Heterologous expression of pediocin/papA in Bacillus subtilis

Abstract

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.

High-efficiency production of the antimicrobial peptide pediocin PA-1 in metabolically engineered Corynebacterium glutamicum using a microaerobic process at acidic pH and elevated levels of bivalent calcium ions

“…6 Benchmarking of microbial pediocin PA-1 production in natural and recombinant cell factories. The data show previous efforts (from top to bottom in the upper part) using L. lactis CL1 [ 107 ], B. subtilis [ 108 ], P. acidilactici 347 [ 109 ], E. coli producing the M31L version of the peptide [ 110 ], P. acidilactici 347 [ 111 ], C. glutamicum CR099 pXMJ19 P tac pedACD Cg [ 23 ], and P. acidilactici 1521 [ 112 ] in shake flasks and test tubes. In addition, the results of the optimization from this work are shown, using C. glutamicum CR099 pXMJ19 P tac pedACD Cg .…”

“…The medium of the letter additionally contained 2 mM IPTG. The pediocin PA-1 concentration obtained with B. subtilis was estimated from the reported biological activity [ 108 ] using the recently obtained conversion factor [ 23 ] …”

Bacillus subtilis as a host for natural product discovery and engineering of biosynthetic gene clusters

“…The pediocin produced in B. subtilis showed similar thermotolerant properties as reported in previous studies. 148,181,182 Rhizocticins are antifungal phosphonate oligopeptide antibiotics produced by B. subtilis ATCC 6633 (Table 1). 149,183 These antibiotics consist of di-and tripeptides with the unique nonproteinogenic amino acid (Z)-L-2-amino-5-phosphono-3-pentenoic acid (APPA) at their C-terminus.…”

“…170,171 Heterologous production of this bacteriocin has been achieved in various hosts, such as E. coli, 172,173 Lactobacillus lactis, 174,175 and Corynebacterium glutamicum. 176 A study by Wang et al investigated if the pediocin peptide (PapA) could be heterologously produced in B. subtilis WB800N and secreted without the need for accessory protein PedC/PapC and ABC transporter PedD/PapD, as previous research had shown that these proteins impose a metabolic burden to the host 148,176 (Table 1). The study used a shuttle vector to express both the original and a codon-optimized version of the papA gene as a fusion protein with an a-amylase signal peptide for secretion.…”

The Progress of the Biotechnological Production of Class IIa Bacteriocins in Various Cell Factories and Its Future Challenges

“…Bacillus subtilis , similar to C. glutamicum , exhibits remarkable secretory characteristics and possesses a well-established fermentation process and genetic manipulation techniques. Currently, active bacteriocin expression in B. subtilis has also been successfully achieved by Li et al [ 34 ]. In that study, the structural gene encoding mature pediocin PA-1 was connected with vector pHT43 and then introduced into B. subtilis WB800N.…”