In recent years, genome mining has become a powerful strategy for the discovery of new specialized metabolites from microorganisms. However, the discovery of new groups of ribosomally synthesized and post-translationally modified peptides (RiPPs) by employing the currently available genome mining tools has proven challenging due to their inherent biases towards previously known RiPP families. In this chapter we provide detailed guidelines on using RiPPER, a recently developed RiPP-oriented genome mining tool conceived for the exploration of genomic database diversity in a flexible manner, thus allowing the discovery of truly new RiPP chemistry. In addition, using TfuA proteins of Alphaproteobacteria as an example, we present a complete workflow which integrates the functionalities of RiPPER with existing bioinformatic tools into a complete genome mining strategy. This includes some key updates to RiPPER (updated to version 1.1), which substantially simplify implementing this workflow.
Keywords: Antibiotics; Bioinformatics; Biosynthetic gene cluster; Genome mining; Natural products; Peptides; RiPP; Specialized metabolites.