Cloning, production, and functional expression of the bacteriocin sakacin A (SakA) and two SakA-derived chimeras in lactic acid bacteria (LAB) and the yeasts Pichia pastoris and Kluyveromyces lactis

Abstract

Mature sakacin A (SakA, encoded by sapA) and its cognate immunity protein (SakI, encoded by sapiA), and two SakA-derived chimeras mimicking the N-terminal end of mature enterocin P (EntP/SakA) and mature enterocin A (EntA/SakA) together with SakI, were fused to different signal peptides (SP) and cloned into the protein expression vectors pNZ8048 and pMG36c for evaluation of their production and functional expression by different lactic acid bacteria. The amount, antimicrobial activity, and specific antimicrobial activity of SakA and its chimeras produced by Lactococcus lactis subsp. cremoris NZ9000 depended on the SP and the expression vector. Only L. lactis NZ9000 (pNUPS), producing EntP/SakA, showed higher bacteriocin production and antimicrobial activity than the natural SakA-producer Lactobacillus sakei Lb706. The lower antimicrobial activity of the SakA-producer L. lactis NZ9000 (pNUS) and that of the EntA/SakA-producer L. lactis NZ9000 (pNUAS) could be ascribed to secretion of truncated bacteriocins. On the other hand, of the Lb. sakei Lb706 cultures transformed with the pMG36c-derived vectors only Lb. sakei Lb706 (pGUS) overproducing SakA showed a higher antimicrobial activity than Lb. sakei Lb706. Finally, cloning of SakA and EntP/SakA into pPICZαA and pKLAC2 permitted the production of SakA and EntP/SakA by recombinant Pichia pastoris X-33 and Kluyveromyces lactis GG799 derivatives although their antimicrobial activity was lower than expected from their production.

Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli

“…acidilactici (4,625 Da). The 16 Da molecular mass difference is explained by the oxidation of methionine residues to MetSO during the recombinant production as it has been described in other heterologous hosts such as yeasts 43, 44 and LABs 45–47 . To protect the peptide from oxidation, Met could be replaced by either Ala, Ile or Leu, with minor effects on the bacteriocin activity 48 .…”

Recombinant pediocin in Lactococcus lactis: increased production by propeptide fusion and improved potency by co‐production with PedC

“…The general secretion (Sec) system of L. lactis was widely used to improve secretion of recombinant proteins including bacteriocins (Herranz and Driessen, ; Bermúdez‐Humarán et al ., ; Borrero et al ., 2011a,b; Jiménez et al ., ) such as the bacteriocin pediocin PA‐1 (Martín et al ., ; Li et al ., ). In order to allow secretion via the Sec system in L. lactis , the signal peptide of the Usp45 protein SP usp45 is commonly fused to the N‐terminus of recombinant proteins (Morello et al ., ).…”

Genetic characterization and expression of leucocin B, a class IId bacteriocin from Leuconostoc carnosum 4010

“…The signal peptide of Usp45 has been used previously and shown to be an effective signal peptide for heterologous production of bacteriocins. For instance, class IIa bacteriocins enterocins A and P, pediocin, sakacin A and class IId bacteriocin hiracin JM79 have been functionally cloned as SSusp45 fusions in Lactococcus and other lactic acid bacteria [21,32,33]. Here, leucocins A and B were cloned and expressed in L. lactis and their antimicrobial activities were compared with previously cloned LecC (Figs.…”