Abstract: This invention provides a recombinant Pichia pastoris strain which has been genetically modified for highly expressing pediocin PA-1 and secreting it into the medium so that the pediocin PA-1 can be easily collected and purified from the culture medium. In this invention, we also demonstrated that the obtained pediocin PA-1 had specific antimicrobial activities against Listeria monocytogenes ATCC 13932 and several other bacteria strains. Therefore, pediocin PA-1 produced from this recombinant Pichia pastoris strain has a great potential for the application in food preservation and pharmaceutical industry.

Abstract: The present invention provides novel reagents and a cloning procedure based on homologous recombination for the site-directed cloning of a DNA fragment to a vector at designed site(s). The cloning reagents are made of mixture of extracts from at least two different cell types, preferably a mixture made of extracts from wild-type E. coli and S. Cerevisiae. Due to the activity of the mixture of cell extracts, recombination occurs between the 3? and 5?-ends of the target DNA and at the ends of linearized vector, which facilitates in-frame construction of expression vectors.