One-pot synthesis of class II lanthipeptide bovicin HJ50 via an engineered lanthipeptide synthetase

Abstract

Lanthipeptides are a large class of bacteria-produced, ribosomally-synthesized and post-translationally modified peptides. They are recognized as peptide antibiotics because most of them exhibit potent antimicrobial activities against Gram-positive bacteria especially those that are phylogenetically related to producers. Maturation of class II lanthipeptide like bovicin HJ50 undergoes precursor modification by LanM and a subsequent leader peptide cleavage by LanT. Herein, via co-expression of precursor gene bovA, modification gene bovM and transporter gene bovT in Escherichia coli C43 (DE3), bioactive bovicin HJ50 was successfully produced and secreted. To further achieve in vitro one-pot synthesis of bovicin HJ50, an engineered bovicin HJ50 synthetase BovT150M was obtained by fusing the peptidase domain of BovT (BovT150) to the N-terminus of BovM. BovT150M exhibited dual functions of precursor modification and leader peptide cleavage to release mature bovicin HJ50. Under the guidance of BovA leader peptide, BovT150M exhibited substrate tolerance to modify non-native substrates including suicin and lacticin 481. This work exemplifies the feasibility of enzyme chimera of peptidase domain (LanT150) and modification enzyme (LanM) as a one-pot lanthipeptide synthetase.

Insights into AMS/PCAT transporters from biochemical and structural characterization of a double Glycine motif protease

“…As a step toward advancing a biochemical and structure-function understanding of AMS/PCAT transporters and providing a tool for removing leader peptides from RiPP products, we sought to identify protease domains that retained catalytic activity in the absence of the TMD. Previous studies have demonstrated that the N-terminal 150 amino acids of AMS/PCAT proteins constitute peptidase C39 family members that can be expressed as individual active domains (Håvarstein et al, 1995; Furgerson Ihnken et al, 2008; Ishii et al, 2006; Wu and Tai, 2004; Wang et al, 2016). In search of a substrate tolerant protease domain, we first surveyed AMS transporters encoded in gene clusters containing multiple genes for precursor peptides with diverse core peptide sequences, because these proteases are expected to be inherently tolerant with respect to residues in the P’ positions.…”

Roseocin, a novel two‐component lantibiotic from an actinomycete

“…The α‐component is 4 times dehydrated peptide with four lanthionine rings ( m/z 3,457.23, Figure f) and the β‐component is 9 times dehydrated with six lanthionine rings ( m/z 3,108.22 without the glycine residue trace, Figure e). For the structure elucidation of RosA peptides, we aim to co‐express RosT p along with recombinant plasmids generated here (Wang, Ge, Zhang, Teng, & Zhong, ), for obtaining roseocin in yield suitable for NMR. However, the purification strategy for the untagged peptides will have to be worked out accordingly.…”

Self‐immunity to antibacterial peptides by ABC transporters

“…The maturation starts by dehydration of Ser and Thr residues resulting in Dha or Dhb, which is catalysed by LanB, in the case of class I lanthipeptides, by a bifunctional enzyme termed LanM, for class II, the trifunctional LanKC, or LanL in class III and class IV or triprotein synthetases (LanK, LanX and LanY) in class V, respectively ( Fig. 1) [20,[29][30][31][32]. As a second step, the lanthionine rings are formed by a Michael-type addition with a cysteine side chain.…”