Production of enterocin P, an antilisterial pediocin-like bacteriocin from Enterococcus faecium P13, in Pichia pastoris

Abstract

The gene encoding mature enterocin P (EntP), an antimicrobial peptide from Enterococcus faecium P13, was cloned into the pPICZalphaA expression vector to generate plasmid pJC31. This plasmid was integrated into the genome of P. pastoris X-33, and EntP was heterologously secreted from the recombinant P. pastoris X-33t1 derivative at a higher production and antagonistic activity than from E. faecium P13.

FIG. 1.

Direct antimicrobial activities of P. pastoris X-33 (spot 1) and P. pastoris X-33t₁ (spot 2) by the stab-on-agar test using (A) E. faecium T136 or (B) E. faecium P13 as the indicator strain.

FIG. 2.

Agar well diffusion test for detection of enterocin P activity against E. faecium T136. Wells contain supernatants of P. pastoris X-33t₁ cultures, grown in the minimal medium BMM (A) or the complex medium BMMY (B), after 0 (d), 4 (e), 6 (f), 8 (g), 10 (h), 12 (i), or 24 (j) hours of incubation. Supernatants of P. pastoris X-33 cultures propagated in BMM or BMMY after 0 (a), 6 (b), or 12 (c) hours of growth were used as negative controls. Supernatants of E. faecium P13 containing 8 (l), 2 (m), and 0.5 (n) μg/ml of EntP were used as positive controls, and the supernatant of E. faecium T136 (k) was used as a negative control.

FIG. 3.

Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of different bacteriocins after (A) silver staining, (B) overlay with the indicator strain E. faecium T136, and (C) Western blotting with specific anti-EntP antibodies. M, molecular weight markers, with sizes (in kDa) given in the left margin. Lanes 1, 1 μg of pure enterocin Q; lanes 2, 1 μg of pure pediocin PA-1; lanes 3, 1 μg of pure enterocin P from E. faecium P13; lanes 4, 1 μg of pure enterocin P from P. pastoris X-33t₁.