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Development of a Food-Grade Integration Vector for Heterologous Gene Expression and Protein Secretion in Lactococcus lactis  

Jeong, Do-Won (Department of Agricultural Biotechnology, Seoul National University)
Lee, Jong-Hoon (Department of Food Science and Biotechnology, Kyonggi University)
Kim, Kyoung-Heon (Division of Food Science, College of Life Sciences and Biotechnology, Korea University)
Lee, Hyong-Joo (Department of Agricultural Biotechnology, Seoul National University)
Publication Information
Journal of Microbiology and Biotechnology / v.16, no.11, 2006 , pp. 1799-1808 More about this Journal
Abstract
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 ${\alpha}$-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 $X-{\alpha}-gal$ 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 ${\alpha}-amylase$ 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. ${\alpha}-Amylase$ was successfully produced and secreted by the recombinant L. lactis, controlled by the addition and concentration of nisin.
Keywords
Lactococcus lactis; food-grade integration expression/secretion vector; site-specific integration; ${\alpha}$-agalactosidase; nisA promoter;
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Times Cited By Web Of Science : 5  (Related Records In Web of Science)
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