Development of a food-grade integration vector for heterologous gene expression and protein secretion in lactococcus lactis

A food-grade integration vector based on site-specific recombination was constructed. The 5.7-kb vector, pIMA20, contained an integrase gene and a phage attachment site originating from bacteriophage A2, with the α-galactosidase gene from Lactobacillus plantarum KCTC 3104 as a selection marker. pIMA20 was also equipped with a controllable promoter of nisA (P_nisA) and a signal peptide-encoding sequence of usp45 (SP_usp45) for the production and secretion of foreign proteins. pIMA20 and its derivatives mediated site-specific integration into the attB-like site on the Lactococcus lactis NZ9800 chromosome. The vector-integrated recombinant lactococci were easily detected by the appearance of blue colonies on a medium containing X-α-gal and also by their ability to grow on a medium containing melibiose as the sole carbon source. Recombinant lactococci maintained these traits in the absence of selection pressure during 100 generations. The α-amylase gene from Bacillus licheniformis, lacking a signal peptide-encoding sequence, was inserted downstream of P_nisA and SP_usp45 in pIMA20, and the plasmid was integrated into the L. lactis chromosome. α-Amylase was successfully produced and secreted by the recombinant L. lactis, controlled by the addition and concentration of nisin.