title: Analyzing the engineering potential of subtilosin A by probing the substrate promiscuity of the thioether forming sactisynthase AlbA
Creators: name: Ali, Ataurehman
Publisher: name: UNSPECIFIED
Year: 2022

description: Sactipeptides are members of a small but growing class of ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are characterized by unique cysteine-sulfur-to-α-carbon crosslinks. This thioether pattern between the donor cysteine side chains and the corresponding acceptors, catalyzed by so called sactisynthases, confers increased structural, thermal, and proteolytic stability as well as a rigid structure to sactipeptides. These traits render these unique scaffolds very attractive for the development of novel biotherapeutics. This work aimed to analyze the substrate promiscuity of the sactisynthase AlbA from the subtilosin A gene cluster in detail. In particular, the ability of the sactisynthase to tolerate major sequence substitutions and insertions was investigated towards the formation of so called sactionine bridges, which were identified and verified mass-spectrometrically. To analyze these exotic scaffolds, the designed sactipeptide variants were expressed in E. coli BL23(DE3). The first part of the work assessed the capability of AlbA to modify non-native sactipeptides. For this purpose, two strategies were followed and the sactisynthase successfully modified two unfamiliar sactipeptides, albeit with a decreased efficiency. Moreover, sactipeptide hybrid peptides consisting of the donor and loop regions of subtilosin A and the C-terminal acceptor region of four different Type I sactipeptides were designed and analyzed by mass spectrometry. These results provide novel insights into the regioselective performance of AlbA. In the second part of the present work, the ability of AlbA to tolerate larger amino acid sequences into the loop region of subtilosin A was scrutinized with respect to the introduced thioether bonds. This allowed the identification of a suitable position within the loop region of subtilosin A for the insertion of various foreign sequences without hampering the AlbA mediated post-translational modifications. The number of installed thioethers varied depending on whether the incorporated sequences met one of the two requirements: (i) a relatively short sequence or (ii) a structural pre-assembled sequence. The third part of the work was devoted to the functionalization of the sactipeptide subtilosin A. By successfully introducing biologically active sequences into the identified position in the loop region of the sactipeptide, the intrinsic traits of the generated thioether-constrained subtilosin A variants were next analyzed in more detail. In summary, specific receptor and pocket binding activities as well as a protease inhibitory property were successfully introduced, which however, suffered from low potency. In the present proof-of-concept work, a strategy was established for the functionalization of the sactipeptide subtilosin A. This approach could enable the functionalization of other sactipeptides as well as the generation of tailor-made subtilosin A variants with improved biological properties.