源对接结构域对来构建新的PKS通路。类似类型的偶联发生在非核糖体多肽合成酶(NRPS)的相互作用模块之间。潜在的，新的PKS-NRPS杂化通路可以通过特定的偶联来调节蛋白质之间的相互作用。一个关键的问题仍然是，来自系统发育不同来源(例如放线菌、海洋蓝藻、粘细菌)的模块化PKS和NPR蛋白如果通过强大的结合界面相互作用，是否能够更有效地相互作用。这个问题提供了一个令人信服的动机来探索人工合成的高亲和力DNA结合域(DBD)介导PKS模块相互作用的能力，从而有效地组装新型聚酮类天然产物分子。DBD广泛存在于细菌、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，这使得它们对蛋白质工程非常有吸引力。值得注意的特征是它们与序列特异的双链DNA结合的亲和力高(Kd&lt；50 nm)。因此，DBDS是取代相对低亲和力的PKS对接结构域(Kd~505M)的一个有吸引力的靶点。这项提议的目的是探索使用DBDS作为一种手段，在保持有效催化活性的同时，建立PKS模块之间的高亲和力相互作用。尽管人们对DBD有了很多了解，但它们作为人工对接领域的应用还没有被探索过。DBD的许多功能可能需要优化，包括DBD的选择、结构域的大小以及用于将两个不同的DBD结合在一起的DNA序列。将测试DBD融合蛋白的功能，并检查严格控制相互作用的能力。最后，将测试一些混合PKS系统产生新型聚酮分子的潜力。这项拟议的工作为释放PKS和NRPS系统的模块潜力以产生新的生物活性天然产物提供了巨大的潜力。

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.