1. Leucine-zipper system을 이용하여 바닐린 생산 유전자인 fcs와 ech의 fusion protein을 구축하여 바닐린 생산에 substrate channeling 효과를 적용하였다. 이렇게 구축한 fusion protein인 pTBE-FP는 2.2 g/L의 바닐린을 생산하는 것을 확인 하였고 pTAHEF의 0.9 g/L에 비해 약 2.4배 많은 바닐린을 생산하였다. 2. 바닐린 생산량과 생산속도가 증가한 pTBE-FP에서 Leucine-zipper system의 효과를 확인 하기 위해 BE-FP를 pET21b ...
1. Leucine-zipper system을 이용하여 바닐린 생산 유전자인 fcs와 ech의 fusion protein을 구축하여 바닐린 생산에 substrate channeling 효과를 적용하였다. 이렇게 구축한 fusion protein인 pTBE-FP는 2.2 g/L의 바닐린을 생산하는 것을 확인 하였고 pTAHEF의 0.9 g/L에 비해 약 2.4배 많은 바닐린을 생산하였다. 2. 바닐린 생산량과 생산속도가 증가한 pTBE-FP에서 Leucine-zipper system의 효과를 확인 하기 위해 BE-FP를 pET21b 벡터에 클로닝해 Fcs의 C-말단에 His-Tag을 붙여 니켈컬럼으로 정제 한 후 Fcs와 Ech 단백질을 SDS-PAGE에서 확인 하였다. 3. 값싼 기질인 포도당과 글리세롤등을 이용하여 바닐린을 만들기 위해 그 전단계인 타이로신을 기질로 하여 페룰산을 만드는 pST5M과 pTT5M을 구축 하였다 4. pST5M이 0.5 g/L의 타이로신으로부터 1.0 g/L 페룰산을 생산하는 것을 확인하였고 타이로신을 공급하지 않은 배양에서도 0.26 g/L 페룰산이 생산 되는 것을 확인 하였다. 5. pST5M과 pTAHEF-gltA를 도입해서 형질전환 시킨 대장균에 0.5 g/L 타이로신을 공급하여 배양하였을 때 바닐린을 0.22 g/L를 생산하였고 타이로신을 1 g/L공급 하였을 때 0.32 g/L 바닐린을 생산하는 것을 확인 하였다.
1. Leucine-zipper system을 이용하여 바닐린 생산 유전자인 fcs와 ech의 fusion protein을 구축하여 바닐린 생산에 substrate channeling 효과를 적용하였다. 이렇게 구축한 fusion protein인 pTBE-FP는 2.2 g/L의 바닐린을 생산하는 것을 확인 하였고 pTAHEF의 0.9 g/L에 비해 약 2.4배 많은 바닐린을 생산하였다. 2. 바닐린 생산량과 생산속도가 증가한 pTBE-FP에서 Leucine-zipper system의 효과를 확인 하기 위해 BE-FP를 pET21b 벡터에 클로닝해 Fcs의 C-말단에 His-Tag을 붙여 니켈컬럼으로 정제 한 후 Fcs와 Ech 단백질을 SDS-PAGE에서 확인 하였다. 3. 값싼 기질인 포도당과 글리세롤등을 이용하여 바닐린을 만들기 위해 그 전단계인 타이로신을 기질로 하여 페룰산을 만드는 pST5M과 pTT5M을 구축 하였다 4. pST5M이 0.5 g/L의 타이로신으로부터 1.0 g/L 페룰산을 생산하는 것을 확인하였고 타이로신을 공급하지 않은 배양에서도 0.26 g/L 페룰산이 생산 되는 것을 확인 하였다. 5. pST5M과 pTAHEF-gltA를 도입해서 형질전환 시킨 대장균에 0.5 g/L 타이로신을 공급하여 배양하였을 때 바닐린을 0.22 g/L를 생산하였고 타이로신을 1 g/L공급 하였을 때 0.32 g/L 바닐린을 생산하는 것을 확인 하였다.
Vanillin (4-hydroxy-3-metoxybenzaldehyde) is one of the world's principal flavoring compounds, and has been used extensively in food industry. Natural vanillin is derived from vanilla pod. Because of the climate-limited cultivation of vanillin pod and labor-intensive production process of natural va...
