Glycosphingolipids from the Soil Microbiome, Understanding Structure and Biosynthesis

来自土壤微生物组的鞘糖脂，了解结构和生物合成

基本信息

批准号：
10836832
负责人：
Paul Davis Boudreau
金额：
$ 27.49万
依托单位：
UNIVERSITY OF MISSISSIPPI
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2025-03-31
项目状态：
未结题

项目摘要

The production of the glycosphingolipid 􀀂-􀀃􀀄􀀅􀀄􀀆􀀇􀀈􀀉􀀊􀀅􀀆􀀋􀀌􀀄􀀍􀀎􀀏􀀋􀀁􀀐􀀂-Gal) by a member of the human gut microbiome was an intriguing result because these lipids are known to be immune stimulating antigens, and their production by the gut microbiome suggests a role in host-microbiome signaling.1 􀀂-Gal is the canonical agonist for the immune system’s CD1d receptor,2–4 but synthetic work has shown that when the 􀀂-linked galactose is replaced with novel sugars, or sugar bioisosteres, the activity of the glycosphingolipid in immune signaling can change dramatically.5–7 These results suggest that bacteria which produce these glycosphingolipids, such as soil dwelling members of the order Sphingomonadales,8–10 might be a source of novel bioactive metabolites. In this project we have designed a soil enrichment screen using PCR amplification of serine palmitoyltransferase (SPT) gene, the first gene involved in sphingolipid synthesis,11,12 to identify sphingolipid producers. Follow-on lipidomic screening of SPT+ organisms on our laboratory’s QTOF LC-MS system will identify novel glycosphingolipids. By utilizing MS/MS fragment spectra analysis we will be able to identify sugar headgroups in our glycosphingolipids from neutral losses of the sugar monomers or the sugar fragment ions. Using GNPS-based molecular networking we will also be able to rapidly dereplicate known glycosphingolipid molecules, speeding up the process of identifying known chemistry to allow us to focus our efforts on novel sugar headgroups. With the novel organisms we isolate we will conduct Whole Genome Sequencing (WGS) with the Oxford Nanopore Technology’s nanopore platform to create a genomic data set that can be searched for the SPT gene. Inspired by the “glycogenomic” approach of mapping sugar chemistry in secondary natural products to biosynthetic gene clusters,13 we will also interrogate our genomes compared against the glycosphingolipids identified by LC-MS/MS analysis to identify candidate genes in the biosynthetic pathway after the SPT gene. Though this poses some unique challenges as sphingolipids are primary metabolites and their biosynthesis is not organized in tight biosynthetic gene clusters as is common in secondary natural products, the use of gene knockouts or heterologous expression can help confirm the role of these genes in the production of complex glycosphingolipids. We will also be able to utilize the known promiscuity of bacterial SPT genes to feed in unnatural lipid molecules,1 using LC-MS/MS monitoring to detect the novel glycosphingolipids produced by the incorporation of these feedstocks, demonstrating what strains might be able to be manipulated into producing compounds with desirable changes to the lipid tail of the glycosphingolipids. Glycosphingolipids isolated from scale up of the cultures will be further characterized by NMR analysis to confirm our structure assignment by MS/MS fragmentation analysis. At the end of the project, our glycosphingolipids will be submitted to a bioassay for cytokine elicitation from macrophages as a first step towards showing the clinical relevance of our glycosphingolipid library.

人类肠道微生物组的糖磷脂果􀀂-gal的产生是一个有趣的结果激动剂或免疫系统的CD1D受体，2-4，但合成工作已经显示出那时，然后将􀀂连接的银河替换为新糖或糖生物散热器，糖磷脂在免疫信号罐中的活性很大程度上。建议产生这些糖磷酸化的细菌，例如鞘氨质级别的成员，8-10可能是新型生物活性代谢物（SPT）基因的来源。跟随我们在我们的实验室系统上的SPT+生物的脂质筛选将通过使用糖单质糖单体或GNPS中性损失的GNPS中的糖头组来识别MS/MS片段的新型糖脂。 - 基于分子网络，我们还将快速降低已知的糖圈分子，加快识别自己的化学的过程，以便使用牛津纳米波尔技术平台进行整个基因组测序的新糖头组。可以搜索SPT基因的基因组数据集。通过LC-MS/MS分析与糖磷脂相比，辅助产物中的糖化化学方法与生物合成基因簇相比，糖合成基因簇的“糖基础”方法。在紧密的生物气体基因中，在Secury Products中sters sters是常见的，使用基因敲除或异源表达可以帮助确认该基因在复杂糖果的产物中的作用。在不自然的脂质分子中进行进食。 MS碎片分析。