fORPHAN · From protein sequence to function – computational and experimental de-orphanization of uncharacterized enzymes in fungi

From protein sequence to function – computational and experimental de-orphanization of uncharacterized enzymes in fungi

Fast advances in genome sequencing and sequence processing technology leave ~30% of predicted proteins as orphans, meaning without known function or closely related enzymes. Being able to assign a function to such orphans opens avenues to select for and design powerful biocatalysts – individual enzymes, biosynthetic pathways or entire organisms. The goal of the fORPHAN project was to develop a de-orphanizing pipeline based on computational and experimental characterization of enzymes from the fungal kingdom. Fungi are known to be prolific producers of secondary metabolites, however, the study of the underlying biosynthetic gene clusters is still a fairly young field. The use of automated search and annotation pipelines is currently limited by the small number of experimentally characterized gene clusters that can be used to train the algorithms. Therefore, we used deep, targeted database searches to identify key biosynthetic enzymes in the publicly available genomes of fungi. As first targets we focused on fungal type 3 polyketide synthases and putative chalcone isomerases to investigate their family-wide sequence-function relationships. By the end of the funding period we have made significant progress in establishing our pipeline and have gained important insight into the substrate scope of fungal type 3 polyketide synthases. We have furthermore explored several microbial expression hosts for type 3 polyketide synthases. We will continue this research by further experimental characterization of the pre-screened enzymes and use them in novel combinatorial biosynthetic pathways. Two manuscripts related to the fORPHAN project are in preparation.

Data: CORDIS, © European Union