Engineering hybrid polyketide synthase systems using high affinity DNA binding do

使用高亲和力 DNA 结合工程杂化聚酮合酶系统

• 批准号: 8124006
• 负责人: Joseph Anthony Chemler
• 金额: $ 4.84万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8124006
• 关键词: Actinobacteria class; Affinity; Agriculture; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Drug Resistance; Binding; Binding Sites; Biological; Biological Factors; Biomedical Engineering; Characteristics; Chemicals; Chemistry; Chimeric Proteins; Collection; Complex; Coupling; Cyanobacterium; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; Development; Docking; Engineering; Enzymes; Erythromycin; Generations; Genes; Head; Health; Hybrids; In Vitro; Individual; Macrolide Antibiotics; Macrolides; Mammals; Marines; Mediating; Microbe; Modeling; Modification; Motivation; Myxococcales; Nature; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Play; Process; Production; Protein Binding; Protein Engineering; Proteins; Public Health; Screening procedure; Shapes; Single-Stranded DNA; Source; Specific qualifier value; Specificity; Structure; System; Tail; Tertiary Protein Structure; Testing; Therapeutic Agents; Time; Training; Tylosin; Virus; Work; antimicrobial; avermectin; base; biological systems; combat; combinatorial chemistry; design; design and construction; drug resistant microorganism; ds-DNA; fungus; genetic manipulation; improved; link protein; microbial; novel; optimism; pathogen; pharmacophore; picromycin; polyketide synthase; polypeptide; protein complex; protein protein interaction; small molecule; synthetic biology

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.

描述（由申请方提供）：I型模块化PKS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的模块化性质使它们成为酶生物工程工作的特别有吸引力的靶标，建立了一种新的和令人兴奋的方法来发现和开发天然产物药物。有显着的乐观，创造非天然的杂合PKS可以使这些天然产物的结构修饰和发展成为治疗剂，并已使用了几种策略。了解多组分聚酮化合物合酶（PKS）中的单个蛋白质如何相互作用以创建功能性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称为对接结构域的卷曲螺旋末端基序介导的，所述对接结构域能够实现有效催化活性的特定成对相互作用。人们可以设想一种合成生物学方法，其利用各种各样的同源对接结构域对来构建新的PKS途径。类似类型的偶联发生在非核糖体多肽链转移酶（NRPS）的相互作用模块之间。潜在的，新的PKS-NRPS杂合途径可以通过介导蛋白质-蛋白质相互作用，通过特定的耦合工程。一个关键的问题仍然是模块化的PKS和NPRS蛋白质从遗传上不同的来源（如放线菌，海洋蓝藻，粘细菌）是否可以更有效地相互作用，如果从事通过强结合界面。这个问题提供了一个令人信服的动机，探索合成的高亲和力DNA结合结构域（DBD）的能力，介导PKS模块的相互作用，有效地组装新的聚酮化合物天然产物分子。 DBD普遍存在于生物系统中，包括细菌、真菌、哺乳动物和病毒。DBD是指独立折叠的蛋白质结构域，其含有至少一个识别双链或单链DNA的基序。DBD具有许多特性，使其对蛋白质工程极具吸引力。值得注意的特征是它们与序列特异性双链DNA结合的高亲和力（KD < 50 nM）。因此，DBDs是一个有吸引力的目标，以取代相对低亲和力PKS对接结构域（KD ~ 50 5 M）。 该提案的目的是探索使用DBD作为在PKS模块之间建立高亲和力相互作用的手段，同时保持有效的催化活性。尽管人们对DBD有很多了解，但它们作为人工对接域的应用还没有被探索。DBD的许多特征可能需要优化，包括DBD的选择、结构域的大小以及用于将两个不同DBD结合在一起的DNA序列。将测试DBD融合蛋白的功能性，并将检查严格控制相互作用的能力。最后，一些混合PKS系统将被测试其潜力，以产生新的聚酮分子。拟议的工作提供了巨大的潜力，解锁PKS和NRPS系统的模块化潜力，以产生新的生物活性的天然产物。 公共卫生相关性：抗生素耐药微生物的迅速崛起使得新型抗菌剂的发现和开发成为国民健康的优先事项。聚酮化合物天然产物，如红霉素，已被证明是抗微生物生物活性的丰富来源;然而，由于其结构复杂性，新型聚酮化合物的合成是一项具有挑战性的、昂贵的和耗时的奋进。PKS系统的模块化性质对于发现和开发新的大环内酯类抗生素是一个有吸引力的特征。生物活性聚酮化合物天然产物分子的合成由多组分复合物介导，所述复合物由以类似于客运列车的方式彼此连接的蛋白质组成，每节车厢代表一种蛋白质，其顺序由蛋白质之间的独特偶联机制决定。每个蛋白质“车”执行一个特定的修饰的聚酮分子，因为它通过蛋白质“列车”的头到尾。蛋白质“汽车”的顺序决定了聚酮化合物分子的最终大小和形状。通过重新设计蛋白质之间的偶联机制，我们计划重新排列蛋白质“火车”内组装蛋白质的顺序，从而产生具有不同生物活性的新的聚酮化合物分子。