Exploiting the polysaccharide breakdown capacity of the human gut microbiome to develop environmentally sustainable dishwashing solutions

盘子上的食物污渍是脂肪、蛋白质和碳水化合物的复杂混合物。这些污渍中的许多碳水化合物是植物多糖，去除这些污渍通常需要高温和大量的化学洗涤剂。在洗碗过程中，为了有效地去除污渍，减少能量和洗涤剂的需求，一个潜在的解决方案是使用特定的碳水化合物活性酶（CAZymes）将这些多糖分解成更容易从盘子上去除的可溶性低聚糖。发现有可能用于洗碗机配方的CAZymes的途径之一是开发大量未开发的新型植物多糖资源，降解由我们肠道微生物群编码的CAZymes。我们肠道菌群的主要营养来源是饮食中的多糖（通常称为膳食纤维），因为这些分子不会被宿主消化酶分解。由于这种进化压力，由肠道微生物群编码的降解多糖的酶的数量是巨大的。在这个项目中，我们的目标是利用肠道微生物群的巨大能力来有效地降解各种植物多糖，以发现新的酶，用于开发更环保的可持续的洗碗机配方。方法：拟杆菌属是微生物群中存在的主要属之一，并且已知特别擅长多糖降解，许多物种编码数百种CAZymes，使它们能够完全解构各种不同的多糖。我们的酶发现方法将是首先筛选我们内部的人类肠道拟杆菌库，以便在与不同问题食物污渍相关的一系列可溶性多糖上生长（例如，木聚糖，甘露聚糖，葡聚糖；这些将根据宝洁的优先级进行选择）。然后，蛋白质组学将用于鉴定与目标多糖生长良好的物种有关的特定降解装置，包括通过远同源检测和使用AlphaFold进行结构建模鉴定的潜在新CAZyme家族的令人兴奋的可能性。最初，我们将瞄准可能的表面内作用酶，因为这些酶是参与细胞外目标多糖最初快速分解为低聚糖的关键酶。人类肠道的快速运输时间意味着来自人类微生物组的酶已经进化到能够非常有效地分解这些植物多糖。这种高活性，再加上这些酶通常位于细胞外环境中，因此在一系列物理条件下高度稳定和健壮，最大限度地发挥了它们在洗碗机配方中的潜在用途。目标酶的重组版本将使用宿主实验室中可用的一系列生化技术进行表征，以确定纯化CAZymes的活性和特异性及其在多糖分解中的作用。针对目标的活性CAZymes将用宝洁提供的模型土壤进行测试。最初，我们将采用微型系统来评估潜在有用的酶和有希望的候选酶，然后在宝洁扩大到整个洗碗周期，以了解机械搅拌，t -坡道和动力学对整体性能的作用。在项目的过程中，新的和有趣的酶靶点将被推进到结构研究中，以确定底物特异性的分子基础。总体而言，该项目产生的数据将为肠道微生物群主要成员降解多糖的机制提供重要见解，并为开发新型酶策略提供基础，以增加新型洗涤产品的可持续性。

Food stains on dishes are complex mixtures of fat, protein and carbohydrates. Many of the carbohydrates in such stains are plant polysaccharides and removal of these stains often requires high temperature and significant levels of chemical detergents. A potential solution to reducing the energy and detergent requirements for effective stain removal during dishwashing is to use specific carbohydrate-active enzymes (CAZymes) to break these polysaccharides down into more soluble oligosaccharides that can be removed from dishes more easily. One route to discovery of CAZymes with the potential to be useful in dishwashing formulations is to exploit the huge untapped resource of novel plant polysaccharide degrading CAZymes encoded by our gut microbiota.The major nutrient source for the our gut microbiota are polysaccharides from the diet (often called dietary fibre) as these molecules are not broken down by host digestive enzymes. Because of this evolutionary pressure, the number of CAZymes encoded by the gut microbiota to degrade polysaccharides is vast. In this project we aim to exploit the enormous capacity of the gut microbiota to efficiently degrade a wide range of plant polysaccharides to discover novel CAZymes for use in development of more environmentally sustainable dishwashing formulations. Approach:Bacteroides spp are one of the main genera present in the microbiota and are known to be particularly adept at polysaccharide degradation, with many species encoding hundreds of CAZymes to enable them to fully deconstruct a wide range of different polysaccharides. Our approach for enzyme discovery will be to initially screen our in-house human gut Bacteroides library for growth on a range of soluble polysaccharides associated with different problem food stains (e.g., xylans, mannans, glucans; these will be chosen based on P&G priorities). Proteomics will then be used to identify the specific degradative apparatus involved in species that grow well on target polysaccharides, including the exciting possibility of potentially novel CAZyme families identified by distant homology detection and structural modelling using AlphaFold. Initially we will target likely surface endo-acting enzymes as these are the key enzymes involved in the initial rapid breakdown of extracellular target polysaccharides into oligosaccharides. The rapid transit time of the human gut means that CAZymes from the human microbiome have evolved to break these plant polysaccharides down very efficiently. This high activity, coupled to the fact these enzymes are often located in the extracellular milieu and so highly stable and robust in a range of physical conditions, maximises their potential use in dishwashing formulations.Recombinant versions of target enzymes will be characterised using a range of biochemical techniques available in the host lab to determine the activity and specificity of the purified CAZymes and their role in polysaccharide breakdown. CAZymes active against the targets will be tested with model soils provided by P&G. Initially, we will employ miniaturised systems to evaluate potentially useful enzymes and promising candidates will then be scaled up at P&G to full dishwashing cycles to understand the role of mechanical agitation, T-ramps and kinetics on the overall performance.Over the course of the project, novel and interesting enzyme targets will be advanced into structural studies to define the molecular basis for substrate specificity. Overall, the data generated in this project will provide significant insight into the mechanism of polysaccharide degradation by prominent members of the gut microbiota and underpin the development of novel enzyme strategies to increase the sustainability of novel dishwashing products.