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

菜肴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。减少洗碗过程中有效去除污渍所需的能量和洗涤剂需求的一个潜在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易溶的低聚糖，更容易从盘子中去除。发现有潜力用于洗碗剂的CAZymes的一条途径是开发由我们的肠道微生物编码的大量未开发的新型植物多糖资源来降解CAZymes。我们肠道微生物区系的主要营养来源是来自饮食的多糖(通常称为膳食纤维)，因为这些分子不被宿主消化酶分解。由于这种进化压力，肠道微生物区系编码的降解多糖的CAZyme数量巨大。在这个项目中，我们的目标是利用肠道微生物区系的巨大能力，有效地降解各种植物多糖，以发现新的CAZyme，用于开发更环保的洗碗剂配方。方法：类杆菌是微生物区系中的主要属之一，已知特别擅长多糖的降解，许多物种编码数百个CAZyme，使它们能够完全分解多种不同的多糖。我们的酶发现方法将是初步筛选我们内部的人类肠道类杆菌文库，以寻找与不同问题食物污渍相关的一系列可溶性多糖(例如，木聚糖、甘露聚糖、葡聚糖；这些将根据宝洁的优先顺序进行选择)。然后，蛋白质组学将被用来鉴定在目标多糖上生长良好的物种所涉及的特定降解装置，包括通过远距离同源性检测和使用AlphaFold进行结构建模来鉴定潜在的新CAZyme家族的令人兴奋的可能性。首先，我们将针对可能的表面内切酶，因为这些酶是参与胞外目标多糖最初快速分解为低聚糖的关键酶。人体肠道的快速转运时间意味着来自人类微生物组的CAZymes已经进化成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，在一系列物理条件下非常稳定和健壮，最大限度地发挥了它们在洗碗剂中的潜在用途。重组的靶酶版本将使用宿主实验室提供的一系列生化技术来表征，以确定纯化的CAZymes的活性和特异性及其在多糖分解中的作用。对目标有效的CAZymes将用宝洁提供的模型土壤进行测试。最初，我们将使用微型系统来评估潜在的有用的酶，然后在宝洁将有希望的候选酶放大到完整的洗碗周期，以了解机械搅拌、T形坡道和动力学对整体性能的作用。在项目过程中，新的和有趣的酶目标将进入结构研究，以确定底物专一性的分子基础。总体而言，该项目产生的数据将为肠道微生物群中重要成员的多糖降解机制提供重要的洞察，并为开发新的酶策略以增加新的洗碗产品的可持续性奠定基础。

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.