Engineering Novel Polyketide Antibiotics

工程新型聚酮化合物抗生素

• 批准号: 9256742
• 负责人: Benjamin Brandsen
• 金额: $ 5.67万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9256742
• 关键词: Acyltransferase; Anabolism; Antibiotic Resistance; Antibiotics; Azithromycin; Bacterial Antibiotic Resistance; Binding; Biosensor; Cells; Chemicals; Chimera organism; Clarithromycin; Coupled; DNA Binding Domain; Development; Engineering; Enzymes; Erythromycin; Escherichia coli; Fluorescence-Activated Cell Sorting; Food; Fresh Water; Gatekeeping; Genes; Genetic Transcription; Genome engineering; Inclusion Bodies; Laboratories; Libraries; Ligand Binding Domain; Ligands; Link; Location; Macrolide Antibiotics; Macrolides; Measurement; Metagenomics; Methodology; Methods; Monitor; Mutagenesis; Mutation; Natural Products; Nature; Pathway interactions; Peptide Hydrolases; Preparation; Production; Protein Binding Domain; Protein Hybridization; Proteins; Reporter; Reporter Genes; Sampling; Site; Source; Therapeutic; Variant; Vertebral column; bacterial resistance; base; biosynthetic product; design; fight against; gene synthesis; hybrid protein; novel; polyketide synthase; scale up; screening

Project Summary Bacterial resistance to antibiotics is a serious and growing problem, and the development of new antibiotics is critical in the fight against antibiotic resistance. One important class of antibiotics are polyketide compounds, which are produced by modular polyketide synthase enzymes. I will identify novel polyketide antibiotics by generating a polyketide biosensor and by applying large-scale mutagenesis, thereby working towards a general strategy to identify novel biosynthetic products. An E. coli biosensor will be constructed by first fusing a ligand-binding domain that binds to diverse polyketide compounds to required bacterial transcriptional component. The hybrid protein will then be engineered for ligand-dependent stability, such that it is stabilized in the presence of ligand and destabilized in the absence of ligand. In this design, increased biosensor stability is linked to increased reporter gene transcription, enabling the rapid measurement of ligand levels. In parallel, gene synthesis methods will be used to construct a synthetic polyketide pathway, incorporating restriction sites in key locations within the relevant genes. This synthetic pathway will be used to construct chimeric pathways in which acyltransferase domains from other polyketide synthase pathways are substituted for the native domain. Acyltransferase domains control which new substrates are incorporated into the growing polyketide backbone, and substitution of acyltransferase domains with unique substrate tolerance will enable the biosynthesis of diverse polyketide compounds. These chimeric pathways will be evaluated for production of their polyketide product. Finally, large-scale mutagenesis and biosensor-based selection will be used to rescue the biosynthesis activity of poorly performing chimeric pathways to produce novel polyketide antibiotics. This strategy of biosensor-based selection coupled with large-scale mutagenesis can be broadly applied to other biosynthetic pathways to produce novel compounds.

项目摘要 细菌对抗生素的抗药性是一个严重且日益严重的问题，新抗生素的开发是 在对抗抗生素耐药性方面至关重要。一类重要的抗生素是聚酮化合物， 它们是由模块化聚酮合成酶产生的。我将通过以下方法鉴定新型聚酮抗生素 产生聚酮生物传感器并应用大规模诱变，从而致力于 确定新的生物合成产品的一般策略。一种新型的大肠杆菌生物传感器将首先将一种 配体结合域，与不同的聚酮化合物结合到所需的细菌转录 组件。然后，杂交蛋白将被设计成依赖于配体的稳定性，这样它就稳定在 配基的存在和没有配基时的不稳定。在此设计中，提高了生物传感器的稳定性 与报告基因转录增加有关，使配基水平能够快速测量。同时， 基因合成方法将被用来构建一个合成的聚酮途径，包括限制位点 在相关基因内的关键位置。这种合成途径将被用来构建嵌合途径 其中来自其他聚酮合成酶途径的酰基转移酶结构域被天然的 域。酰基转移酶结构域控制哪些新底物被结合到生长中的聚酮中 骨架，取代具有独特底物耐受性的酰基转移酶结构域，将使 不同聚酮化合物的生物合成。这些嵌合途径将被评估为生产 他们的聚酮产品。最后，将使用大规模诱变和基于生物传感器的选择来拯救 表现不佳的嵌合途径的生物合成活性，以产生新型聚酮抗生素。这 基于生物传感器的选择与大规模诱变相结合的策略可以广泛应用于其他 产生新化合物的生物合成途径。