Screening of endogenous strong promoters of Leuconostoc citreum EFEL2700 based on transcriptome analysis and its application for food-grade production of β-galactosidase

Abstract

Leuconostoc citreum is a heterofermentative lactic acid bacterium frequently found in the various fermented foods. L. citreum EFEL2700 isolated from Korean kimchi has been used as a host strain for biotechnological applications. For the use as a food-grade host to over-produce food ingredients or enzymes, strong endogenous promoters guarantying high expression levels of target genes are necessary. In this study, transcriptomic analysis of L. citreum EFEL2700 was performed using RNA-Seq and three promoters of the most highly expressed genes were selected: glyceraldehyde 3-phosphate dehydrogenase (G3PD), 6-phosphogluconate dehydrogenase (6PGD), and phosphoketolase (PPK). Thereafter, they were used as promoters to express β-galactosidase gene from Lactobacillus plantarum WCFS1 in L. citreum EFEL2700 and the levels were compared with the control promoter P710 from L. mesenteroides ATCC 8293. As results, the β-galactosidase activities of the transformants were 2.73, 0.27, 37.43, and 9.25 units/mg under the P710, G3PD, 6PGD, and PPK promoters, respectively. The expression level of endogenous promoter 6PGD was superior to the heterologous P710 promoter previously used in a Leuconostoc-Escherichia coli shuttle vector. The 6PGD developed in this study can be used as the most suitable promoter for β-galactosidase expression in L. citreum EFEL2700.

Keywords: Endogenous promoter screening; Leuconostoc citreum; Transcriptome analysis; β-galactosidase expression.