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Genetic DIversity of AVIdins for Novel Biotech Applications

Final Report Summary - DIVA (Genetic DIversity of AVIdins for Novel Biotech Applications)

The use of artificial metalloenzymes based on biotin-streptavidin technology has increased enormously in the past few years. The creation of artificial metalloenzymes provides a "bottom-up" approach to test and increase our understanding of fundamental aspects of biological catalysis, a quest that may lead to the development of improved and useful catalysts. The great potential in the field of artificial metalloenzymes is in the combination of apparently distant disciplines, including evolutionary biology, structural biology, computational chemistry and inorganic chemistry. The project funded by this ERG illustrates the progress and general challenges in this interdisciplinary field, as presented in several international conferences and summarised in a book-chapter co-written by the Marie-Curie fellow.

During this project, new avidins were investigated. Avidins are amongst the most widely used proteins in biotechnology, with applications encompassing molecular labelling, purification & detection, as well as uses in diagnostics, targeted drug delivery and nanotechnology, such as enantioselective catalysis by artificial catalysts. Avidins also serve as outstanding model systems for our understanding of biomolecular interactions, through their extremely high affinity for biotin (dissociation constant, Kd˜10-14-10-16 M). and biotinylated molecules. However, despite their importance in biotechnology, only two avidins have found widespread and routine use amongst the scientific community: chicken avidin and bacterial streptavidin.

During this project, we investigated avidins in the human pathogen and potential bioterrorism agent B. pseudomallei. To our knowledge, we provided the first demonstration of an avidin in a human pathogen having strong biotin-binding activity, with potential important implications in the medical field. Recombinant expression of this highly stable protein in E. coli required periplasmic secretion (achieved using the native Burkholderia secretion signal) and formation of an intramonomeric disulphide bridge. This new avidin may also prove useful in many biotechnological applications, including as an artificial metalloenzyme, as shown in proof-of-principle studies using hydrogenation catalysis.

Finally, the work with novel avidins of microbial human pathogens has opened-up a new line of research within the group, in particular looking at new potential targets for antibiotics. The emergence of bacterial strains that are resistant to virtually all currently available antibiotics underscores the importance of developing new antimicrobial compounds. We focused on DapE, a promising antibiotic target. We discovered that a lead-compound that inhibits DapE in vitro did not show any measurable anti-DapE effect in bacteria, providing a sobering reminder of the difficulty of translating in vitro data to effects in vivo, even in pure microbiological cultures. The development of other (more effective) DapE inhibitors in vitro and in vivo continues to be a very worthy goal and a promising line of research toward new antibiotics, which we will be pursuing further.

In summary, this interdisciplinary project at the boundary of chemistry and biology developed new technologies relevant to the field of biocatalysis (artificial enzymes) as well as in the biomedical field (diagnostic and antimicrobial targets and agents).