Sactipeptide Engineering by Probing the Substrate Tolerance of a Thioether‐Bond‐Forming Sactisynthase

Abstract

Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α‐carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in‐depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides. We identified a possible modification site within the sactipeptide subtilosin A allowing for peptide engineering without compromising formation of thioether bridges. A panel of natural and hybrid sactipeptides was produced to study the AlbA‐mediated formation of thioether bridges, which were identified mass‐spectrometrically. In a proof‐of‐principle study, we re‐engineered subtilosin A to a thioether‐bridged, specific streptavidin targeting peptide, opening the door for the functional engineering of sactipeptides.

Probiogenomic analysis of Lactiplantibacillus plantarum SPS109: A potential GABA-producing and cholesterol-lowering probiotic strain

“…In addition, certain sactipeptides exhibit a narrow antibiotic efficacy against drug-resistant bacteria. This could be crucial in the future, given the imminent threat of a significant antibiotic crisis worldwide [ 36 ].…”

Discovery and engineering of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products

“…AlbA, the sacipeptie synthase responsible for the formation of thioether cross-links in subtilosin A, exhibits remarkable substrate promiscuity, allowing for generation of various subtilosin analogs by modification of precursor peptides with unnatural cores. 179 Although successful engineering cases have been limited, which requires a deeper understanding of substrate recognition and catalytic mechanisms, these efforts have prepared the way for the directed evolution of studies aimed at optimizing sactipeptide properties.…”

Mechanistic and functional aspects of the Ruminococcin C sactipeptide isoforms

“…Among these RiPPs, sactipeptides form a small but growing subfamily currently comprising 11 characterized members. 3 , 4 Sactipeptides are featured by one or several sulfur-to-alpha carbon thioether cross-links introduced by radical SAM enzymes and can be further divided into three subfamilies ( Figures 1 A and 1C). 4 , 5 The first and oldest subclass (Type I) comprises subtilosin A, 6 thurincin H, 7 the sporulation killing factor (SkfA), 8 thuricin CD 9 that consists of two peptides, Trn-α and Trn-β, thuricin Z also named huazacin, 10 , 11 and hyicin 4244 12 ( Figures 1 B and 1C).…”