Engineering Unnatural Variants of Plantazolicin through Codon Reprogramming

Abstract

Plantazolicin (PZN) is a polyheterocyclic natural product derived from a ribosomal peptide that harbors remarkable antibiotic selectivity for the causative agent of anthrax, Bacillus anthracis. To simultaneously establish the structure-activity relationship of PZN and the substrate tolerance of the biosynthetic pathway, an Escherichia coli expression strain was engineered to heterologously produce PZN analogs. Variant PZN precursor genes were produced by site-directed mutagenesis and later screened by mass spectrometry to assess posttranslational modification and export by E. coli. From a screen of 72 precursor peptides, 29 PZN variants were detected. This analog collection provided insight into the selectivity of the posttranslational modifying enzymes and established the boundaries of the natural biosynthetic pathway. Unlike other studied thiazole/oxazole-modified microcins, the biosynthetic machinery appeared to be finely tuned towards the production of PZN, such that the cognate enzymes did not process even other naturally occurring sequences from similar biosynthetic clusters. The modifying enzymes were exquisitely selective, installing heterocycles only at pre-defined positions within the precursor peptides while leaving neighboring residues unmodified. Nearly all substitutions at positions normally harboring heterocycles prevented maturation of a PZN variant, though some exceptions were successfully produced lacking a heterocycle at the penultimate residue. No variants containing additional heterocycles were detected, although several peptide sequences yielded multiple PZN variants as a result of varying oxidation states of select residues. Eleven PZN variants were produced in sufficient quantity to facilitate purification and assessment of their antibacterial activity, providing insight into the structure-activity relationship of PZN.

Elucidating the Specificity Determinants of the AtxE2 Lasso Peptide Isopeptidase

“…E-mail: ajlink@princeton.edu. 3 The abbreviations used are: RiPP, ribosomally synthesized and post-translationally modified peptide; AtxE2, A. excentricus lasso peptide isopeptidase 2; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single quantum correlation; aTc, anhydrotetracycline; AUC, area under the curve; aa, amino acids; RMSD, root-mean-square-deviation.…”

“…Thus, much like the lasso peptide biosynthetic enzymes, lasso peptide isopeptidase has broad substrate specificity. Quantitative analysis of the cleavage reactions indicated that alanine substitutions in loop positions of these peptides led to reduced cleavage, suggesting that the loop is serving as a recognition element for the isopeptidase.Ribosomally synthesized and post-translationally modified peptides (RiPPs) 3 (1) are a diverse set of natural products that are formed by the action of maturation enzymes on a linear peptide substrate. An emerging theme in the biosynthesis of RiPPs is that the maturation enzymes tend to have broad substrate specificity (2-8).…”

“…Ribosomally synthesized and post-translationally modified peptides (RiPPs) 3 (1) are a diverse set of natural products that are formed by the action of maturation enzymes on a linear peptide substrate. An emerging theme in the biosynthesis of RiPPs is that the maturation enzymes tend to have broad substrate specificity (2)(3)(4)(5)(6)(7)(8).…”

“…An emerging theme in the biosynthesis of RiPPs is that the maturation enzymes tend to have broad substrate specificity (2)(3)(4)(5)(6)(7)(8). Lasso peptides are a class of RiPPs, characterized by a threaded structure resembling a slipknot (9 -11).…”

Insights into Methyltransferase Specificity and Bioactivity of Derivatives of the Antibiotic Plantazolicin

“…An alignment of the five known PZN precursor peptides shows that the N -terminal portion is considerably more conserved than the C -terminal portion, which could support the previous conjecture (6). Nevertheless, some modifications to the C -terminus are detrimental to the anti- B. anthracis activity (20). The combined biochemical, and microbiological analyses of PZN methyltransferase specificity sets the framework for future studies aimed to engineering non-natural PZN variants with expanded and/or orthogonal bioactivities.…”