Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

Abstract

Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts.thiopeptide | affinity-tagged intermediates | biosynthesis | antibiotic | RiPP

Promiscuous enzymes cooperate at the substrate level en route to lactazole A

“…36 Using this platform, termed the FIT-Laz system, we showed that selective biosynthesis of lactazole A occurred only when all enzymes were present in the reaction mixture from the beginning, whereas stepwise treatments led to either under-or overdehydrated thiopeptides containing 3 or 5 Dha, respectively. These results suggest that cooperation between Laz enzymes extends beyond the "azoles form first, Dha second" model observed for thiopeptides studied to date, 20,21,37 and indicate that lactazole may be a good model system to study concerted action of multiple enzymes in RiPP biosynthesis.…”

“…The copyright holder for this preprint (which this version posted May 14, 2020. ; https://doi.org/10.1101/2020.05.12.092031 doi: bioRxiv preprint 14 Substrate specificity Primary macrocycle assembly for thiopeptides studied to date separates azole and Dha formation into two stages, where Dha installation follows azole formation. 20,21,37 Such a separation can be due to the specificity of a Dha-installing enzyme toward the native azole pattern, 20 or due to an additional enzyme acting as a gatekeeper and preventing premature Dha synthesis. 21,37 Intertwined enzymatic action observed in the experiments above is distinct from these models, indicating that lactazole biosynthesis integrity is maintained (Fig.…”

“…20,21,37 Such a separation can be due to the specificity of a Dha-installing enzyme toward the native azole pattern, 20 or due to an additional enzyme acting as a gatekeeper and preventing premature Dha synthesis. 21,37 Intertwined enzymatic action observed in the experiments above is distinct from these models, indicating that lactazole biosynthesis integrity is maintained (Fig. 5a, LazA CP1-4 ).…”

Biosynthesis of the nosiheptide indole side ring centers on a cryptic carrier protein NosJ

“…Because of the multiple modes of action and the potent activity toward many drug-resistant pathogenic bacteria 5 , 18 , 41 , 42 , thiopeptides serve as a promising group of leads in antibiotic development. Despite significant progress has been made over the past few years 6 – 12 , many aspects in thiopeptide biosynthesis remain unclear. In this work, we have revealed the function and substrate specificity of NosI, NosJ, NosN and NosK, demonstrating a unique biosynthetic strategy that centers on the Ppant arm of a carrier protein NosJ (Fig.…”

“…Among the most extensively modified RiPPs are thiopeptides, a class of sulfur-rich, polyazole-containing macrocyclic peptides featuring a central six-membered nitrogen-containing heterocycle 5 . Similar to all RiPPs, thiopeptides are produced from a ribosomally synthesized precursor peptide, involving actions of highly divergent biosynthetic enzymes that are organized in a well-orchestrated process 6 – 12 . The complex nature of thiopeptide biosynthesis is further demonstrated by the fact that some thiopeptides, such as nocathiacin and nosiheptide, contain additional macrocycle rings that are not derived from the ribosomally synthesized precursor peptides 13 – 16 .…”