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KMID : 0545120060160111799
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Journal of Microbiology and Biotechnology
2006 Volume.16 No. 11 p.1799 ~ p.1808
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Development of a Food-Grade Integration Vector for Heterologous Gene Expression and Protein Secretion in Lactococcus lactis
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Jeong Do-Won
Lee Jong-Hoon
Kim Kyoung-Heon
Lee Hyong-Joo
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Abstract
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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 -galactosidase gene from Lactobacillus plantarum KCTC 3104 as a selection marker. pIMA20 was also equipped with a controllable promoter of nisA () and a signal peptide-encoding sequence of 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 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 gene from Bacillus licheniformis, lacking a signal peptide-encoding. sequence, was inserted downstream of in pIMA20, and the plasmid was integrated into the L. lactis chromosome. was successfully produced and secreted by the recombinant L. lactis, controlled by the addition and concentration of nisin.
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KEYWORD
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Lactococcus lactis, food-grade integration expression/secretion vector, site-specific integration, α-galactosidase, nisA promoter
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