Heterologous Expression of the Class IIa Bacteriocins, Plantaricin 423 and Mundticin ST4SA, in Escherichia coli Using Green Fluorescent Protein as a Fusion Partner

Vermeulen, Ross R.; Staden, Anton D. van; Dicks, Leon M. T.

doi:10.3389/fmicb.2020.01634

Cited by 17 publications

(17 citation statements)

References 72 publications

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“…Bacteriocin screening via BAGEL4 revealed that AGA58 predicted to carry 3 core bacteriocin biosynthesis genes in the same operon (Fig 4). The protein sequence encoding A2 lantipeptide did also show around 80% similarity with Plantaricin_423 peptide which is known to harbor anti-listerial activity [35]. However, performing a protein blast of the sequence [ MKKEIELSEKELVRII GG KYYGNGVSCTKKHGCKVNWGQAFTCSVNRFANFGHGNC] in NCBI showed that it has the highest sequence homology with A2 lantipeptide (Table S2, supplemental).…”

Section: Resultsmentioning

confidence: 99%

Metabolic potentials of Liquorilactobacillus nagelii AGA58 isolated from Shalgam based on genomic and functional analysis

Yetiman

Ortakci

2021

Preprint

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The aim of present study was to perform functional and genomic characterization of a novel Liquorilactobacillus nagelii AGA58 isolated from Shalgam to understand its metabolic potentials. AGA58 is gram-positive, catalase-negative and appears as short-rods under light-microscope. The AGA58 chromosome composed of a single linear chromosome of 2,294,535 bp that is predicted to carry 2151 coding sequences, including 45 tRNA genes, 4 rRNA operons. Genome has a GC content of 36.9% includes 45 pseudogenes, 32 transposases and one intact-prophage. AGA58 is micro-anaerobic owing to shorter doubling time and faster growth rate achieved compared microaerofilic condition. It carries flagellar biosynthesis protein-encoding genes predicting motile behavior. AGA58 is an obligatory homofermentative where hexose sugars such as galactose, glucose, fructose, sucrose, mannose, N-acetyl glucosamine, maltose, trehalose are fermented to lactate thru glycolysis and no acid production from pentose sugars achieved due to lack of key enzyme namely phosphoketolase in pentose phosphate pathway. Carbohydrate fermentation tests showed AGA58 cannot ferment pentoses which was also confirmed in silico. Putative pyruvate metabolism revealed formate, malate, oxaloacetate, acetate, acetaldehyde, acetoin and lactate forms from pyruvate. AGA58 predicted to carry bacteriocin genes for type A2 lantipeptide, Blp family class II bacteriocins showing antimicrobial potential of this bacterium which can be linked to antagonism tests that AGA58 can inhibit E. coli O157:H7, S. Typhimurium ATCC14028, and K. pneumonia ATCC13883. Moreoever, AGA58 is tolerant to acid and bile concentrations simulating the human gastrointestinal conditions. L. nagelii AGA58 depicting the probiotic potential of AGA58 as a first report in literature within same species.

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Section: Resultsmentioning

confidence: 99%

Metabolic potentials of Liquorilactobacillus nagelii AGA58 isolated from Shalgam based on genomic and functional analysis

Yetiman

Ortakci

2021

Preprint

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“…The fused proteins were inactive, and it was necessary to purify and cleave the fused proteins to obtain pure PapA mature peptide. Then, the pure PapA peptide can inhibit L. monocytogenes [ 25 , 27 , 28 ]. The class IIα bacteriocins are produced as prebacteriocin with an N-terminal extension, which is removed by site-specific proteolytic cleavage during export.…”

Section: Discussionmentioning

confidence: 99%

“…In E. coli , a periplasmic leaky E. coli strain had to be used to express and secrete the chimeric protein into the medium, which displayed listericidal activity. The pedA gene was also fused with the dihydrogolate reductase ( DHFR ) gene [ 25 ], the thioredoxin ( Trx ) gene [ 26 , 27 ], and the green fluorescent protein ( GFP ) gene [ 28 ] on the 5´ side. The fused protein had no biological activity after IPTG induction.…”

