Site‐Directed and Global Incorporation of Orthogonal and Isostructural Noncanonical Amino Acids into the Ribosomal Lasso Peptide Capistruin

Abstract

Expansion of the structural diversity of peptide antibiotics was performed through two different methods. Supplementation-based incorporation (SPI) and stop-codon suppression (SCS) approaches were used for co-translational incorporation of isostructural and orthogonal noncanonical amino acids (ncAAs) into the lasso peptide capistruin. Two ncAAs were employed for the SPI method and five for the SCS method; each of them probing the incorporation of ncAAs in strategic positions of the molecule. Evaluation of the assembly by HR-ESI-MS proved more successful for the SCS method. Bio-orthogonal chemistry was used for post-biosynthetic modification of capistruin congener Cap_Alk10 containing the ncAA Alk (Nε-Alloc-L-lysine) instead of Ala. A second-generation Hoveyda-Grubbs catalyst was used for an in vitro metathesis reaction with Cap_Alk10 and an allyl alcohol, which offers options for post-biosynthetic modifications. The use of synthetic biology allows for the in vivo production of new peptide-based antibiotics from an expanded amino acid repertoire.

A new genome-mining tool redefines the lasso peptide biosynthetic landscape

“…Lasso peptide BGCs frequently feature transporters and a RiPP recognition element (RRE, E-protein), which binds the leader peptide and directs enzymatic modification 11 . Interest in these scaffolds is evident from the numerous studies geared toward generating unnatural lasso peptides 14–16 .…”

Recombinant thiopeptides containing noncanonical amino acids

“…However, the total synthesis of variants in sufficient quantities for mechanistic or therapeutic purposes is often time-consuming and costly. The ability to genetically encode noncanonical amino acids (ncAAs) in bacteria has provided an alternative approach to incorporating building blocks with novel structures and chemistries into ribosomally biosynthesized proteins (11), cyclic peptides (12,13), lanthipeptides (14,15), and lasso peptides (16,17). However, methods to heterologously express thiopeptides in Escherichia coli, where the genetic incorporation of ncAAs with orthogonal nonsense or frameshift suppressor aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs is well-established (11), have not yet been reported.…”

Reprogramming the Biosynthesis of Cyclodepsipeptide Synthetases to Obtain New Enniatins and Beauvericins

“…[7] This has been realizedf or proteins and ribosomally synthesized and post-translationally modified peptides (RiPPs), preferably by site-directed mutagenesis. [12,13] Similar approaches for the design of artificial non-ribosomally synthesized peptides appear more difficult because of the sophisticated framework of interacting enzymatic domains or modules of nonribosomal peptides ynthetases (NRPSs). [12,13] Similar approaches for the design of artificial non-ribosomally synthesized peptides appear more difficult because of the sophisticated framework of interacting enzymatic domains or modules of nonribosomal peptides ynthetases (NRPSs).…”

“…The new combinationso fN RPS modules yielded novel molecules and could provides access to molecules otherwise difficult and laborious to synthesize. [11,13] Hence, the substrate tolerances of ESYN and PSYN previously determined in vitro [27,32] could be adapted to an in vivo approachb yP DB and MBS. [43,44] Our approachp rovides ar obust in vivo system for the generation of structurally diverseC PDs, and we have significantly expandedt he possibilities for the design and engineering of CDP synthetasesw ith novel biosynthetic capabilities.…”

“…[8][9][10] Further expansion towards non-canonical amino acids is achieved by supplementationbased incorporation [11] or stop-codons uppression. [12,13] Similar approaches for the design of artificial non-ribosomally synthesized peptides appear more difficult because of the sophisticated framework of interacting enzymatic domains or modules of nonribosomal peptides ynthetases (NRPSs). In NPRSs,t he specific recognition and activation of amino acids is conducted by adenylation (A) domains;t ransfer of activated amino acids onto the phosphopantetheinyl carrier protein domainsi sf ollowed by amide bond formation in the condensation (C) domains.…”