Heterologous Expression of Plantaricin 423 and Mundticin ST4SA in Saccharomyces cerevisiae

Abstract

Antimicrobial peptides or bacteriocins are excellent candidates for alternative antimicrobials, but high manufacturing costs limit their applications. Recombinant gene expression offers the potential to produce these peptides more cost-effectively at a larger scale. Saccharomyces cerevisiae is a popular host for recombinant protein production, but with limited success reported on antimicrobial peptides. Individual recombinant S. cerevisiae strains were constructed to secrete two class IIa bacteriocins, plantaricin 423 (PlaX) and mundticin ST4SA (MunX). The native and codon-optimised variants of the plaA and munST4SA genes were cloned into episomal expression vectors containing either the S. cerevisiae alpha mating factor (MFα1) or the Trichoderma reesei xylanase 2 (XYNSEC) secretion signal sequences. The recombinant peptides retained their activity and stability, with the MFα1 secretion signal superior to the XYNSEC secretion signal for both bacteriocins. An eight-fold increase in activity against Listeria monocytogenes was observed for MunX after codon optimisation, but not for PlaX-producing strains. After HPLC-purification, the codon-optimised genes yielded 20.9 mg/L of MunX and 18.4 mg/L of PlaX, which displayed minimum inhibitory concentrations (MICs) of 108.52 nM and 1.18 µM, respectively, against L. monocytogenes. The yields represent a marked improvement relative to an Escherichia coli expression system previously reported for PlaX and MunX. The results demonstrated that S. cerevisiae is a promising host for recombinant bacteriocin production that requires a simple purification process, but the efficacy is sensitive to codon usage and secretion signals.

Keywords: Saccharomyces cerevisiae; Antimicrobial peptides; Class IIa bacteriocins; Heterologous expression.

Fig. 1

Antimicrobial activity of the recombinant S. cerevisiae strains against L. monocytogenes EDG-e. a Agar-overlay assays with yeast strains and b agar well-diffusion assays with CFS from strains producing plantaricin 423 (PlaX) peptide (left) or mundticin ST4SA (MunX) peptide (right). The negative controls are BBH1, BBH4 and MR

Fig. 2

Bacteriocin activity for native and codon-optimised a PlaX and b MunX strains, and c dry cell weight (DCW) for the negative control Y294[MR] (C), Y294[MFa1-PlaX_Opt] (P) and Y294[MFa1-MunX_Opt] (M) strains after 72 h

Fig. 3

Tricine SDS-PAGE analysis of equal volumes of 20-fold concentrated supernatant containing recombinant a–c plantaricin 423 (PlaX) or d–f mundticin ST4SA (MunX) peptides. Images a and d represent silver-stained gels; b and e are overlay gels with L. monocytogenes; c and f represent the superimposed gels. Arrows indicate the recombinant PlaX and MunX peptides, respectively. The MFα1 and XYN controls refer to the supernatant of the Y294[MR] and Y294[BBH4] control strains containing the MFα1 and XYNSEC secretion signals, respectively

Fig. 4

SEM images of L. monocytogenes cells after 18 h a without treatment, b treated with PlaX_Opt and c treated with MunX_Opt. The untreated L. monocytogenes cells appear smooth and intact, but shrivelled after treatment with PlaX_Opt or MunX_Opt. The arrows indicate intracellular debris released from cells due to pore formation in the cell wall

Fig. 5

HPLC purification, accurate mass determination and peptide sequence confirmation of PlaX_Opt. a HPLC fractionation (C8 column) of PlaX_Opt with anti-listerial activity identified in fractions 8–12, with fraction 9 displaying the highest activity and subsequently analysed with LC–MS/MS. Monoisotopic ions (red arrows) were observed for PlaX_Opt carrying b + 5 charges ([M + 5H]⁺⁵ with an expected m/z 786.7493) and c + 4 charges ([M + 4H]⁺⁴ with an expected m/z 983.1848). d Collision-induced peptide fragmentation spectra confirmed the PlaX_Opt peptide sequence and the two disulphide bonds represented as sections of no fragmentation

Fig. 6

HPLC purification, accurate mass determination and peptide sequence confirmation of MunX_Opt. a HPLC fractionation (C8 column) of MunX_Opt with anti-listerial activity identified in fractions 8–17, with fraction 8 displaying the greatest antimicrobial activity and subsequently analysed with LC–MS/MS. Monoisotopic ions (red arrows) were observed for MunX_Opt carrying b + 6 charges ([M + 6H]⁺⁶ with an expected m/z 715.1898), c + 5 charges ([M + 5H]⁺⁵ with an expected m/z 858.0264 and d + 4 charges ([M + 4H]⁺⁴ with an expected m/z 1072.2811). e Collision-induced peptide fragmentation spectra confirmed the peptide sequence of MunX_Opt and single disulphide bond represented as the section of no fragmentation