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Production of lantipeptides in Escherichia coli.

Shi Y, Yang X, Garg N, van der Donk WA - J. Am. Chem. Soc. (2010)

Bottom Line: Lantipeptides are ribosomally synthesized and posttranslationally modified peptides containing thioether cross-links.We describe the preparation of seven different lantipeptides in Escherichia coli and demonstrate that this methodology can be used to incorporate nonproteinogenic amino acids.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

ABSTRACT
Lantipeptides are ribosomally synthesized and posttranslationally modified peptides containing thioether cross-links. We describe the preparation of seven different lantipeptides in Escherichia coli and demonstrate that this methodology can be used to incorporate nonproteinogenic amino acids.

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(A) Sequences of the ProcA lantipeptide core peptides used in this study. The residue replaced by pBpa in this work is highlighted in red. For the sequences of the leader peptides, see Figure S1. (B) Representative structures of prochlorosins 1.7 and 3.3. Abu, 2-aminobutyric acid; Dha, dehydroalanine; Dhb, dehydrobutyrine.
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fig2: (A) Sequences of the ProcA lantipeptide core peptides used in this study. The residue replaced by pBpa in this work is highlighted in red. For the sequences of the leader peptides, see Figure S1. (B) Representative structures of prochlorosins 1.7 and 3.3. Abu, 2-aminobutyric acid; Dha, dehydroalanine; Dhb, dehydrobutyrine.

Mentions: We first focused our efforts on the prochlorosins. These lantipeptides were recently discovered as products of the ubiquitous marine cyanobacterium Prochlorococcus.(8) The production levels are less than 10 μg of prochlorosin from 20 L of culture of Prochlorococcus MIT 9313, insufficient to investigate their function. Prochlorosins are members of the class II lantipeptides that are modified by a single bifunctional lanthionine synthetase that carries out both dehydration and cyclization reactions,(8) but they have unusually large leader peptides (Figure S1). We selected prochlorosin 1.7, 2.11, 3.2, and 3.3 (Figure 2) for investigation because their biosynthesis has been reconstituted in vitro.(8) The genes for their precursor peptides and the synthetase ProcM were cloned into the pRSFDUET-1 vector as a biscistronic construct. For rapid purification, an N-terminal hexahistidine tag was encoded at the N-termini of the ProcA peptides, and artificial protease cleavage sites were introduced between the leader and core peptides to allow in vitro removal of the leader sequence (Figure S1).(9)E. coli BL21 (DE3) cells were transformed with the plasmids, and the cells were grown in Luria−Bertani broth and induced with IPTG. After harvest and lysis of the cells followed by immobilized metal affinity chromatography (IMAC) purification of the ProcA peptides from the soluble protein fraction, the fully modified peptides were isolated in yields ranging from 10 to 35 mg per liter of culture, significantly more than the 1.5 mg/L of modified NukA reported previously using a different expression system.(7) This production level was surprising because when lantipeptide precursor peptides are expressed in the absence of ProcM, they are almost exclusively present in inclusion bodies.8,10 Hence, posttranslational modifications improve the solubility of the products. The purified peptides were then treated with the appropriate commercial protease (trypsin for ProcA1.7, Lys-C for ProcA 2.11 and 3.3, and TEV protease for ProcA3.2, Figure 3A−C) and purified by HPLC. Although, in principle, the proteolytic step could be carried out in vivo by introduction of the gene for the bifunctional protease transporter, we elected to remove the leader peptide in vitro to avoid complications often encountered in heterologous expression of proteases and to avoid any potential cytotoxic effects of the final posttranslationally modified products.(11) After HPLC purification, pure prochlorosins were obtained (Figures S2 and S3). The purified, modified peptides were analyzed next by tandem mass spectrometry (MS) displaying the same fragmentation patterns as observed previously(8) (Figure S4) demonstrating that the ring topology of the products is the same as that of the natural products.(12)


Production of lantipeptides in Escherichia coli.

