Cloning, production, and functional expression of the bacteriocin enterocin A, produced by Enterococcus faecium T136, by the yeasts Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Arxula adeninivorans

Abstract

The bacteriocin enterocin A (EntA) produced by Enterococcus faecium T136 has been successfully cloned and produced by the yeasts Pichia pastoris X-33EA, Kluyveromyces lactis GG799EA, Hansenula polymorpha KL8-1EA, and Arxula adeninivorans G1212EA. Moreover, P. pastoris X-33EA and K. lactis GG799EA produced EntA in larger amounts and with higher antimicrobial and specific antimicrobial activities than the EntA produced by E. faecium T136.

Fig 1

Antimicrobial activities of recombinant yeasts as determined by a SOAT. Panels: A, P. pastoris X-33 (pPICZαA) and X-33EA (pPICEA); B, K. lactis GG799 (pKLAC2) and GG799EA (pKLEA); C, H. polymorpha KL8-1 (YRC-ALEU2m) and KL8-1EA (YRC-BTEA); D, A. adeninivorans G1212 (YRC-ATRP1m) and G1212EA (YRC-BYEA). The P. pastoris X-33 derivatives were streaked onto the surfaces of plates containing 1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6), 1.34% yeast nitrogen base without amino acids, 4 × 10⁻⁵% biotin, and 0.5% methanol; the K. lactis GG799 derivatives were streaked onto plates containing 1% yeast extract, 2% peptone, and 2% galactose; the H. polymorpha KL8-1 derivatives were streaked onto yeast extract-peptone-dextrose (Sigma-Aldrich Inc.) plates; and the A. adeninivorans G1212 derivatives were spotted onto YMM-glucose plates containing 20 mM NaNO₃ (YMM-NO₃) (6). All plates were incubated at 30°C. After growth of the yeast hosts, 40 ml of MRS (Oxoid Ltd.) soft agar containing the indicator microorganism E. faecium P13 at about 1 × 10⁵ CFU/ml was poured over the plates, which were then incubated at 30°C overnight for development of halos of inhibition.

Fig 2

Mass spectrometry analysis of purified EntA from P. pastoris X-33EA (A), K. lactis GG799EA (B), and H. polymorpha KL8-1EA (C).