A strategy to construct vector-free amylolytic strains through nondisruptive homologous recombination: application to Enterococcus faecalis

Abstract

In order to direct chromosomal integration of the α-amylase-encoding gene from Bacillus licheniformis ( amyL) under the control of expression and secretion signals from Enterococcus faecalis, the chromosomal fragment (named AB) from the pGIP3124 plasmid [Hols et al., Gene 118 (1992) 21–30] was chosen and split into two fragments (A and B). A translation fusion between the A fragment and ' amyL, deleted of its expression and secretion signals, was made and this fusion was flanked with the AB fragment at its right end. The A::' amyL:AB integration module was cloned into a thermosensitive pE194 replicon (chloramphenicol resistant; Cm R) and electro-transformed into E. faecalis OG1X. After an overnight culture in selective liquid medium, the offspring from the amylolytic transformants obtained was shown to yield Cm R colonies with two distinct halo sizes on iodine-stained starch plates. Southern analysis clearly showed that the smaller halos corresponded to descendants in which the plasmid had integrated into the chromosome through homologous recombination. One such Amy + integrant in the AB site was further cultured under nonselective conditions at 42°C for about 20 generations, and the offspring was screened for Amy +/Cm S clones. Such revertants were indeed found, and Southern analysis clearly showed that the vector matrix had been excised through homologous recombination between the redundant A sites, leaving the integrated amy gene intact.