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, 48 (5), 1309-18

Incorporation of Tryptophan Analogues Into the Lantibiotic Nisin

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Incorporation of Tryptophan Analogues Into the Lantibiotic Nisin

Liang Zhou et al. Amino Acids.

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.

Keywords: Biosynthetic incorporation; Lantibiotics; Nisin; Non-canonical amino acids; Tryptophan analogues.

Figures

Fig. 1
Fig. 1
a Structures of tryptophan and its analogues used in this study. 5FW, 5-fluorotryptophan; 5HW, 5-hydroxytryptophan; 5MeW, 5-methyltryptophan. b Incorporation of tryptophan analogues into nisin A. Dha, dehydroalanine; Dhb, dehydrobutyrine; Ala-S-Ala, lanthionine; Abu-S-Ala, methyllanthionine. Ile1, Ile4, Met17 and Val32 (green label) were mutated and replaced by Trp or Trp analogues (red label). The colour depth of the red label indicates the yield of the variant and darker colour means higher yield. The antimicrobial activity of some variants compared to wild-type nisin is indicated. The yield of other variants was too low to purify enough material for accurate minimum inhibitory concentration (MIC) determination. In general, incorporation of a Trp analogue in nisin gave lower production yields and equal or higher MIC values than natural Trp incorporation
Fig. 2
Fig. 2
Map of the plasmid pCZnisA–trpRS. sczA, encoding the repressor of PczcD; PczcD, a zinc-inducible promoter; nisA, encoding NisA; trpRS, encoding tryptophanyl-tRNA synthetase; T, terminator; BamHI and XhoI, restriction sites; repA and repC, encoding plasmid replication proteins; cmR, chloramphenicol resistance gene. Partly referred to (Mu et al. 2013)
Fig. 3
Fig. 3
Coomassie blue-stained tricine SDS-PAGE gel. Each well contained TCA-precipitated prepeptides from 600 μl supernatant. M1: protein marker (Biolabs); WT: wild type; Trp: tryptophan; F: 5-fluoroTrp; OH: 5-hydroxyTrp; Me: 5-methylTrp
Fig. 4
Fig. 4
Purified tryptophan analogues-containing variants shown by Coomassie blue-stained tricine SDS-PAGE gel
Fig. 5
Fig. 5
Nisin variant production and tryptophan analogue incorporation systems. Olive line the conventional NICE system; aqua line the tryptophan analogues incorporation system constructed by Petrović et al.; blue line the new system constructed in this research, which combines the NICE system, zirex system and the tryptophan analogues incorporation system

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References

    1. Bierbaum G, Sahl H-G. Lantibiotics: mode of action, biosynthesis and bioengineering. Curr Pharm Biotechnol. 2009;10:2–18. doi: 10.2174/138920109787048616. - DOI - PubMed
    1. Bosma T, Kanninga R, Neef J, et al. Novel surface display system for proteins on non-genetically modified gram-positive bacteria. Appl Environ Microbiol. 2006;72:880–889. doi: 10.1128/AEM.72.1.880-889.2006. - DOI - PMC - PubMed
    1. Breukink E, de Kruijff B. Lipid II as a target for antibiotics. Nat Rev Drug Discov. 2006;5:321–332. doi: 10.1038/nrd2004. - DOI - PubMed
    1. Breukink E, van Kraaij C, van Dalen A, et al. The orientation of nisin in membranes. Biochemistry (Mosc) 1998;37:8153–8162. doi: 10.1021/bi972797l. - DOI - PubMed
    1. Demel RA, Peelen T, Siezen RJ, et al. Nisin Z, mutant nisin Z and lacticin 481 interactions with anionic lipids correlate with antimicrobial activity. A monolayer study. Eur J Biochem FEBS. 1996;235:267–274. doi: 10.1111/j.1432-1033.1996.00267.x. - DOI - PubMed

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