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

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.

This project is focussed on a study of a new class of outer membrane-anchored vitamin B12-binding proteins, with femtomolar affinities for their B12 ligands, that has been described in the common commensal gut bacterium Bacteroides thetaiotamicron (Bt). Recognition of the physiochemical mechanisms that underpin high affinity protein-ligand interactions are key to understanding what drives specificity and how enthalpic and entropic processes contribute to such uncompromised binding. This is particularly important for high affinity binding with very fast on rates since these systems can be exploited in biotechnological applications including rapid diagnostics. This project will elucidate the structural basis for such a strong binding interaction: one that takes place between B12 and proteins such as BtuG, where the observed tight binding ultimately results in the uptake of the nutrient from the environment. There are more B12-binding proteins encoded within Bt than previously thought and we will compare their physical and structural properties against a range of cobamides that represent the full biological range of vitamin B12 analogues. This project will generate new fundamental insights into how Bt acquires B12 from the environment and how it uses this capacity to compete against other bacteria within crowded environments. Binding of B12 to these B12-binding proteins is on a similar scale to that observed between biotin and avidin, another vitamin-vitamin binding protein interaction. For this reason, we will explore how the B12-B12 binding proteins can be manipulated so that they can be used in similar applications. Moreover, we will explore how to use the B12 binding proteins to improve the efficiency of B12 extraction from large scale fermentations, which is how B12 is produced industrially. The project will keep the UK at the cutting edge of research in the area of basic and applied biology.