Section: Introductionmentioning

confidence: 99%

Heterologous expression of pediocin/papA in Bacillus subtilis

et al. 2022

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Listeria monocytogenes is a food-borne pathogen. Pediocin is a group IIα bacteriocin with anti-listeria activity that is naturally produced by Pediococcus acidilactic and Lactobacillus plantarum. The pedA/papA gene encodes pediocin/plantaricin. In native hosts, the expression and secretion of active PedA/PapA protein rely on the accessory protein PedC/PapC and ABC transporter PedD/PapD on the same operon. The excretion machines were also necessary for pediocin protein expression in heterologous hosts of E. coli, Lactobacillus lactis, and Corynebacterium glutamicum. In this study, two vectors carrying the codon sequence of the mature PapA peptide were constructed, one with and one without a His tag. Both fragments were inserted into the plasmid pHT43 and transformed into Bacillus subtilis WB800N. The strains were induced with IPTG to secrete the fused proteins PA1 and PA2. Supernatants from both recombinant strains can inhibit Listeria monocytogenes ATCC54003 directly. The fused protein possesses inhibition activity as a whole dispense with removal of the leading peptide. This is the first report of active pediocin/PapA expression without the assistance of PedCD/PapCD in heterogeneous hosts. In addition, the PA1 protein can be purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography.

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“…In E. coli, papA in fused protein was inactive, which had to be cut from the fused protein to get pure papA mature peptide [9,11,17]. The pure papA peptide possessed anti-listeria active.…”

Section: Discussionmentioning

confidence: 99%

“…Pediocin gene has been introduced to lactococcus lactis [6], and E. coli [7,8], where the ABC transporter protein and secretion apparatus-genes or other secretion apparatus were necessary for active peptide. To overcome this shortage, pedA gene was fused with dihydrogolate reductase (DHFR) gene [9], thioredoxin (Trx) gene [10], green uorescent protein (GFP) gene [11] on the 5´ side. The fused protein had no biological activity after IPTG induction.…”

Section: Introductionmentioning

confidence: 99%

Heterologous Expression of Pediocin/Papa in Bacillus Subtilis

Wang¹,

Guo²,

Zhang³

et al. 2021

Preprint

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Listeria moniocytogenes is food-borne pathogen. Pediocin is group II α bacteriocin with anti-listeria activity, naturally produced by Pediococuus acidilactic and Lactobacillus plantarum. Gene pepA/papA encode for pediocin. Expression and secretion of active papA was relayed on transporter papC and accessary protein papD on the same operon in native host. The excretion machines were also necessary for pediocin protein expression in heterologous host of E. coli, Lactobacillus lactis, and Corynebacterium glutamicum. In this study, two vector carrying codon sequence of papA mature peptide was constructed, with or without His tag. Both fragments were inserted into plasmid pHT43 and transformed Bacillus subtilis WB800N. The strains were induced with IPTG to secrete recombination protein PA1 and PA2 respectively. Supernatant from both recombination strains can inhibit Listeria monocytogenes ATCC54003 directly. The fused protein possesses inhibition activity as a whole, exempting from cleavage of leading peptide. Protein PA1 can be purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography. This is the first report for active pediocin expression without assistance of papCD in heterogenous host.

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Heterologous Expression of the Class IIa Bacteriocins, Plantaricin 423 and Mundticin ST4SA, in Escherichia coli Using Green Fluorescent Protein as a Fusion Partner

Cited by 17 publications

References 72 publications

Metabolic potentials of Liquorilactobacillus nagelii AGA58 isolated from Shalgam based on genomic and functional analysis

Metabolic potentials of Liquorilactobacillus nagelii AGA58 isolated from Shalgam based on genomic and functional analysis

Heterologous expression of pediocin/papA in Bacillus subtilis

Heterologous Expression of Pediocin/Papa in Bacillus Subtilis

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