Harnessing the gut microbiome to detect and quantify glycans

项目摘要 检测异质混合物中存在的各种组成相似但结构不同的聚糖， 由于当前糖组学方法面临的限制，传统上是具有挑战性的。这些限制 阻碍了我们对同时存在于细胞中的单个聚糖的可用性和丰度的理解。 哺乳动物肠道，其中含有微生物、哺乳动物和植物来源的寡核苷酸和 多糖此外，肠聚糖是肠道微生物组组成的主要驱动因素， 代谢，这代表了一个越来越重要的人类健康决定因素，并需要一个深刻的 了解糖-微生物界面，以确定重要的生物相互作用和微生物 开发针对特定微生物活性的聚糖衍生疗法。因此，需要新的工具， 检测和测量存在于异质性细胞中的单个聚糖底物的相对丰度， 由生物来源如哺乳动物肠内容物制备的混合物。我们已经利用了 由肠道微生物群的主要成员使用的聚糖检测机制，以检测、测量和分离 存在于从哺乳动物肠中提取的异源混合物中的单个聚糖底物。在此， 我们展示了强大的，具体的和可扩展的方法，通过这些方法，工程微生物报告了 以难以置信的灵敏度对单个聚糖底物进行分析。此外，我们证明，这种方法可以 在宽线性范围内准确测量混合物中存在的单个聚糖的丰度， 这些测量的特异性和灵敏度可以通过修饰特定的微生物聚糖 利用基因最后，我们证明了微生物编码的聚糖结合蛋白可用于分离 用于下游组成和结构测定的混合物中的单个目标聚糖。我们提出 至1.）开发不同肠道微生物物种的阵列库，每一个都被设计成报告独特的 靶聚糖，2.）开发快速聚糖分离管道，以纯化感兴趣的单个底物， 下游结构和功能表征，以及3.）开发转基因微生物菌株， 增强的灵敏度或目标特异性。除了提供一个快速和廉价的替代量化 已知的聚糖，我们相信这些工具的进一步发展将揭示存在和丰富的 以前检测不到的聚糖，并大大提高了我们对肠道之间相互作用的理解， 微生物及其哺乳动物宿主。

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.