Chimeric Leader Peptides for the Generation of Non-Natural Hybrid RiPP Products

[Image: see text] Combining biosynthetic enzymes from multiple pathways is an attractive approach for producing molecules with desired structural features; however, progress has been hampered by the incompatibility of enzymes from unrelated pathways and intolerance toward alternative substrates. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a diverse natural product class that employs a biosynthetic logic that is highly amenable to engineering new compounds. RiPP biosynthetic proteins modify their substrates by binding to a motif typically located in the N-terminal leader region of the precursor peptide. Here, we exploit this feature by designing leader peptides that enable recognition and processing by multiple enzymes from unrelated RiPP pathways. Using this broadly applicable strategy, a thiazoline-forming cyclodehydratase was combined with enzymes from the sactipeptide and lanthipeptide families to create new-to-nature hybrid RiPPs. We also provide insight into design features that enable control over the hybrid biosynthesis to optimize enzyme compatibility and establish a general platform for engineering additional hybrid RiPPs.