Translocation of the thioesterase domain for the redesign of plipastatin synthetase.

Ling Gao,Hongxia Liu,Xiaomei Bie,Jinzhi Han,Zhi Ma,Zhaoxin Lu,Fengxia Lv,Chen Dai

doi:10.1038/srep38467

Abstract

Non-ribosomal peptide synthetases (NRPSs) are large enzymatic complexes that catalyse the synthesis of biologically active peptides in microorganisms. Genetic engineering has recently been applied to reprogram NRPSs to produce lipopeptides with a new sequence. The carboxyl-terminal thioesterase (TE) domains from NRPSs catalyse cleavage products by hydrolysis or complex macrocyclization. In this study, we modified plipastatin synthetase by moving the intrinsic TE region to the end of the internal thiolation (T) domains, thus generating Bacillus subtilis strains that could produce new truncated cyclic or linear peptides of the predicted sequence, which further provided an important insight into the regioselectivity of plipastatin TE. The TE was capable of recognizing and catalysing the lactone formation between L-Try3 with the last few residues L-Pro7 and L-Gln8 at the C-terminus. Additionally, the unmatched linkers connecting the TE region and T domain resulted in nonproduction strains, suggesting that the native T–TE linker is necessary and sufficient for the TE domain to release the products from the hybrid enzymes. This is the first report to demonstrate truncated cyclic lipopeptides production and module skipping by simply moving the TE domain forward in an NRPS system.

Highlights

Non-ribosomal peptide synthetases (NRPSs) are large enzymatic complexes that catalyse the synthesis of biologically active peptides in microorganisms
We found that deletion of the TE domain of plipastatin synthetase results in the synthesis of truncated enzymes that were unable to produce the corresponding peptides
This result suggested that the TE domain is necessary for plipastatin production, which is consistent with a study on the surfactin TE domain reported by de Ferra et al.[23]

Summary

Introduction

Non-ribosomal peptide synthetases (NRPSs) are large enzymatic complexes that catalyse the synthesis of biologically active peptides in microorganisms. Previous studies have established that plipastatin is assembled on five giant NRPS multi-enzymes, PPSA, PPSB, PPSC, PPSD, and PPSE7,8, encoded by the ppsA, ppsB, ppsC, ppsD, and ppsE genes, respectively, in the plipastatin synthetase operon (Fig. 1) According to their biosynthetic function, the NRPSs can be subdivided into distinct modules responsible for initiation, elongation, and termination. The sequence of the peptide product directly corresponds to the linear arrangement of modules and domains within the biosynthetic template This structure enables the development of straightforward strategies for the rational design of NRPSs via the recombination of gene fragments to generate arrays of peptide derivatives and expand the diversity of microbial-produced lipopeptides. We further investigated the structure of the novel peptides generated to analyse the catalytic activity and selectivity of the TE domain in the new hybrid enzymes

Methods

Results

Conclusion