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Applied and Environmental Microbiology, August 2007, p. 5292-5299, Vol. 73, No. 16
0099-2240/07/$08.00+0     doi:10.1128/AEM.00558-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development of Innovative Pediocin PA-1 by DNA Shuffling among Class IIa Bacteriocins{triangledown} ,{dagger}

Tatsuya Tominaga1* and Yoshinori Hatakeyama2

Saitama Industrial Technology Center North Institute, 2-133 Suehiro, Kumagaya, Saitama 360-0031,1 Laboratory of Applied Entomology, Department of Plant Science and Resources, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan2

Received 10 March 2007/ Accepted 19 June 2007

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.

* Corresponding author. Mailing address: Saitama Industrial Technology Center North Institute, 2-133 Suehiro, Kumagaya, Saitama 360-0031, Japan. Phone: 81 (485) 210614. Fax: 81 (485) 256052. E-mail: tominaga{at}saitama-itcn.jp

{triangledown} Published ahead of print on 25 June 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, August 2007, p. 5292-5299, Vol. 73, No. 16
0099-2240/07/$08.00+0     doi:10.1128/AEM.00558-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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