Generation of thiocillin ring size variants by prepeptide gene replacement and in vivo processing by Bacillus cereus

Abstract

The thiocillins from Bacillus cereus ATCC 14579 are natural products from the broader class of thiazolyl peptides. Their biosynthesis proceeds via extensive post-translational modification of a ribosomally encoded precursor peptide. This post-translational tailoring involves a key step formal cycloaddition between two distal serine residues. In the wild-type structure, this cycloaddition forms a major macrocycle circumscribed by 26-atoms (shortest path). Results presented herein demonstrate the promiscuity of this last step by means of a set of "competition" experiments. Cyclization proceeds in many cases to provide altered ring sizes, giving access to several variant rings sizes that have not previously been observed in nature.

Figure 1

Structures of naturally occurring thiazolyl peptide ring size variants and fragment of thiocillin gene cluster from B. cere- us ATCC 14579.

Figure 2

Overlayed extracted ion chromatograms (EICs) for expected thiazolyl peptide masses in isolates from ring size competition mutants; peaks of compounds with 26-membered rings in blue, 23-memebered rings in red, and 29-membered in black. C11S depicts the total ion count (TIC) as no expected products were detected. Multiple peaks for each ring size were detected due to stochastic modification at Val6 and Thr8 and combined to create a single chromatogram. A detailed breakdown of ions present in the composite peaks above is given in the supporting information.

Figure 3

MS/MS fragmentation pattern of 26-member and 29-member ring compounds from S9-10 insertion mutant. Two key fragmentations are indicated on structures above and according ions are labeled below in an overlayed spectra; derivatives of these ions are further labeled with asterisks. The same spectral data is also presented in the supporting information with a more detailed fragment assignment.