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

In order to direct chromosomal integration of the alpha-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; CmR) 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 CmR 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 degrees C for about 20 generations, and the offspring was screened for Amy+/CmS 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.