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Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli.

ABSTRACT: The bacteriocins bactofencin A (class IId) and pediocin PA-1 (class IIa) are encoded by operons with a similarly clustered gene organization including a structural peptide, an immunity protein, an ABC transporter and accessory bacteriocin transporter protein. Cloning of these operons in E. coli Tuner^TM (DE3) on a pETcoco-2 derived vector resulted in successful secretion of both bacteriocins. A corresponding approach, involving the construction of vectors containing different combinations of these genes, revealed that the structural and the transporter genes alone are sufficient to permit heterologous production and secretion in this host. Even though the accessory protein, usually associated with optimal disulfide bond formation, was not required for bacteriocin synthesis, its presence did result in greater pediocin PA-1 production. The simplicity of the system and the fact that the associated bacteriocins could be recovered from the extracellular medium provides an opportunity to facilitate protein engineering and the overproduction of biologically-active bacteriocins at industrial scale. Additionally, this system could enable the characterization of new bacteriocin operons where genetic tools are not available for the native producers.

SUBMITTER: Mesa-Pereira B

PROVIDER: S-EPMC5465099 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli.

Mesa-Pereira Beatriz B O'Connor Paula M PM Rea Mary C MC Cotter Paul D PD Hill Colin C Ross R Paul RP

Scientific reports 20170608 1

The bacteriocins bactofencin A (class IId) and pediocin PA-1 (class IIa) are encoded by operons with a similarly clustered gene organization including a structural peptide, an immunity protein, an ABC transporter and accessory bacteriocin transporter protein. Cloning of these operons in E. coli Tuner<sup>TM</sup> (DE3) on a pETcoco-2 derived vector resulted in successful secretion of both bacteriocins. A corresponding approach, involving the construction of vectors containing different combinati ...[more]

PMID: 28596555

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Project description:Bacteriocins are ribosomally synthesized bacterial antimicrobial peptides that have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like (or class IIa) bacteriocins (PLBs) exhibit antibacterial activity against several Gram-positive bacterial strains by forming pores in the cytoplasmic membrane of target cells with a specific receptor, the mannose phosphotransferase system (man-PTS). In this study, we report the cryo-electron microscopy structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1, the first and most extensively studied representative PLB, at resolutions of 3.12 and 2.45 Å, respectively. The structures revealed that the binding of pediocin PA-1 opens the Core domain of man-PTS away from its Vmotif domain, creating a pore through the cytoplasmic membranes of target cells. During this process, the N-terminal β-sheet region of pediocin PA-1 can specifically attach to the extracellular surface of the man-PTS Core domain, whereas the C-terminal half penetrates the membrane and cracks the man-PTS like a wedge. Thus, our findings shed light on a design of novel PLBs that can kill the target pathogenic bacteria. IMPORTANCE Listeria monocytogenes is a ubiquitous microorganism responsible for listeriosis, a rare but severe disease in humans, who become infected by ingesting contaminated food products (i.e., dairy, meat, fish, and vegetables): the disease has a fatality rate of 33%. Pediocin PA-1 is an important commercial additive used in food production to inhibit Listeria species. The mannose phosphotransferase system (man-PTS) is responsible for the sensitivity of Listeria monocytogenes to pediocin PA-1. In this study, we report the cryo-EM structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1 at resolutions of 3.12 and 2.45 Å, respectively. Our results facilitate the understanding of the mode of action of class IIa bacteriocins as an alternative to antibiotics.

Project description:BackgroundRecombinant protein-based therapeutics have become indispensable for the treatment of many diseases. They are produced using well-established expression systems based on bacteria, yeast, insect and mammalian cells. The majority of therapeutic proteins are glycoproteins and therefore the post-translational attachment of sugar residues is required. The development of an engineered Escherichia coli-based expression system for production of human glycoproteins could potentially lead to increased yields, as well as significant decreases in processing time and costs.ResultsThis work describes the expression of functional human-derived glycosyltransferase UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAcT2) in a recombinant E. coli strain. For expression, a codon-optimised gene encoding amino acids 52-571 of GalNAcT2 lacking the transmembrane N-terminal domain was inserted into a pET-23 derived vector encoding a polyhistidine-tag which was translationally fused to the N-terminus of the glycosyltransferase (HisDapGalNAcT2). The glycosyltransferase was produced in E. coli using a recently published expression system. Soluble HisDapGalNAcT2 produced in SHuffle® T7 host cells was purified using nickel affinity chromatography and was subsequently analysed by size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and circular dichroism spectroscopy to determine molecular mass, folding state and thermal transitions of the protein. The activity of purified HisDapGalNAcT2 was monitored using a colorimetric assay based on the release of phosphate during transfer of glycosyl residues to a model acceptor peptide or, alternatively, to the granulocyte-colony stimulating growth factor (G-CSF). Modifications were assessed by Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry analysis (MALDI-TOF-MS) and Electrospray Mass Spectrometry analysis (ESI-MS). The results clearly indicate the glycosylation of the acceptor peptide and of G-CSF.ConclusionIn the present work, we isolated a human-derived glycosyltransferase by expressing soluble HisDapGalNAcT2 in E. coli. The functional activity of the enzyme was shown in vitro. Further investigations are needed to assess the potential of in vivo glycosylation in E. coli.

Project description:Sialyltransferases (STs) are disulfide-containing, type II transmembrane glycoproteins that catalyze the transfer of sialic acid to proteins and lipids and participate in the synthesis of the core structure oligosaccharides of human milk. Sialic acids are found at the outermost position of glycostructures, playing a key role in health and disease. Sialylation is also essential for the production of recombinant therapeutic proteins (RTPs). Despite their importance, availability of sialyltransferases is limited due to the low levels of stable, soluble and active protein produced in bacterial expression systems, which hampers biochemical and structural studies on these enzymes and restricts biotechnological applications. We report the successful expression of active human sialyltransferases ST3Gal1 and ST6Gal1 in commercial Escherichia coli strains designed for production of disulfide-containing proteins. Fusion of hST3Gal1 with different solubility enhancers and substitution of exposed hydrophobic amino acids by negatively charged residues (supercharging-like approach) were performed to promote solubility and folding. Co-expression of sialyltransferases with the chaperon/foldases sulfhydryl oxidase, protein disulfide isomerase and disulfide isomerase C was explored to improve the formation of native disulfide bonds. Active sialyltransferases fused with maltose binding protein (MBP) were obtained in sufficient amounts for biochemical and structural studies when expressed under oxidative conditions and co-expression of folding factors increased the yields of active and properly folded sialyltransferases by 20%. Mutation of exposed hydrophobic amino acids increased recovery of active enzyme by 2.5-fold, yielding about 7 mg of purified protein per liter culture. Functionality of recombinant enzymes was evaluated in the synthesis of sialosides from the β-d-galactoside substrates lactose, N-acetyllactosamine and benzyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl)-α-d-galactopyranoside.

Dataset Information

Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli.

Publications

Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli.

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