Incorporation of tryptophan analogues into the lantibiotic nisin.

Liang Zhou,Jinfeng Shao,Oscar P Kuipers,Marcel P De Vries,Jaap Broos,Auke J Van Heel,Qian Li

doi:10.1007/s00726-016-2186-3

Liang Zhou, Jinfeng Shao + Show 5 more

Open Access

https://doi.org/10.1007/s00726-016-2186-3

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Abstract

Lantibiotics are posttranslationally modified peptides with efficient inhibitory activity against various Gram-positive bacteria. In addition to the original modifications, incorporation of non-canonical amino acids can render new properties and functions to lantibiotics. Nisin is the most studied lantibiotic and contains no tryptophan residues. In this study, a system was constructed to incorporate tryptophan analogues into nisin, which included the modification machinery (NisBTC) and the overexpression of tryptophanyl-tRNA synthetase (TrpRS). Tryptophan and three different tryptophan analogues (5-fluoroTrp (5FW), 5-hydroxyTrp (5HW) and 5-methylTrp (5MeW)) were successfully incorporated at four different positions of nisin (I1W, I4W, M17W and V32W). The incorporation efficiency of tryptophan analogues into mutants I1W, M17W and V32W was over 97 %, while the mutant I4W showed relatively low incorporation efficiency (69–93 %). The variants with 5FW showed relatively higher production yield, while 5MeW-containing variants showed the lowest yield. The dehydration efficiency of serines or threonines was affected by the tryptophan mutants of I4W and V32W. The affinity of the peptides for the cation-ion exchange and reverse phase chromatography columns was significantly reduced when 5HW was incorporated. The antimicrobial activity of IIW and its 5FW analogue both decreased two times compared to that of nisin, while that of its 5HW analogue decreased four times. The 5FW analogue of I4W also showed two times decreased activity than nisin. However, the mutant M17W and its 5HW analogue both showed 32 times reduced activity relative to that of nisin.

Highlights

As the traditional antibiotics are not sufficient to deal with several drug-resistant pathogens (Ferri et al 2015), many efforts have been made in mining new antimicrobial agents and engineering pre-existing antimicrobial peptides
The LC–MS data show that the incorporation efficiency of tryptophan analogues into mutants I1W, M17W and V32W was more than 97 %, as the peaks of peptides containing Trp were almost undetectable
Incorporation of non-canonical amino acids into natural products can be a strategy to improve the diverse properties of these compounds

Summary

Introduction

As the traditional antibiotics are not sufficient to deal with several drug-resistant pathogens (Ferri et al 2015), many efforts have been made in mining new antimicrobial agents and engineering pre-existing antimicrobial peptides. Several kinds of lacticin 481 mutants containing ncAAs have been generated by in vitro mutasynthesis, i.e. the chemically synthesized substrate peptides containing ncAAs were modified by the enzyme LctM in vitro Two of these lacticin 481 mutants containing Trp19Nal (Nal = naphthylalanine) or Phe23hPhe (hPhe = homophenylalanine) showed enhanced activity against L. lactis HP (Levengood et al 2009). In the L. lactis tryptophan auxotroph strain (PA1002), a native tryptophanyl-tRNA synthetase (lacTrpRS) was overexpressed This TrpRS has a relaxed substrate specificity, which can increase the incorporation efficiency of Trp analogues and improve the yield of the target protein. We used one new (I4) and three previously published positions (1, 17 and 32) for the Trp codons The latter ones have slightly higher MIC values than wild-type nisin. The new properties and antimicrobial activities of the variants are described

Materials and methods

Results

Discussion

Compliance with ethical standards

Findings

Methods