Isolation and Characterization of Some Promoter Sequences from Leuconostoc mesenteroides SY2 Isolated from Kimchi

Abstract

Some promoters were isolated and characterized from the genome of Leuconostoc mesenteroides SY2, an isolate from kimchi, a Korean traditional fermented vegetable. Chromosomal DNA of L. mesenteroides SY2 was digested with Sau3AI and ligated with BamHI-cut pBV5030, a promoter screening vector containing a promoterless cat-86. Among E. coli transformants (TFs) resistant against Cm (chloramphenicol), 17 were able to grow in the presence of 1,000 mu g/ml Cm and their inserts were sequenced. Transcription start sites were examined for three putative promoters (P04C, P25C, and P33C) by primer extension. Four putative promoters were inserted upstream of a promoterless a-amylase reporter gene in pJY15 alpha. alpha-Amylase activities of E. coli TFs containing pJY15a (control, no promoter), pJY03 alpha (pJY15 alpha with P03C), pJY04 alpha (with P04C), pJY25 alpha (with P25C), and pJY33 alpha (with P33C) were 66.9, 78.7, 122.1, 70.8, and 99.3 U, respectively. Cells harboring pJY04 alpha showed 1.8 times higher activity than the control. Some promoters characterized in this study might be useful for construction of food-grade expression vectors for Leuconostoc sp. and related lactic acid bacteria.