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. 2017 Aug 30;139(34):11646-11649.
doi: 10.1021/jacs.7b04159. Epub 2017 Aug 15.

Recombinant Macrocyclic Lanthipeptides Incorporating Non-Canonical Amino Acids

Claudio Zambaldo  1 , Xiaozhou Luo  1 , Angad P Mehta  1 , Peter G Schultz  1
Affiliations

Recombinant Macrocyclic Lanthipeptides Incorporating Non-Canonical Amino Acids

Claudio Zambaldo et al. J Am Chem Soc. .

Abstract

Nisin is a complex lanthipeptide that has broad spectrum antibacterial activity. In efforts to broaden the structural diversity of this ribosomally synthesized lantibiotic, we now report the recombinant expression of Nisin variants that incorporate noncanonical amino acids (ncAAs) at discrete positions. This is achieved by expressing the nisA structural gene, cyclase (nisC) and dehydratase (nisB), together with an orthogonal nonsense suppressor tRNA/aminoacyl-tRNA synthetase pair in Escherichia coli. A number of ncAAs with novel chemical reactivity were genetically incorporated into NisA, including an α-chloroacetamide-containing ncAA that allowed for the expression of Nisin variants with novel macrocyclic topologies. This methodology should allow for the exploration of lanthipeptide variants with new or enhanced activities.

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Figures

Figure 1
Figure 1
Wild type mNisA. Highlighted thioether rings and key residues for this study (residues 30-34 omitted for simplicity).
Figure 2
Figure 2
Altered ring structures of Nisin created by ncAA incorporation.
Figure 3
Figure 3
(A) ncAAs used in this study (B) Biorthogonal chemical handles incorporated into Ser5TAG NisA.
Figure 4
Figure 4
(A) SDS-PAGE mNisA ring variants incorporating 1. Lane 1: ring A, lane 2: ring B, lane 3: ring C. (B-D) QTOF-ESI for mNisA ring variants incorporating 1: (B) Ser3TAG, ring A. (C) Thr8TAG, ring B. (D) Thr13TAG, ring C. (E)C-NMR of labeled 1 (red) and 2 (green). (F) 13C-NMR mNisA Cys to Ala mutants incorporating labeled 1, non-cyclized (red) and cyclized (green) peptides. Asterisks indicate GSH addition products.
See this image and copyright information in PMC

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