Vanillin (4-hydroxy-3-metoxybenzaldehyde) is one of the world's principal flavoring compounds, and has been used extensively in food industry. Natural vanillin is derived from vanilla pod. Because of the climate-limited cultivation of vanillin pod and labor-intensive production process of natural vanillin, vanillin is mainly of synthetic origin. In recent years, owing to the increasing demand for healthy and natural food, biovanillin produced from natural raw materials by metabolically engineered microorganism or plant tissue culture is expected to substitute for natural vanillin. Vanillin production was tested in E. coli harboring plasmid pTAHEF containing fcs (feruloyl-CoA synthase) and ech (enoyl-CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104 with different concentrations of added ferulic acid. The production of vanillin was found to be 0.9 g/L with addition of 3.0 g/L ferulic acid for 48 hours culture. In order to obtain substrate channeling effect of ferulic acid to vanillin, Fcs and Ech proteins were combined by using Leucine-zipper system, which significantly increased vanillin production. Bait and prey of Leucine-zipper system were introduced to N-terminal of Ech and C-terminal of Fcs, respectively, resulting into pTBE-FP plasmid. E. coli harboring pTBE-FP produced 2.2 g/L of vanillin at an initial ferulic acid concentration of 3 g/L for 48 hours of culture, which was 2.3-fold higher than vanillin production of 0.9 g/L from E. coli harboring pTAHEF with no Leucine-zipper system. The E. coli (pTBE-FP) could be a potential strain for increased vanillin production by substrate channeling. For production of vanillin from abundant and cheap substrates such as glucose and glycerol, it was designed artificial synthesis pathway of L-tyrosine to ferulic acid, a precursor for vanillin production since E. coli has an efficient synthesis pathway of L-tyrosine amino acid, introduction of the artificial ferulic acid synthesis pathway makes E. coli produce ferulic acid from glycerol. The pathway of L-tyrosine to ferulic acid is composed of three enzymatic steps of L-Tyrosine ammonia lyase (TAL), 4-Coumarate 3-hydroxylase (Sam5), Caffeic acid O-methyltransferase (COM). Plasmids of pST5M and pTT5M containing 3genes encoding the above enzymes were construed by using weak and strong expression vectors of pSTV28 and pTrc99A, respectively. The E. coli DH5α harboring pST5M produced 1 g/L of ferulic acid for 48 hours culture with supplementation of 0.5 g/L of L-tyrosine and 0.26 g/L of ferulic acid was also obtained even in culture with no supplementation of L-tyrosine. Higher production of ferulic acid was observed in E. coli when using pST5M rather then pTT5M. Vanillin production from L-tyrosine was performed using E. coli DH5α harboring vanillin plasmids of pTAHEF-gltA and ferulic acid plasmid of pST5M. The recombination E. coli produced 0.22 g/L of vanillin for 48 hours culture with addition of 0.5 g/L of L-tyrosine.
Vanillin (4-hydroxy-3-metoxybenzaldehyde) is one of the world's principal flavoring compounds, and has been used extensively in food industry. Natural vanillin is derived from vanilla pod. Because of the climate-limited cultivation of vanillin pod and labor-intensive production process of natural vanillin, vanillin is mainly of synthetic origin. In recent years, owing to the increasing demand for healthy and natural food, biovanillin produced from natural raw materials by metabolically engineered microorganism or plant tissue culture is expected to substitute for natural vanillin. Vanillin production was tested in E. coli harboring plasmid pTAHEF containing fcs (feruloyl-CoA synthase) and ech (enoyl-CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104 with different concentrations of added ferulic acid. The production of vanillin was found to be 0.9 g/L with addition of 3.0 g/L ferulic acid for 48 hours culture. In order to obtain substrate channeling effect of ferulic acid to vanillin, Fcs and Ech proteins were combined by using Leucine-zipper system, which significantly increased vanillin production. Bait and prey of Leucine-zipper system were introduced to N-terminal of Ech and C-terminal of Fcs, respectively, resulting into pTBE-FP plasmid. E. coli harboring pTBE-FP produced 2.2 g/L of vanillin at an initial ferulic acid concentration of 3 g/L for 48 hours of culture, which was 2.3-fold higher than vanillin production of 0.9 g/L from E. coli harboring pTAHEF with no Leucine-zipper system. The E. coli (pTBE-FP) could be a potential strain for increased vanillin production by substrate channeling. For production of vanillin from abundant and cheap substrates such as glucose and glycerol, it was designed artificial synthesis pathway of L-tyrosine to ferulic acid, a precursor for vanillin production since E. coli has an efficient synthesis pathway of L-tyrosine amino acid, introduction of the artificial ferulic acid synthesis pathway makes E. coli produce ferulic acid from glycerol. The pathway of L-tyrosine to ferulic acid is composed of three enzymatic steps of L-Tyrosine ammonia lyase (TAL), 4-Coumarate 3-hydroxylase (Sam5), Caffeic acid O-methyltransferase (COM). Plasmids of pST5M and pTT5M containing 3genes encoding the above enzymes were construed by using weak and strong expression vectors of pSTV28 and pTrc99A, respectively. The E. coli DH5α harboring pST5M produced 1 g/L of ferulic acid for 48 hours culture with supplementation of 0.5 g/L of L-tyrosine and 0.26 g/L of ferulic acid was also obtained even in culture with no supplementation of L-tyrosine. Higher production of ferulic acid was observed in E. coli when using pST5M rather then pTT5M. Vanillin production from L-tyrosine was performed using E. coli DH5α harboring vanillin plasmids of pTAHEF-gltA and ferulic acid plasmid of pST5M. The recombination E. coli produced 0.22 g/L of vanillin for 48 hours culture with addition of 0.5 g/L of L-tyrosine.
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