Leader- and Terminal Residue Requirements for Circularin A Biosynthesis Probed by Systematic Mutational Analyses

Abstract

Circularin A is a circular bacteriocin belonging to a subgroup of the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. The post-translational biosynthesis of circular bacteriocins primarily consists of leader cleavage, core peptide circularization, and bacteriocin secretion. However, none of these processes have been fully elucidated due to the complex biosynthesis of such bacteriocins and the lack of homology to the functions of other known biosynthetic enzymes. In this study, we investigated the leader-and terminal residue requirements for the biosynthesis of circularin A by systematic mutational analyses, including the mutational effects of variable leader lengths, as well as site-directed substitutions of residues at positions near the leader cleavage site and the circularization site. Results show that the leader with only one Met residue, the shortest leader possible, is sufficient to produce mature circularin A; helixforming short-sidechain hydrophobic residues are required at positions Val1 and Ala2 of the N-terminus to form active peptide derivatives, indicating the possible steric hindrance effect at these two positions; and an aromatic residue is required at the Cterminal Tyr69 position to produce a mature circular derivative. However, the requirements for residues at position Ala68 are much more relaxed relative to the positions of Val1 and Ala2, since even substitution with the largest possible residue, i.e., tryptophan, still allows the generation of an active Ala68Trp derivative. Our findings provide new perspectives for the biosynthesis of this short-leader circular bacteriocin, which enables the application of circular bacteriocin biosynthesis in rational modified peptide engineering.

Engineering circular bacteriocins: structural and functional effects of α-helix exchanges and disulfide introductions in circularin A

“…Notably, discrepancies of up to 18 Daltons were observed between the observed and theoretical masses for several peptide variants, including the wild-type circularin A (WT). As reported previously (Liu et al, 2023), we confirmed the simultaneous existence of three mature circularin A forms in the WT strain due to oxidation (Supplementary Figure S2). The differences in observed mass via MALDI-TOF analysis were attributed to the mass averaging of these three forms (WT, WT+16, WT+32).…”

“…The differences in observed mass via MALDI-TOF analysis were attributed to the mass averaging of these three forms (WT, WT+16, WT+32). Importantly, in the α1 and α5 replacement variants, we observed partial peptide degradation involving cleavage between M44 and T45 (Figure 2C, Table 2), similar to observations previously reported for some single residue substitutions of CirA (Liu et al, 2023).…”

“…For bacteriocin production, slight modifications were made to the expression conditions of cirA mutants compared to the previously reported method (Liu et al, 2022), and two rounds of nisin induction were employed during peptide expression (Liu et al, 2023). Briefly, the expression host (L. lactis) was first grown overnight in the GM17 medium at 30 • C. The following day, cells from the 5-mL fresh overnight culture were spun down and transferred into 100-mL minimal medium supplemented with 2.2% glucose and 5 ng/mL nisin for the initial induction.…”

“…After purification, MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and tricine-SDS-PAGE (tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were employed to evaluate the production of peptide variants as previously reported (Liu et al, 2022). For MALDI-TOF analysis, 1-µl concentrated peptide sample was applied, using the same method as previously described (Liu et al, 2023). The amount of peptide used for tricine-SDS-PAGE analysis was purified from 20 mL of cell culture, i.e., 20-µl concentrated peptide sample.…”

“…It has been reported that the biosynthetic machinery of circularin A prefers small hydrophobic residues at the N-terminus and aromatic residues at the C-terminus for circularization (Liu et al, 2023). Taking this into account, we designed two mutational variations of α-helix replacement between circularin A and acidocin B.…”