Biosynthetic engineering of plantazolicin natural products
Plantazolicin (PZN) is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product that exhibits extraordinarily narrow-spectrum antibacterial activity towards the causative agent of anthrax, Bacillus anthracis. During PZN biosynthesis, a cyclodehydratase catalyzes cyclization of cysteine, serine, and threonine residues in the PZN precursor peptide (BamA) to azolines. Subsequently, a dehydrogenase then oxidizes most of these azolines to thiazoles and (methyl)oxazoles. The final biosynthetic steps consist of leader peptide removal and dimethylation of the nascent N-terminus. 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. 72 variant BamA peptides were screened by mass spectrometry to assess post-translational modification and export by E. coli, from which 29 PZN variants were detected. The modifying enzymes were exquisitely selective, installing heterocycles only at predefined positions within the precursor peptides while leaving neighboring residues unmodified. Nearly all substitutions at positions normally harboring heterocycles prevented maturation of a PZN variant. 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. Using heterologously expressed and purified heterocycle synthetase, the BamA peptide was processed in vitro concordant with the pattern of post-translational modification found in the naturally occurring compound. Using a suite of BamA-derived peptides, including amino acid substitutions as well as contracted and expanded substrate variants, the substrate tolerance of the heterocycle synthetase was elucidated in vitro, and the residues crucial for leader peptide binding were identified. Despite increased promiscuity compared to what was previously observed in E. coli, the synthetase retained selectivity in cyclization of unnatural peptides only at positions which correspond to those cyclized in the natural product. A cleavage site was subsequently introduced to facilitate leader peptide removal, yielding mature PZN variants after enzymatic or chemical dimethylation. In addition, we report the isolation and characterization of two novel PZN-like natural products whose existence was predicted by genome sequencing.
Natural products, Biosynthesis, Ribosomally synthesized and post-translationally modified peptide (RiPP)
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign