AIMS: To develop a method and plasmid vectors suitable for expression of class II bacteriocins from Escherichia coli. METHODS AND RESULTS: The expression vector pSuV1 was constructed by inserting the PelB secretion signal coding sequence and a number of restriction endonuclease sites for cloning, into pTYB1. Codon optimized genes encoding the active mature region of each bacteriocin were constructed and inserted into pSuV1. Transfer of these constructs to a host expressing T7 RNA polymerase allowed for expression of secreted mature or fusion forms of the bacteriocins. Generation of the fusion, to the adjacent intein-chitin-binding domain gene, was achieved by removal of a small intervening BseRI fragment. The bacteriocins BacR1, divercin V41, enterocin P, pediocin PA-1 and piscicolin 126 were expressed from this system. For piscicolin 126, expression levels of 200 microg l(-1) in the mature form and 1100 microg l(-1) when cleaved from the fusion partner were achieved. All expressed bacteriocins displayed antimicrobial activity. CONCLUSIONS: Several class II bacteriocins have been expressed in E. coli using purpose designed plasmid vectors described here. SIGNIFICANCE AND IMPACT OF THE STUDY: This method provides a common expression system capable of producing a range of different class II bacteriocins. It allows researchers to study class II bacteriocins without access to the original producer strain, the native bacteriocin gene, or highly specific heterologous producing strains. Resulting expression levels are as high or higher than those previously reported for related bacteriocins.
AIMS: To develop a method and plasmid vectors suitable for expression of class II bacteriocins from Escherichia coli. METHODS AND RESULTS: The expression vector pSuV1 was constructed by inserting the PelB secretion signal coding sequence and a number of restriction endonuclease sites for cloning, into pTYB1. Codon optimized genes encoding the active mature region of each bacteriocin were constructed and inserted into pSuV1. Transfer of these constructs to a host expressing T7 RNA polymerase allowed for expression of secreted mature or fusion forms of the bacteriocins. Generation of the fusion, to the adjacent intein-chitin-binding domain gene, was achieved by removal of a small intervening BseRI fragment. The bacteriocins BacR1, divercin V41, enterocin P, pediocin PA-1 and piscicolin 126 were expressed from this system. For piscicolin 126, expression levels of 200 microg l(-1) in the mature form and 1100 microg l(-1) when cleaved from the fusion partner were achieved. All expressed bacteriocins displayed antimicrobial activity. CONCLUSIONS: Several class II bacteriocins have been expressed in E. coli using purpose designed plasmid vectors described here. SIGNIFICANCE AND IMPACT OF THE STUDY: This method provides a common expression system capable of producing a range of different class II bacteriocins. It allows researchers to study class II bacteriocins without access to the original producer strain, the native bacteriocin gene, or highly specific heterologous producing strains. Resulting expression levels are as high or higher than those previously reported for related bacteriocins.
Authors: Beatriz Mesa-Pereira; Paula M O'Connor; Mary C Rea; Paul D Cotter; Colin Hill; R Paul Ross Journal: Sci Rep Date: 2017-06-08 Impact factor: 4.379
Authors: Agnieszka K Olejnik-Schmidt; Marcin T Schmidt; Anna Sip; Tomasz Szablewski; Włodzimierz Grajek Journal: Ann Microbiol Date: 2013-11-27 Impact factor: 2.112