Production of Lantipeptides in Escherichia coli

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111

167

The biosynthesis of several classes of ribosomally synthesized and posttranslationally modified peptides involves dehydration of serine and threonine residues. For class I lantibiotics, thiopeptides, and goadsporin, this dehydration is catalyzed by lanthionine biosynthetic enzyme B (LanB) or LanB-like proteins. Although LanB proteins have been studied since 1992, in vitro reconstitution of their dehydration activity has been elusive. We show here the in vitro activity of the dehydratase involved in the biosynthesis of the food preservative nisin (NisB). In vitro, NisB dehydrated its substrate peptide NisA eight times in the presence of glutamate, ATP, Mg 2+ , and the ribosomal/ membrane fraction of bacterial cell extract. 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 during both Escherichia coli coexpression of NisA with these mutants and in vitro assays. Treatment of the glutamylated substrate with WT NisB results 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 latter step has precedent in the virginiamycin resistance protein virginiamycin B lyase. These studies will facilitate investigation of other LanB proteins involved in the biosynthesis of lantibiotics, thiopeptides, and goadsporin.

Section: Resultsmentioning

confidence: 99%

“…We recently reported the successful preparation of nisin in Escherichia coli by coexpression of the precursor peptide NisA with NisB and NisC (21). Hence, the NisB protein must be functional in E. coli, prompting renewed attempts at establishing an in vitro reconstituted activity.…”

mentioning

confidence: 99%

In vitro activity of the nisin dehydratase NisB

Garg¹,

Salazar-Ocampo

Donk

2013

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111

167

“…Our ProcM directionality study focused on one of the substrates, ProcA3.2 (Fig. 6A), which has four Ser/Thr residues but is dehydrated only three times (25,31). HSEE analysis of the onefold and twofold dehydrated ProcA3.2 indicated that ProcM initially dehydrated the third Thr in the core peptide followed by the second Thr (Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural investigation of ribosomally synthesized natural products by hypothetical structure enumeration and evaluation using tandem MS

Zhang

Ortega

Shi

et al. 2014

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Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a growing class of natural products that are found in all domains of life. These compounds possess vast structural diversity and have a wide range of biological activities, promising a fertile ground for exploring novel natural products. One challenging aspect of RiPP research is the difficulty of structure determination due to their architectural complexity. We here describe a method for automated structural characterization of RiPPs by tandem mass spectrometry. This method is based on the combined analysis of multiple mass spectra and evaluation of a collection of hypothetical structures predicted based on the biosynthetic gene cluster and molecular weight. We show that this method is effective in structural characterization of complex RiPPs, including lanthipeptides, glycopeptides, and azole-containing peptides. Using this method, we have determined the structure of a previously structurally uncharacterized lanthipeptide, prochlorosin 1.2, and investigated the order of the posttranslational modifications in three biosynthetic systems.dehydration | genome mining | lantibiotics | directionality R ibosomally synthesized and posttranslationally modified peptides (RiPPs) are a major class of natural products as revealed by the genome-sequencing efforts of the past decade (1). RiPPs are biosynthesized from genetically encoded and ribosomally produced precursor peptides, which typically consist of a core peptide that is transformed to the final product and an N-terminal extension called the leader peptide that is usually important for recognition by the posttranslational modification (PTM) enzymes (1). Because of the highly diverse PTMs, these compounds possess vast structural diversity and have a wide range of biological activities, thus representing a fertile ground for exploration. Furthermore, the ribosomal origin of RiPPs makes them particularly well suited for genome mining efforts. By using genome mining to explicitly avoid species harboring biosynthetic gene clusters identical to those that produce known compounds, a combination of strain prioritization and mass spectrometry (MS)-based analysis offers a new route to discovering natural products that can overcome the burden of rediscovery that has increasingly hampered discovery efforts (2, 3). One challenging aspect of high-throughput genome mining for new natural products is the difficulty to determine their molecular structures in high throughput. We present here a method that allows automated RiPP structure elucidation.In contrast to nonribosomal peptides that have an average molecular weight of less than 1,000 Da, as documented in the NORINE database (4), RiPPs in many cases have molecular weights larger than 2,500 Da. Molecules of this size are difficult to rapidly analyze by NMR spectroscopy, rendering MS the most convenient tool for RiPP structural characterization. Even when the precursor peptide sequences are known and the types of PTMs can be predicted based on the sequen...

“…Electrocompetent BL21(DE3) cells were cotransformed with the plasmids. A single colony was picked to inoculate a culture used to overexpress modified GeoAI as described for other lantibiotics (28). The cells were lysed, and modified GeoAI was purified using nickel affinity chromatography and reversed phase (RP) HPLC (48).…”

Section: Methodsmentioning

confidence: 99%

Lantibiotics from Geobacillus thermodenitrificans

Garg¹,

Tang²,

Goto³

et al. 2012

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128

126

The lantibiotic nisin has been used as an effective food preservative to combat food-borne pathogens for over 40 y. Despite this successful use, nisin’s stability at pH 7 is limited. Herein, we describe a nisin analog encoded on the genome of the thermophilic bacterium Geobacillus thermodenitrificans NG80-2. This analog termed geobacillin I was obtained by heterologous expression in Escherichia coli and subsequent purification. Extensive NMR characterization demonstrated that geobacillin I contains seven thioether cross-links, two more than the five cross-links found in nisin and the most cross-links found in any lantibiotic to date. The antimicrobial spectrum of geobacillin I was generally similar to that of nisin A, with increased activity against Streptococcus dysgalactiae , one of the causative agents of bovine mastitis. Geobacillin I demonstrated increased stability compared to nisin A. In addition to geobacillin I, the genome of G. thermodenitrificans NG80-2 also contains a class II lantibiotic biosynthetic gene cluster. The corresponding compound was produced in E. coli , and has a ring topology different than that of any known lantibiotic as determined by tandem mass spectrometry. Interestingly, geobacillin II only demonstrated antimicrobial activity against Bacillus strains. Seven Geobacillus strains were screened for production of the geobacillins using whole-cell MALDI-MS and five were shown to produce geobacillin I, but none produced geobacillin II.