Recognition Sequences and Substrate Evolution in Cyanobactin Biosynthesis

Ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products are of broad interest because of their intrinsic bioactivities and potential for synthetic biology. The RiPP cyanobactin pathways <i>pat</i> and <i>tru</i> have been experimentally shown to be extremely tolerant of mutations. In nature, the pathways exhibit “substrate evolution”, where enzymes remain constant while the substrates of those enzymes are hypervariable and readily evolvable. Here, we sought to determine the mechanism behind this promiscuity. Analysis of a series of different enzyme–substrate combinations from five different cyanobactin gene clusters, in addition to engineered substrates, led us to define short discrete recognition elements within substrates that are responsible for directing enzymes. We show that these recognition sequences (RSs) are portable and can be interchanged to control which functional groups are added to the final natural product. In addition to the previously assigned N- and C-terminal proteolysis RSs, here we assign the RS for heterocyclization modification. We show that substrate elements can be swapped <i>in viv</i>o leading to successful production of natural products in <i>E. coli.</i> The exchangeability of these elements holds promise in synthetic biology approaches to tailor peptide products <i>in vivo</i> and <i>in vitro</i>

Aestuaramides, a Natural Library of Cyanobactin Cyclic Peptides Resulting from Isoprene-Derived Claisen Rearrangements

We report 12 cyanobactin cyclic peptides, the aestuaramides, from the cultivated cyanobacterium <i>Lyngbya aestuarii</i>. We show that aestuaramides are synthesized enzymatically as reverse <i>O</i>-prenylated tyrosine ethers that subsequently undergo a Claisen rearrangement to produce forward <i>C</i>-prenylated tyrosine. These results reveal that a nonenzymatic Claisen rearrangement dictates isoprene regiochemistry in a natural system. They also reveal one of the mechanisms that organisms use to generate structurally diverse compound libraries starting from simple ribosomal peptide pathways (RiPPs)