Vitamin scavenging in the gut: Structure/function of the tight-binding B12 foraging machinery in Bacteroides - and its biotechnological applications

该项目的目的是阐明在肠道细菌的一个常见属中发现的一系列显着的维生素B12结合蛋白的详细分子特征，并将这些信息用于生物技术和工业过程。维生素B12不仅在人体健康中起着关键作用，它也是人体胃肠道中发现的许多细菌的必需营养素。这些肠道细菌中的许多似乎已经进化出了复杂而创新的方式来竞争这种稀缺的商品。多形拟杆菌（Bacteroides thetaiotamicron，Bt）是一种常见的细菌，它已经开发出一系列B12结合蛋白，对营养素具有极高的亲和力;该系统也存在于拟杆菌属的其他成员中。这种细菌利用位于表面的高亲和力结合蛋白从环境中获取营养，甚至能够从人体内因子中剥离营养，这是人类获得B12的主要途径。位于外表面的蛋白质将营养素转移到使其内化的运输系统。细菌细胞外囊泡（BEV）也会产生，并含有这些高亲和力的B12结合蛋白; BEV既能吸收营养，又能作为杀菌剂阻止竞争细菌获得营养。我们的首要目标是发现更多关于这些惊人的B12结合蛋白的信息，并开发利用它们的方法。本研究中的B12结合蛋白包括多种形式的BtuG、BtuH和BtuI，虽然结构基因组计划已经产生了btu操纵子内编码的蛋白质的几种apo结构，但以如此大的亲和力结合B12的机制尚不清楚。在这个项目中，我们将详细研究Bt中所有潜在的B12结合蛋白，确定它们的整体结构和它们对B12（和相关类似物）的结合亲和力，并阐明它们的释放机制。我们将通过靶向敲除实验结合荧光B12类似物的高分辨率荧光显微镜研究，研究该挽救系统的各个组分在体内的功能。我们将开发化学生物学方法，使我们能够将B12与一系列生物分子结合，以便B12结合蛋白的精致亲和力和特异性可以以与生物素-抗生物素蛋白生产系统相同的方式在商业上实现;这将使这些蛋白质用作探针和亲和基质，用于一系列生物技术和医学应用。最后，我们还将开发B12结合蛋白，用于从大型发酵中提取和快速分离B12，因为B12仍然是通过细菌发酵生产的少数维生素之一。该项目将为肠道微生物组的关键组成部分B12的获取和运输提供基础和根本的见解。它将提供关于一类新的维生素B12结合蛋白的基本分子细节，并产生用于抢救目的的紧密结合的新概念。它将允许利用这种非凡的结合能力来解决当前现实世界的问题;这样做，它将提供一个对生物技术和工业都有巨大好处的系统。

The aim of this project is to elucidate the detailed molecular characteristics of a range of remarkable vitamin B12 binding proteins that are found in a common genus of gut bacteria, and to exploit this information for biotechnological and industrial processes. Not only does vitamin B12 play a key role in human health, it is also an essential nutrient for many bacteria found in the human gastrointestinal tract. Many of these gut bacteria appear to have evolved elaborate and innovative ways to outcompete each other for this scarce commodity. One such common commensal, Bacteroides thetaiotamicron (Bt), has developed an array of B12-binding proteins with an extremely high affinity for the nutrient; the system is also present within other members of the Bacteroides genus. The bacterium uses surface-located high affinity binding proteins to acquire the nutrient from the environment and is even able to strip the nutrient from human intrinsic factor, which is the main route by which humans acquire B12. Proteins located on the outer surface transfer the nutrient to a transport system that enables its internalisation. Bacterial extracellular vesicles (BEVs) are also produced and contain these high affinity B12 binding proteins; BEVs both scavenge for the nutrient and also act as bactericidal agents that prevent competing bacteria from accessing the nutrient.Our overarching aim is to discover more about these astonishing B12-binding proteins and develop methods to exploit them for useful purposes. The B12 binding proteins in this study include multiple forms of BtuG, BtuH and BtuI, and while structural genomic projects have generated several apo-structures of the proteins encoded within the btu operons, but the mechanism for binding B12 with such great affinity is not known. In this project we will characterise, in detail, all the potential B12 binding proteins within Bt, determine their holo-structures and their binding affinities for B12 (and related analogues), and elucidate their mechanism of release. We will investigate the function of the individual components of this salvage system in vivo through targeted knock out experiments coupled with high resolution fluorescent microscopic investigation of fluorescent B12 analogues. We will develop chemical biology approaches that will enable us to conjugate B12 with a range of biomolecules so that the exquisite affinity and specificity of B12-binding proteins can be achieved commercially in the same way as the biotin-avidin production system; this will allow these proteins to be used as probes and affinity matrices for a range of biotechnological and medical applications. Finally, we will also develop the B12-binding proteins for extraction and rapid isolation of B12 in the industrial production of B12 from large fermentations, as B12 remains one of the few vitamins that is produced through bacterial fermentation.This project will provide both basic and fundamental insights into the acquisition and trafficking of B12 within a key component of the gut microbiome. It will provide essential molecular detail on a new class of vitamin B12 binding protein and generate new concepts on tight binding for salvaging purposes. It will allow exploitation of this remarkable binding capacity to address current real-world problems; in so doing, it will deliver a system that will have enormous benefit to both biotech and industry.