Challenges and advances in genome mining of ribosomally synthesized and post-translationally modified peptides (RiPPs)

Ribosomally synthesized and post-translationally modified peptides(RiPPs)are a class of cyclic or linear peptidic natural products with remarkable structural and functional diversity.Recent advances in genomics and synthetic biology,are facilitating us to discover a large number of new ribosomal natural products,including lanthipeptides,lasso peptides,sactipeptides,thiopeptides,microviridins,cyanobactins,linear thiazole/oxazole-containing peptides and so on.In this review,we summarize bioinformatic strategies that have been developed to identify and prioritize biosynthetic gene clusters(BGCs)encoding RiPPs,and the genome mining-guided discovery of novel RiPPs.We also prospectively provide a vision of what genomics-guided discovery of RiPPs may look like in the future,especially the discovery of RiPPs from dominant but uncultivated microbes,which will be promoted by the combinational use of synthetic biology and metagenome mining strategies.

Mechanistic Investigations into the Catalytic Mode of a Dehydratase Complex Involved in the Biosynthesis of Lantibiotic Cacaoidin

Dehydration of serine/threonine residues necessitates the activity of a dehydratase enzyme(domain)during the biosynthesis of RiPP.Recently,it was reported that dehydration in the thioviridamide pathway relies on a distinct dehydratase complex that showcases the activities of a phosphotransferase TvaC for serine/threonine phosphorylation and a lyase TvaD for subsequent phosphate elimination.Herein,we report that dehydration reactions in the pathway of lantibiotic cacaoidin involves a similar dehydratase complex,CaoK/CaoY.Remarkably,this dehydratase complex exhibits flexible enzymatic activity and tolerates significant variations in its substrate peptide sequence.By binding with the leader peptide(LP)sequence of precursor peptide CaoA,the dehydration reactions proceed in a directional manner from the C-terminus of the core peptide(CP)to the N-terminus,and C-terminally truncated variants of CP are acceptable.We show that fusing CaoK to CaoY in a 1:1 molar ratio enables the resulting enzyme CaoYK to exert enhanced dehydration activity.CaoK binds with the LP to improve its own solubility and to ensure the phosphate transfer activity,while CaoY functions in a manner independently of LP.This work advances our understanding of the dehydration process during cacaoidin formation,and provides useful enzymes and methods for the studies of the rapidly emerging RiPPs.

Complex peptide natural products: Biosynthetic principles, challenges and opportunities for pathway engineering

Complex peptide natural products exhibit diverse biological functions and a wide range of physico-chemical properties.As a result,many peptides have entered the clinics for various applications.Two main routes for the biosynthesis of complex peptides have evolved in nature:ribosomally synthesized and post-translationally modified peptide(RiPP)biosynthetic pathways and non-ribosomal peptide synthetases(NRPSs).Insights into both bioorthogonal peptide biosynthetic strategies led to the establishment of universal principles for each of the two routes.These universal rules can be leveraged for the targeted identification of novel peptide biosynthetic blueprints in genome sequences and used for the rational engineering of biosynthetic pathways to produce non-natural peptides.In this review,we contrast the key principles of both biosynthetic routes and compare the different biochemical strategies to install the most frequently encountered peptide modifications.In addition,the influence of the fundamentally different biosynthetic principles on past,current and future engineering ap-proaches is illustrated.Despite the different biosynthetic principles of both peptide biosynthetic routes,the arsenal of characterized peptide modifications encountered in RiPP and NRPS systems is largely overlapping.The continuous expansion of the biocatalytic toolbox of peptide modifying enzymes for both routes paves the way towards the production of complex tailor-made peptides and opens up the possibility to produce NRPS-derived peptides using the ribosomal route and vice versa.