Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker

Construction of a food-grade expression vector for application to lactic acid bacteria (LAB) is of importance for dairy fermentation system. An alpha-galactosidase (aga) gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid pRAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36e to substitute the primary antibiotic selectable marker of pMG36e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long alpha-amylase (amy) gene from Bacillus licheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. The selection efficiency of aga was 8.71 x 10(3) CFU with a standard deviation of 9.1 x 10(2) CFU mu g(-1) DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putative molecular weight of alpha-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of alpha-amylase by expressing of amy gene of pMG36-aga-amy.