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.

Development of Class IIa Bacteriocins as Therapeutic Agents

“…Some chimeras of pediocin PA-1 with other class IIa bacteriocins showed either comparable or greater bactericidal activities to the corresponding natural bacteriocins against certain indicator strains [43, 51]. …”

Natural Preservatives-Research Progress in Class II a Bacteriocins

“…DNA shuffling technology has revealed that replacing residues in the N-terminus of pediocin PA-1 with the corresponding residues from other class Ⅱa bacteriocins did not affect the antimicrobial activity [5]. Whereas, the Cterminus regions among different class Ⅱa bacteriocins are relatively various which decide the antimicrobial spectrum [6].…”

Other Organism‐Based Processes and Products

“…Considerable efforts have been made to functionally characterize bacteriocin operons and to express them in heterologous systems (Tominaga and Hatakeyama, 2007). The genes responsible for bacteriocin production are frequently associated with mobilizable elements, or in the chromosome in association with transposons or plasmids.…”