Harnessing the gut microbiome to detect and quantify glycans

项目摘要 检测多相混合物中存在的各种成分相似但结构不同的多糖 由于目前糖组学方法面临的限制，传统上一直具有挑战性。这些限制具有 阻碍了我们对同时存在的单个多糖的可用性和丰富性的理解 哺乳动物肠道，包含微生物、哺乳动物和植物来源的寡聚和 多糖类。此外，肠道多糖是肠道微生物群组成的主要驱动力， 新陈代谢，这代表着一个日益重要的人类健康决定因素，并需要一个深层次的 了解血糖-微生物界面，以确定重要的生物相互作用和推测 开发针对特定微生物活动的葡聚糖衍生疗法。因此，需要新的工具来 检测和测量存在于非均相中的单个多糖底物的相对丰度 由生物来源制成的混合物，如哺乳动物的肠道内容物。我们已经利用了 肠道微生物区系的优势成员用来检测、测量和分离的葡聚糖检测机械 单独的多糖底物存在于从哺乳动物肠道中提取的异质混合物中。在这里， 我们展示了强大的、特定的和可扩展的方法，工程微生物通过这些方法报告存在 以令人难以置信的敏感度对单个多糖底物进行分析。此外，我们还证明了这种方法可以 在广泛的线性范围内准确测量混合物中存在的单个多糖的丰度，并且 这些测量的特异度和灵敏度可以通过修饰特定的微生物多糖来调节 利用基因。最后，我们证明了微生物编码的糖链结合蛋白可以用来分离 从混合物中分离出单独的目标多糖，用于下游的组成和结构测定。我们建议 设置为1。)开发不同肠道微生物物种的排列文库，每个文库都经过改造以报告独特的 靶向多糖，2。)开发一种快速分离葡聚糖的管道，以纯化单个感兴趣的底物 3.下游结构和功能表征。)开发转基因微生物菌株 增强的敏感度或靶标特异性。除了提供一种快速且廉价的量化替代方案 我们相信，这些工具的进一步开发将揭示多糖的存在和丰富 以前检测不到的多糖，并极大地增强了我们对肠道之间相互作用的理解 微生物和它们的哺乳动物宿主。

Project Summary Detecting various compositionally similar but structurally distinct glycans present in heterogenous mixtures has traditionally been challenging due to the limitations facing current glycomics approaches. These limitations have hindered our understanding of the availability and abundance of individual glycans simultaneously present in the mammalian intestine, which contains complex mixtures of microbial, mammalian, and plant-derived oligo- and polysaccharides. Moreover, intestinal glycans are a primary driver of gut microbiome composition and metabolism, which represents an increasingly important human health determinant, and necessitates a deep understanding of the glycomic-microbial interface to identify important biological interactions and putatively develop glycan-derived therapeutics to target specific microbial activities. Therefore, new tools are necessary to detect and measure the relative abundance of individual glycan substrates present in the heterogeneous mixtures prepared from biological sources such as mammalian intestinal contents. We have harnessed the glycan detection machinery employed by dominant members of the gut microbiota to detect, measure and isolate individual glycan substrates present in heterogenous mixtures extracted from the mammalian intestine. Herein, we demonstrate robust, specific, and scalable approaches by which engineered microbes report the presence of individual glycan substrates with incredible sensitivity. Furthermore, we demonstrate that this approach can accurately measure the abundance of individual glycans present in mixtures across wide linear ranges and that the specificity and sensitivity of these measurements can be tuned by modifying particular microbial glycan utilization genes. Finally, we demonstrate that microbially-encoded glycan-binding proteins can be used to isolate individual target glycans from mixtures for downstream compositional and structural determination. We propose to 1.) develop arrayed libraries of distinct gut microbial species, each engineered to report the presence of unique target glycans, 2.) develop a rapid glycan isolation pipeline to purify individual substrates of interest for downstream structural and functional characterization, and 3.) develop genetically modified microbial strains with enhanced sensitivity or target specificity. In addition to offering a rapid and inexpensive alternative to quantifying known glycans, we believe that further development of these tools will reveal the presence and abundance of previously undetectable glycans and dramatically enhance our understanding of the interactions between gut microbes and their mammalian hosts.