1. Seung Kee Cho, Hye Hyeong Kim and Nam Soo Han* Department of Food Science and Technology, Chungbuk National University, Cheongju, 361-763, Korea e. mail : chosk@cbnu.ac.kr Promoter Sequences for Strong and Constitutive Gene Expression in Leuconostoc mesenteroides ATCC 8293 and Leuconostoc citreum 95 For construction of gene expression vector for Leuconostoc spp., that are important lactic acid bacteria for fermented vegetables and milk, strong and constitutive promoters are necessary. Transcriptomic analysis of L. mesenteroides ATCC 8293 using microarrays revealed that genes of Leum_209, 710, 866, 1326, 1506 and 1694, were expressed most strongly and constitutively in both glucose- and sucrose-MRS medium. For constitutive over-expression of β-galactosidase gene in L. mesenteroides and L. citreum, the six promoter sequences were fused to the promoterless β-gal gene from Lactobacillus plantarum to construct an Escherichia coli- Leuconostoc shuttle vectors, pEK32progals, by using pLeuCM42. When six vectors were transformed into Leuconostoc spp. and the transformants were cultivated in glucose-MRS medium, total β-gal activities of L. mesenteroides in culture medium and cytoplasm were 1.37, 0.86, 0.81, 0.37, 0.90 and 0.04 Units/mg for Leum_710, 209, 1694, 866, 1326 and 1506 promoter sequences, respectively, and in case of L. citreum, these activities were 4.37, 2.21 and 1.47 units/mg for Leum_710, 1694 and 209 promoter sequences, respectively. The transcription levels of promoters show that the promoter sequence of transglycosylase-associated protein (Leum_866) and ribosomal protein S8 (Leum_209) can be used as a strong constitutive promoter in the expression vector for the study of functional genomics and metabolic engineering of Leuconostoc spp.. Abstract 4. Conclusion Industrial application of Leuconostoc 1. Research Goals (a ) Leuconostoc 2. 1. Screening of strongly and constitutively putative promoter 3. Results  The strong contstitutive promoters from L. mesenteroides ATCC 8293 were identified via a transcriptome approach.  Among the promoters, P710 showed the highest promoter activity in Leuconostoc.  The promoter sequence of transglycosylase-associated protein (Leum_866) and ribosomal protein S8 (Leum_209) showed the highest transcription levels in L. mesenteroides and L. citreum, respectively.  Although galactosidase activity using promoter P710 characterized in the present study was low, it can be utilized for the construction of food-grade expression vector in Leuconostoc, which is eventually for the heterologous protein production. NoGene Name.KEGG No. 150S Ribosomal protein L24LEUM_0207 230S Ribosomal protein S8LEUM_0209 350S Ribosomal protein L6PLEUM_0210 450S Ribosomal protein L36LEUM_0218 5Pseudouridylate synthase, 23S RNA-specificLEUM_0680 6NUDIX family hydrolaseLEUM_0704 7Integral membrane protein, interacts with FtsHLEUM_0710 8Predicted membrane proteinLEUM_0866 950S Ribosomal protein L21LEUM_1326 1050S Ribosomal protein L33LEUM_1361 1150S Ribosomal protein L28LEUM_1506 12Phosphoenolpyruvate carboxylase (archaeal type)LEUM_1694 Transcription analysis a. Prediction Softberry Hind111 Sal1 Bgl1 1 b. Putative promoter Fig. 1. Strategy of strong promoter selection 2.2. Strategy of strong promoter selection 3.1. Construction of promoterless vector Promoterless Gal- For LacL LacM Genomic DNA of Lb. plantarum Gal-Re pEK32 cloning 2.3. Layouts of selected genes. Fig. 2. Layouts of selected genes. Promoters are shown as black arrows; and Rho-independent terminators shown as stem loops. Fig. 4. Construction of the pEK32ΔGal plasmid for strong promoter isolation 3. 2. Screening of strong promoter in L. mesenteroides Hind111 Sal1 Bgl11 Putative promoter pEK32ΔGal Cloning ONPG (o-nitrophenyl-, β-D-galactopyranoside) + o - nitrophenol β-D- galactose β-D-galactosidase Table 1. List of constitutive promoter in L. mesenteroides on both glucose- and sucrose-MRS medium 5. References 1.Eom, H.J,·J. M. Park, · M. J. Seo, · M. D. Kim and N. S Han. 2008. Monitoring of Leuconostoc mesenteroides DRC starter in fermented vegetable by random integration of chloramphenicol acetyltransferase gene. J Ind Microbiol Biotechnol. 35:953–959 2.Eom H.J, J. S. Moon, E. Y. Seo and N. S. Han. 2009. Heterologous expression and secretion of Lactobacillus amylovorus alpha-amylase in Leuconostoc citreum.. Biotechnol Lett. 31(11):1783-1788 3.Eom, H.J,·J. S. Moon, · S. K.Cho, · J. H. Kim and N. S Han. 2011. Construction of theta-type shuttle vector for Leuconostoc and otherlactic acid bacteria using pCB42 isolated from kimchi.. Plasmid. 67: 35-43 2.4. Nucleotide sequence of select intergenic regions. Fig. 3. Nucleotide sequence of select intergenic regions. Putative -35 and -10 regions are in bold. A; P209, B; P710, C; P866, D; P1326, E; P1506, F; P1694. 3. 3. Specific activity of β-galactosidase from different promoters Fig. 8. Comparison of transcription levels of putative promoters. Transcription level of L. mesenteroides (black) and L. citreum (grey). Fig. 7. Comparison of β-galactosidase activity of constitutive vectors. of β-galactosidase activity of L. mesenteroides (black) and L. citreum (grey). Starter for Kimchi & Sauerkraut (fermented vegetable) Starter for Kefir (fermented milk) Production of Dextran Additives in Animal Feeds Production of Isomalto- oligosaccharides Mannitol production 2. Materials and Methods 1.Screening of strong promoter in Leuconostoc spp.. 2. Development of constitutive gene expression system ⇒ pEk32ΔGal harbouring b-Gal as reporter gene was used for constructions of plasmids with selected promoters Transformant selection (X-gal;Blue colony) ⇒ Screening for different promoter activities was achieved by plating the cells onto agar that contained X-Gal, on which the developing colonies ranged in color from white to blue. 3. 4. Transcription levels of promoters in the Leuconostoc ⇒ The resulting PCR products of promoter sequences were digested with Hind111 and ligated to compatible ends of the digested pEK32ΔGa l. ⇒ Among the promoters, P710 showed the highest promoter activity in L. mesenteroides and L. citreum ⇒ The promoter sequence of transglycosylase-associated protein (Leum_866) and ribosomal protein S8 (Leum_209) showed the highest transcription levels in L. mesenteroides and L. citreum, respectively. Fig. 5. Agarose gel electrophoresis of pEK32ProGal digested with restriction enzyme. Fig. 6. Expression of β-galactosidase in Leuconostoc