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

Jian Wang,Xiaoxuan Ge,Kunling Teng,Jin Zhong,Li Zhang

doi:10.1038/srep38630

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.

Highlights

Drug-resistant bacteria have posed increasing threats to human health and have raised global concern for lack of effective resorts[1,2,3]
Bovicin HJ50 has been produced by way of a two-step semi-in vitro biosynthesis system (SIVB) consisting of (1) in vivo modification of precursor peptide by co-expression of precursor gene bovA and modification gene bovM in E. coli and (2) in vitro digestion of leader peptide by N-terminal peptidase domain of BovT (BovT150) (Fig. 1a)[20]
BovA could be successfully modified by BovM when they are co-expressed in E. coli[20]

Summary

Introduction

Drug-resistant bacteria have posed increasing threats to human health and have raised global concern for lack of effective resorts[1,2,3]. Bovicin HJ50 has been produced by way of a two-step semi-in vitro biosynthesis system (SIVB) consisting of (1) in vivo modification of precursor peptide by co-expression of precursor gene bovA and modification gene bovM in E. coli and (2) in vitro digestion of leader peptide by N-terminal peptidase domain of BovT (BovT150) (Fig. 1a)[20]. An engineered lanthipeptide synthetase BovT150M (BovT150-BovM fusion enzyme) was successfully reconstituted that exerted dual functions of precursor modification and leader peptide cleavage.

Results

Conclusion