Development of Bile Salt-Resistant Leuconostoc citreum by Expression of Bile Salt Hydrolase Gene
0
조회
0
다운로드
2
인용
0
참고문헌
키워드
초록
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 saltresistant 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.
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 saltresistant 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.
참고문헌 (28)
- Allison G, "Functional analysis of the gene encoding immunity to lactacin F, lafI, and its use as a Lactobacillus-specific, food-grade genetic marker", Appl. Environ. Microbiol, 62, pp.4450~4460 (1996)
- Bohmer N, "Recombinant expression, purification and characterisation of the native glutamate racemase from Lactobacillus plantarum NC8", Protein Expr. Purif, 88, pp.54~60 (2013)
- Budde BB, "Leuconostoc carnosum 4010 has the potential for use as a protective culture for vacuum-packed meats: culture isolation, bacteriocin identification, and meat application experiments", Int. J. Food Microbiol, 83, pp.171~184 (2003)
- Cho SK, "Development of food-grade vector for constitutive and inducible gene expression in Leuconostoc citreum", 박사, pp.2015
- Dashkevicz MP, "Development of a differential medium for bile salt hydrolase-active Lactobacillus spp", Appl. Environ. Microbiol, 55, pp.11~16 (1989)
- De Vos WM, "Gene expression systems for lactic acid bacteria", Curr. Opin. Microbiol, 2, pp.289~295 (1999)
- Dickely F, "Isolation of Lactococcus lactis nonsense suppressors and construction of a food-grade cloning vector", Mol. Microbiol, 15, pp.839~847 (1995)
- Eom HJ, "Selection of psychrotrophic Leuconostoc spp. producing highly active dextransucrase from lactate fermented vegetables", Int. J. Food Microbiol, 117, pp.61~67 (2007)
- Eom HJ, "Monitoring of Leuconostoc mesenteroides DRC starter in fermented vegetable by random integration of chloramphenicol acetyltransferase gene", J. Ind. Microbiol. Biotechnol, 35, pp.953~959 (2008)
- Eom HJ, "Construction of theta-type shuttle vector for Leuconostoc and other lactic acid bacteria using pCB42 isolated from kimchi", Plasmid, 67, pp.35~43 (2012)
- "Guidelines for the evaluation of probiotics in food" (2002)[URL]
- Hemme D, "Leuconostoc, characteristics, use in dairy technology and prospects in functional foods", Int. Dairy J, 14, pp.467~494 (2004)
- Johansson P, "Genome sequence of a food spoilage lactic acid bacterium, Leuconostoc gasicomitatum LMG18811T, in association with specific spoilage reactions", Appl. Environ. Microbiol, 77, pp.4344~4351 (2011)
- Kahala M, "Characterization of starter lactic acid bacteria from the Finnish fermented milk product viili", J. Appl. Microbiol, 105, pp.1929~1938 (2008)
- Kim JF, "Complete genome sequence of Leuconostoc citreum KM20", J. Bacteriol, 190, pp.3093~3094 (2008)
- Leenhouts K, "Construction of a food-grade multiple-copy integration system for Lactococcus lactis", Appl. Microbiol. Biotechnol, 49, pp.417~423 (1998)
- Makarova K, "Comparative genomics of the lactic acid bacteria", Proc. Natl. Acad. Sci. USA, 103, pp.15611~15616 (2006)
- Minekus M, "A standardised static in vitro digestion method suitable for food – an international consensus", Food Funct, 5, pp.1113~1124 (2014)
- 문진석, "Genome sequence analysis of potential probiotic strain Leuconostoc lactis EFEL005 isolated from kimchi", The Journal of Microbiology, 53(5), pp.337~342 (2015), 한국미생물학회[DOI]
- Nguyen TT, "A food-grade system for inducible gene expression in Lactobacillus plantarum using an alanine racemase-encoding selection marker", J. Agric. Food Chem, 59, pp.5617~5624 (2011)
- Peterbauer C, "Food-grade gene expression in lactic acid bacteria", Biotechnol. J, 6, pp.1147~1161 (2011)
- Pfaffl MW, "A new mathematical model for relative quantification in real-time RT–PCR", Nucleic Acids Res, 29, pp.e45~ (2001)
- Posno M, "Complementation of the inability of Lactobacillus strains to utilize D-xylose with D-xylose catabolismencoding genes of Lactobacillus pentosus", Appl. Environ. Microbiol, 57, pp.2764~2766 (1991)
- Rodgers S., "Novel applications of live bacteria in food services: probiotics and protective cultures", Trends Food Sci. Technol, 19, pp.188~197 (2008)
- Saarela M, "Probiotic bacteria: safety, functional and technological properties", J. Biotechnol, 84, pp.197~215 (2000)
- Takala T, "A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI", Appl. Microbiol. Biotechnol, 59, pp.467~471 (2002)
- Tannock GW, "Probiotic properties of lactic-acid bacteria:plenty of scope for fundamental R & D", Trends Biotechnol, 15, pp.270~274 (1997)
- Yin S, "A novel vector for lactic acid bacteria that uses a bile salt hydrolase gene as a potential food-grade selection marker", J. Biotechnol, 152, pp.49~53 (2011)