title: Recognition sequences and substrate evolution in cyanobactin biosynthesis.
abstract: 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 pat and tru 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 in vivo leading to successful production of natural products in E. coli. The exchangeability of these elements holds promise in synthetic biology approaches to tailor peptide products in vivo and in vitro.
authors.affiliation: Department of Medicinal Chemistry, University of Utah , Salt Lake City, Utah 84112, United States.
authors.forename: Debosmita
authors.initials: D
authors.lastname: Sardar
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Pierce
authors.forename: John A
authors.initials: JA
authors.lastname: McIntosh
authors.forename: Eric W
authors.initials: EW
authors.lastname: Schmidt
mesh_terms: D000595:Amino Acid Sequence; D001688:Biological Products / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012270:Ribosomes / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001688:Biological Products; D010456:Peptides, Cyclic; D011994:Recombinant Proteins
keywords: cyanobactins; posttranslational modifications; recognition sequences; ribosomally synthesized and posttranslationally modified peptides (RiPP); substrate evolution