Engineering Novel Polyketide Antibiotics

项目摘要 细菌对抗生素的耐药性是一个严重的和日益严重的问题，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， 其由模块化聚酮化合物合酶产生。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，并通过应用大规模诱变，从而致力于 鉴定新型生物合成产物的一般策略。大肠大肠杆菌生物传感器将通过首先融合 配体结合结构域，其结合不同的聚酮化合物以实现所需的细菌转录 成分然后将杂合蛋白质工程化以获得配体依赖性稳定性，使得其在细胞内稳定。 在配体存在下稳定，在配体不存在下不稳定。在该设计中，增加的生物传感器稳定性是 与增加的报告基因转录相关，从而能够快速测量配体水平。同时， 基因合成方法将用于构建合成聚酮途径，引入限制性位点 在相关基因的关键位置。该合成途径将用于构建嵌合途径 其中来自其它聚酮化合物合酶途径的酰基转移酶结构域取代天然的 域酰基转移酶结构域控制哪些新底物被掺入到生长的聚酮化合物中 骨架，并且具有独特底物耐受性的酰基转移酶结构域的取代将使得能够在 生物合成不同的聚酮化合物。将评价这些嵌合途径的产生。 他们的聚酮产品。最后，将采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的聚酮化合物抗生素。这 基于生物传感器选择与大规模诱变相结合的策略可广泛应用于其他 生物合成途径来产生新化合物。

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.