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Development of Bile Salt-Resistant Leuconostoc citreum by Expression of Bile Salt Hydrolase Gene
Development of Bile Salt-Resistant Leuconostoc citreum by Expression of Bile Salt Hydrolase Gene
Journal of Microbiology and Biotechnology. 2015. Dec, 25(12): 2100-2105
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : May 26, 2015
  • Accepted : August 11, 2015
  • Published : December 28, 2015
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Seung Kee Cho
Brain Korea 21 Center for Bio-Resource Development, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea
Soo Jin Lee
Brain Korea 21 Center for Bio-Resource Development, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea
So-Yeon Shin
Brain Korea 21 Center for Bio-Resource Development, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea
Jin Seok Moon
Brain Korea 21 Center for Bio-Resource Development, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea
Ling Li
Brain Korea 21 Center for Bio-Resource Development, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea
Wooha Joo
Brain Korea 21 Center for Bio-Resource Development, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea
Dae-Kyung Kang
Department of Animal Resources Science, Dankook University, Cheonan 31116, Republic of Korea
Nam Soo Han
Brain Korea 21 Center for Bio-Resource Development, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea
namsoo@chungbuk.ac.kr

Abstract
Probiotic bacteria must have not only tolerance against bile salt but also no genes for antibiotic resistance. Leuconostoc citreum is a dominant lactic acid bacterium in various fermented foods, but it is not regarded as a probiotic because it lacks bile salt resistance. Therefore, we aimed to construct a bile salt-resistant L. citreum strain by transforming it with a bile salt hydrolase gene ( bsh ). We obtained the 1,001 bp bsh gene from the chromosomal DNA of Lactobacillus plantarum and subcloned it into the pCB4170 vector under a constitutive P710 promoter. The resulting vector, pCB4170BSH was transformed into L. citreum CB2567 by electroporation, and bile salt-resistant transformants were selected. Upon incubation with glycodeoxycholic acid sodium salt (GDCA), the L. citreum transformants grew and formed colonies, successfully transcribed the bsh gene, and expressed the BSH enzyme. The recombinant strain grew in up to 0.3% (w/v) GDCA, conditions unsuitable for the host strain. In in vitro digestion conditions of 10 mM bile salt, the transformant was over 67.6% viable, whereas only 0.8% of the host strain survived.
Keywords
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( A ) Comparison of transcription levels of the bsh gene in L. citreum. ( B ) Relative intensity of the bsh gene in L. citreum.
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Overnight culture cells were streaked onto MRS agar plates supplemented with 0.3% (w/v) GDCA and incubated for 48 h.
Acknowledgements
This research was supported by funds from iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry of Agriculture, Food and Rural Affairs (914002-04-1-HD020 and 113034-03-2-HD020).
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