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

Do Won Jeong, Jong Hoon Lee, Kyoung Heon Kim, Hyong Joo Lee

Research output: Contribution to journalArticle

5 Citations (Scopus)

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 α-galactosidase gene from Lactobacillus plantarum KCTC 3104 as a selection marker. pIMA20 was also equipped with a controllable promoter of nisA (PnisA) and a signal peptide-encoding sequence of usp45 (SPusp45) 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 PnisA and SPusp45 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.

Original languageEnglish
Pages (from-to)1799-1808
Number of pages10
JournalJournal of microbiology and biotechnology
Volume16
Issue number11
Publication statusPublished - 2006 Nov

Keywords

  • Food-grade integration expression/secretion vector
  • Lactococcus lactis
  • Site-specific integration
  • nisA promoter
  • α-galactosidase

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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