1,4-Dihydroxy-2-naphthoic acid (DHNA), a precursor of menaquinone (vitamin K2), has an effect on growth stimulation of bifidobacteria and prevention of osteoporosis, making it a promising functional food material. Therefore, we tried to clone the menB gene encoding DHNA synthase from Leuconostoc mes...
1,4-Dihydroxy-2-naphthoic acid (DHNA), a precursor of menaquinone (vitamin K2), has an effect on growth stimulation of bifidobacteria and prevention of osteoporosis, making it a promising functional food material. Therefore, we tried to clone the menB gene encoding DHNA synthase from Leuconostoc mesenteroides CJNU 0147. Based on the genome sequence of Leu. mesenteroides ATCC 8293 (GenBank accession no., CP000414), a primer set (Leu_menBfull_F and Leu_menBfull_R) was designed for the PCR amplification of menB gene of CJNU 0147. A DNA fragment (1,190 bp), including the menB gene, was amplified, cloned into pGEM-T Easy vector, and sequenced. The deduced amino acid sequence of MenB (DHNA synthase) protein of CJNU 0147 had a 98% similarity to the corresponding protein of ATCC 8293. The menB gene was subcloned into pCW4, a lactic acid bacteria - E. coli shuttle vector, and transferred to CJNU 0147. The transcription of menB gene of CJNU 0147 (pCW4::menB) was increased, when compared with those of CJNU 0147 (pCW4) and CJNU 0147 (−). The DHNA was produced from it at a detectable level, indicating that the cloned menB gene of CJNU 0147 encoded a DHNA synthase which is responsible for the production of DHNA, resulting in an increase of bifidogenic growth stimulation activity.
1,4-Dihydroxy-2-naphthoic acid (DHNA), a precursor of menaquinone (vitamin K2), has an effect on growth stimulation of bifidobacteria and prevention of osteoporosis, making it a promising functional food material. Therefore, we tried to clone the menB gene encoding DHNA synthase from Leuconostoc mesenteroides CJNU 0147. Based on the genome sequence of Leu. mesenteroides ATCC 8293 (GenBank accession no., CP000414), a primer set (Leu_menBfull_F and Leu_menBfull_R) was designed for the PCR amplification of menB gene of CJNU 0147. A DNA fragment (1,190 bp), including the menB gene, was amplified, cloned into pGEM-T Easy vector, and sequenced. The deduced amino acid sequence of MenB (DHNA synthase) protein of CJNU 0147 had a 98% similarity to the corresponding protein of ATCC 8293. The menB gene was subcloned into pCW4, a lactic acid bacteria - E. coli shuttle vector, and transferred to CJNU 0147. The transcription of menB gene of CJNU 0147 (pCW4::menB) was increased, when compared with those of CJNU 0147 (pCW4) and CJNU 0147 (−). The DHNA was produced from it at a detectable level, indicating that the cloned menB gene of CJNU 0147 encoded a DHNA synthase which is responsible for the production of DHNA, resulting in an increase of bifidogenic growth stimulation activity.
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대상 데이터
mesenteroides CJNU 0147 in which a putative promoter is included, a primer set (Leu_menBfull_F: 5’-TGAGGCTGCTGTTTC AGC-3’; Leu_menBfull_R: 5’-GATCATACCCCGTATT CC-3’) based on the genome sequence of Leu. mesenter- oides ATCC 8293 (GenBank accession no., CP000414) was designed. A commercial PCR premix kit (Bioneer, Korea) was used for amplification of the gene.
A slot blot assay showed the menB gene was well transcribed in the recombinant strain, but was not detected in the transcripts from wild type strain and a control transformant Leu. mesenteroides CJNU 0147 (pCW4) (Fig. 1). The result signifies that the cloned menB gene is transcribed in its original host Leu.
Glycerol stocks (-76℃) for Leu. mesenteroides CJNU 0147, CJNU 0147 (pCW4), and CJNU 0147 (pCW4:: menB) were inoculated into 5 mL MRS broth and incu- bated at 37℃ for 24 h. The cultures were subcultured into 200 mL MRS broth supplemented with 0.
BGS activities of samples for Leu. mesenteroides CJNU 0147, CJNU 0147 (pCW4), and CJNU 0147 (pCW4::menB). B.
데이터처리
(SD). A statistical software (SPSS v. 12.0; SPSS Co., USA) was used for Duncan’s multiple range tests for de- termining significance of difference (p<0.05).
longum, Only the cells were inoculated in 5 mL RCM broth; MRS, cells plus 100 μL MRS broth as a control; others, cells plus 100 μL of culture supernatant. Values are shown as mean±SD (n=3) and means having different letters are significantly different by Duncan’s multiple range tests (p<0.05).
성능/효과
2). The results confirm that the recombinant strain can produce DHNA at a detectable level, which is caused by the re- combinant plasmid vector pCW4::menB; a control recom- binant strain harboring pCW4 did not produce detectable level of DHNA. At the same time, it was also confirmed that the cloned menB gene from Leu.
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