Development of innovative pediocin PA-1 by DNA shuffling among class IIa bacteriocins

Abstract

Pediocin PA-1 is a member of the class IIa bacteriocins, which show antimicrobial effects against lactic acid bacteria. To develop an improved version of pediocin PA-1, reciprocal chimeras between pediocin PA-1 and enterocin A, another class IIa bacteriocin, were constructed. Chimera EP, which consisted of the C-terminal half of pediocin PA-1 fused to the N-terminal half of enterocin A, showed increased activity against a strain of Leuconostoc lactis isolated from a sour-spoiled dairy product. To develop an even more effective version of this chimera, a DNA-shuffling library was constructed, wherein four specific regions within the N-terminal half of pediocin PA-1 were shuffled with the corresponding sequences from 10 other class IIa bacteriocins. Activity screening indicated that 63 out of 280 shuffled mutants had antimicrobial activity. A colony overlay activity assay showed that one of the mutants (designated B1) produced a >7.8-mm growth inhibition circle on L. lactis, whereas the parent pediocin PA-1 did not produce any circle. Furthermore, the active shuffled mutants showed increased activity against various species of Lactobacillus, Pediococcus, and Carnobacterium. Sequence analysis revealed that the active mutants had novel N-terminal sequences; in active mutant B1, for example, the parental pediocin PA-1 sequence (KYYGNGVTCGKHSC) was changed to TKYYGNGVSCTKSGC. These new and improved DNA-shuffled bacteriocins could prove useful as food additives for inhibiting sour spoilage of dairy products.

FIG. 1.

Amino acid sequences of pediocin PA-1 (Ped), enterocin A (Ent), and the constructed chimeric bacteriocins. Residues in common between pediocin PA-1 and enterocin A are in black boxes. Regarding the chimeric bacteriocins, the fragments derived from pediocin PA-1 are in black boxes. The peptide sequences were derived from the NCBI protein database under the following accession numbers: pediocin PA-1, AAA25559, and enterocin A, CAA63890.

FIG. 2.

(A) Multiple sequence alignment of class IIa bacteriocins used in the DNA-shuffling experiment. The common residues in all bacteriocins are in black boxes. The peptide sequences were derived from the NCBI protein database under the following accession numbers: pediocin PA-1, AAA25559; enterocin A, CAA63890; leucocin A, AAA68003; mundticin KS, BAB88211; piscicolin 126, P80569; bavaricin MN, P80493; divercin V41, CAA11804; enterocin P, AAC45870; carnobacteriocin BM1, AAA23014; sakacin A, CAA86942; and carnobacteriocin B2, AAA72431. (B) DNA-shuffling strategy. In the first PCR, CSVDWGKA or CSVNWGKA was used as a forward primer. In the second PCR, Ped1st, Ent1st, LeuA1st, Mund1st, Pis1st, Bav1st, EntP1st, CarBM1st, SakA1st, or CarB2-1st was used as a forward primer. In the third PCR, NGVHC, NGVSC, NGVTC, or NGVYC was used as a forward primer. In the fourth PCR, Bav2nd, EntA2nd, Ped2nd, or CarBM1-2nd was used as a forward primer. All primers used in this study are listed in the supplemental material. There were no degenerate primers.

FIG. 3.

Sequence analysis of highly active (A) and inactive (B) mutants against L. lactis YKLAB10. Only the N-terminal domain of pediocin PA-1 is shown. Mutant names correspond to group B, E, P, or C. For example, B1 belongs to mutants of group B. The diameters of the inhibition circles of the inactive mutants, whose activity was under the detection level, are given as <1.0 mm. WT indicates parental pediocin PA-1. The numbers above the columns on the right correspond to the amino acid numbers of pediocin PA-1.

FIG. 4.

The antimicrobial activities of DNA-shuffled mutants and pediocin PA-1 (Ped), enterocin A (Ent), divercin V41 (Div), and carnobacteriocin BM1 (Car) against L. lactis YKLAB10 (A), L. lactis JCM 6123 (B), L. mesenteroides subsp. mesenteroides IAM 13004 (C), L. curvatus NBRC 15884 (D), P. pentosaceus JCM 2026 (E), and C. maltaromaticum NBRC 15684 (F) as indicated by the diameters of their inhibition circles. Bavaricin MN was not sequenced completely, so divercin V41, which had almost the same sequence as bavaricin MN (Fig. 2), was synthesized instead. The mutant names, as shown also in Fig. 3, are shown under the horizontal axis. The data represent the average values from two independent experiments, and the error bars show standard deviations.