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Title:Exploring and understanding lantibiotic biosynthesis
Author(s):Garg, Neha
Director of Research:van der Donk, Wilfred A.; Nair, Satish K.
Doctoral Committee Chair(s):van der Donk, Wilfred A.
Doctoral Committee Member(s):Nair, Satish K.; Martinis, Susan A.; Zhao, Huimin
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Antibiotic resistance
Lantibiotic
Biosynthesis
Stereochemistry
Abstract:The ribosome translates genomic information into structural and catalytic protein molecules. In addition to its role in protein synthesis, the ribosome also produces small non-catalytic peptides that exert antimicrobial properties. The recent surge in the availability of genome sequences from a wide variety of bacterial sources has led to the rapid discovery of ribosomally-synthesized and post-translationally modified peptides (RiPPs). The biosynthetic machinery of RiPPs offers a new platform for the discovery and engineering of peptidic natural products. Although rapid advances have been made in elucidating biosynthetic pathways generating various RiPPs, investigations with respect to enzyme chemistry and action are still in their nascent stage. The novel chemistry via which post translational modifications (PTMs) are introduced into RiPPs emphasizes the synthetic potential found in nature. One such class of RiPPs are the lantibiotics, lanthipeptides with antimicrobial activity. The unifying PTMs found in lanthipeptides are thioether crosslinks, called lanthionine and methyl lanthionine. The crosslinks are formed via the dehydration of serine and threonine residues to dehydroalanine (Dha) and dehydrobutyrine (Dhb) residues, respectively, followed by Michael-type addition of cysteines to the dehydrated residues. Nisin, a class I lantibiotic produced by Lactococcus lactis, is one of the oldest known antibiotics. In this study, we have developed a production strategy for class I lantibiotics in Escherichia coli and show successful production of nisin as a proof of concept.The dehydration reaction in class I lantibiotics, thiopeptides, and goadsporin is catalyzed by LanB or LanB-like proteins. Although LanB proteins have been studied since 1992, in vitro reconstitution of their dehydration activity has been elusive. Herein, we demonstrate the in vitro activity of NisB, the dehydratase involved in the biosynthesis of nisin. NisB requires glutamate, adenosine-5’-triphosphate, Mg2+, and the ribosomal/membrane fraction of bacterial cell extract to dehydrate its substrate peptide NisA. Mutation of 23 highly conserved residues of NisB identified a number of amino acids that are essential for dehydration activity. In addition, these mutagenesis studies identified three mutants, R786A, R826A, and H961A that result in multiple glutamylations of the NisA substrate. Glutamylation was observed both during E. coli co-expression of NisA with these mutants and during in vitro assays. Treatment of the glutamylated substrate with wild type NisB resulted in dehydrated NisA suggesting that the glutamylated peptide is an intermediate in dehydration. Collectively, these studies suggest that dehydration involves glutamylation of the side chains of Ser and Thr followed by elimination. The proposed reaction mechanism is unprecedented for lanthipeptide dehydration and these findings facilitate mechanistic investigations for other LanB proteins that are involved in the biosynthesis of lantibiotics, thiopeptides, and goadsporin. Nisin is used extensively for food preservation and is also FDA-approved for the treatment of bovine mastitis. Despite its commercial use for more than 55 years, no evidence of significant resistance to nisin has emerged. Unfortunately, the stability of nisin is limited at pH 7.0 and elevated temperatures, which hinders its broader application. Conceivably, lanthipeptides from thermophilic bacteria, or bacteria which live in harsher ecological niches than L. lactis, might not suffer from the limitations of nisin. In this study, using genome mining, we discovered two novel lantibiotics, geobacillin I and geobacillin II, from a thermophilic bacterium Geobacillus thermodenitrificans NG80-2 that has an optimal growth temperature of 65 °C. The heterologous production system that was developed for nisin in E. coli enabled their production, and biochemical and structural characterization. Geobacillin I is a class I lantibiotic and resembles nisin in structure, but it contains seven thioether crosslinks, two more than nisin and the most crosslinks found in any lantibiotic to date. Compared to nisin, geobacillin I displayed increased activity against Streptococcus dysgalactiae, one of the causative agents of bovine mastitis. Geobacillin I also demonstrated increased stability compared to nisin A at temperatures of 37 °C and 60 °C at pH 7.0. Despite the lack of a hinge region that has been reported as important for the pore-formation activity of nisin, geobacillin I was found to form pores in the membranes of Gram-positive bacteria. Geobacillin II is a class II lantibiotic, and was found to have a ring topology unlike any known lantibiotic, as determined by tandem mass spectrometry. It contains unusual stereochemistry at the first lanthionine ring. Interestingly, geobacillin II has a narrow spectrum of antibacterial action as it only demonstrates antimicrobial activity against Bacillus strains amongst the bacteria tested. The activity of the class II lanthionine synthetase catalyzing the PTMs in geobacillin II was reconstituted in vitro and the enzyme was found to be active at temperatures up to 80 °C. Seven Geobacillus strains were screened for production of the geobacillins using whole cell MALDI-MS and five were shown to produce geobacillin I; none produced geobacillin II. In summary, a strategy was developed for the production of class I lantibiotics in E. coli. Using this methodology, two novel lantibiotics, geobacillin I and geobacillin II were produced and characterized. Further, NisB-catalyzed dehydration and GeoM-catalyzed dehydration/cyclization was investigated in vitro and provided insights into the mechanism of catalysis.
Issue Date:2014-01-16
URI:http://hdl.handle.net/2142/46883
Rights Information:Copyright 2013 Neha Garg
Date Available in IDEALS:2014-01-16
2016-01-16
Date Deposited:2013-12


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