Shi Y, Yang X, Garg N, van der Donk WA - J. Am. Chem. Soc. (2010)

(A) Sequences of the ProcA lantipeptide core peptides used in this study. The residue replaced by pBpa in this work is highlighted in red. For the sequences of the leader peptides, see Figure S1. (B) Representative structures of prochlorosins 1.7 and 3.3. Abu, 2-aminobutyric acid; Dha, dehydroalanine; Dhb, dehydrobutyrine.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3044485&req=5

fig2: (A) Sequences of the ProcA lantipeptide core peptides used in this study. The residue replaced by pBpa in this work is highlighted in red. For the sequences of the leader peptides, see Figure S1. (B) Representative structures of prochlorosins 1.7 and 3.3. Abu, 2-aminobutyric acid; Dha, dehydroalanine; Dhb, dehydrobutyrine.
Mentions: We first focused our efforts on the prochlorosins. These lantipeptides were recently discovered as products of the ubiquitous marine cyanobacterium Prochlorococcus.(8) The production levels are less than 10 μg of prochlorosin from 20 L of culture of Prochlorococcus MIT 9313, insufficient to investigate their function. Prochlorosins are members of the class II lantipeptides that are modified by a single bifunctional lanthionine synthetase that carries out both dehydration and cyclization reactions,(8) but they have unusually large leader peptides (Figure S1). We selected prochlorosin 1.7, 2.11, 3.2, and 3.3 (Figure 2) for investigation because their biosynthesis has been reconstituted in vitro.(8) The genes for their precursor peptides and the synthetase ProcM were cloned into the pRSFDUET-1 vector as a biscistronic construct. For rapid purification, an N-terminal hexahistidine tag was encoded at the N-termini of the ProcA peptides, and artificial protease cleavage sites were introduced between the leader and core peptides to allow in vitro removal of the leader sequence (Figure S1).(9)E. coli BL21 (DE3) cells were transformed with the plasmids, and the cells were grown in Luria−Bertani broth and induced with IPTG. After harvest and lysis of the cells followed by immobilized metal affinity chromatography (IMAC) purification of the ProcA peptides from the soluble protein fraction, the fully modified peptides were isolated in yields ranging from 10 to 35 mg per liter of culture, significantly more than the 1.5 mg/L of modified NukA reported previously using a different expression system.(7) This production level was surprising because when lantipeptide precursor peptides are expressed in the absence of ProcM, they are almost exclusively present in inclusion bodies.8,10 Hence, posttranslational modifications improve the solubility of the products. The purified peptides were then treated with the appropriate commercial protease (trypsin for ProcA1.7, Lys-C for ProcA 2.11 and 3.3, and TEV protease for ProcA3.2, Figure 3A−C) and purified by HPLC. Although, in principle, the proteolytic step could be carried out in vivo by introduction of the gene for the bifunctional protease transporter, we elected to remove the leader peptide in vitro to avoid complications often encountered in heterologous expression of proteases and to avoid any potential cytotoxic effects of the final posttranslationally modified products.(11) After HPLC purification, pure prochlorosins were obtained (Figures S2 and S3). The purified, modified peptides were analyzed next by tandem mass spectrometry (MS) displaying the same fragmentation patterns as observed previously(8) (Figure S4) demonstrating that the ring topology of the products is the same as that of the natural products.(12)

Bottom Line: Lantipeptides are ribosomally synthesized and posttranslationally modified peptides containing thioether cross-links.We describe the preparation of seven different lantipeptides in Escherichia coli and demonstrate that this methodology can be used to incorporate nonproteinogenic amino acids.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

ABSTRACT
Lantipeptides are ribosomally synthesized and posttranslationally modified peptides containing thioether cross-links. We describe the preparation of seven different lantipeptides in Escherichia coli and demonstrate that this methodology can be used to incorporate nonproteinogenic amino acids.

Show MeSH
Related in: MedlinePlus