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      배양공정 연계 유전자의 cloning을 통한 lovastatin 고생산성 형질전환 균주개발 및 fill and draw 생물공정 개발

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      https://www.riss.kr/link?id=T10359026

      • 저자
      • 발행사항

        춘천 : 江原大學校 大學院, 2006

      • 학위논문사항

        학위논문(석사) -- 江原大學校 大學院 , 微生物學科 , 2006. 2

      • 발행연도

        2006

      • 작성언어

        한국어

      • KDC

        475.3 판사항(4)

      • 발행국(도시)

        강원특별자치도

      • 기타서명

        Development of high yielding transformants by cloning of key gene related to bioprocess, and application of fill & draw fermentations for enhanced production of lovastatin

      • 형태사항

        127 p. : 삽도 ; 26 cm

      • 일반주기명

        참고문헌 : p.121-124

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      다국어 초록 (Multilingual Abstract)

      Lovastatin, a competitive inhibitor of HMG-CoA reductase, is a powerful anti-hypercholesterolemic agent produced by Aspergillus terreus as a secondary metabolite. It is well known that highly branched filamentous morphology results in significantly high viscosities of culture broth in fungal cell fermentations, leading to significant reduction in mass- and oxygen-transfer capabilities. In our previous research, we also observed that cultures with compact-pelleted forms showed about 3 fold higher oxygen transfer coefficient(kLa), resulting in 1.8 fold increase in lovastatin production as compared to those with filamentous forms. It had been also demonstrated the enhanced oxygen transfer rate in the pellet-formed fermentations was due to lower viscosity of the culture medium with fungal cells of pellet morphology. In this study, for more facilitated utilization of dissolved oxygen by the high-yielding mutants, we constructed and introduced into the high producers the respective expression vector with Vitreoscilla hemoglobin(VHb) gene. Notably, the resulting transformant, SELI8-113/VHb+, harboring the wild VHb gene showed approximately 2.5 fold higher lovastatin productivity than the parallel nontransformed strains in 5L-bioreactor fermentations performed under the relatively low levels of dissolved oxygen environments. Furthermore, production stabilities of most of the strains screened from the SELI8-113/VHb+ were excellent, exhibiting sharp contrast to the results obtained from the nontransformants. Therefore, it was concluded that optimal supply of oxygen was prerequisite for the enhanced biosynthesis of lovastatin as well as production stability of the high-yielding producers.
      In addition, we tested fill and draw mode of fermenter operation in order to maximally utilize the fermentation characteristics of the high-yielding transformants, as observed in the previous experiments. In the fill and draw cultures performed by exchanging 30%, 50%, and 70%, respectively, of the fermention broth with a fresh production medium, notable results were observed that the transformant(SELI8-113/VHb+) maintatined its higher lovastatin productivity as well as cell growth rate until the third round of repeated batch operations. In contrast to these results, the nontransformed strain(SELI8-113) showed significantly reduced level in terms of biosynthetic capability of lovastatin and cell proliferation in the comparative repeated batch fermentations (i.e., fill and draw culture) performed under the identical culture conditions. Also notable was the positive role of glycerol added in the production medium, since it contributed to remarkable enhancement in the lovastatin-biosynthetic capability of the transformant. The optimum concentration of glycerol supplemented to the previously utilized production medium, which did not cause catabolite repression/inhibition phenomenon to the higher producer was determined to be 12 g/L through statistical medium optimization studies. (Response surface method(RSM) based on central composite design(CCD) was adopted.). In another set of fill and draw fermentations performed with medium exchange of 70%, 80% and 90% (v/v) respectively, optimum amount of exchanged medium producing maximum amount of lovastatin was observed to be 70% (v/v), (In theses fermentations, the newly modified medium was adopted, as explained above.).
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      Lovastatin, a competitive inhibitor of HMG-CoA reductase, is a powerful anti-hypercholesterolemic agent produced by Aspergillus terreus as a secondary metabolite. It is well known that highly branched filamentous morphology results in significantly hi...

      Lovastatin, a competitive inhibitor of HMG-CoA reductase, is a powerful anti-hypercholesterolemic agent produced by Aspergillus terreus as a secondary metabolite. It is well known that highly branched filamentous morphology results in significantly high viscosities of culture broth in fungal cell fermentations, leading to significant reduction in mass- and oxygen-transfer capabilities. In our previous research, we also observed that cultures with compact-pelleted forms showed about 3 fold higher oxygen transfer coefficient(kLa), resulting in 1.8 fold increase in lovastatin production as compared to those with filamentous forms. It had been also demonstrated the enhanced oxygen transfer rate in the pellet-formed fermentations was due to lower viscosity of the culture medium with fungal cells of pellet morphology. In this study, for more facilitated utilization of dissolved oxygen by the high-yielding mutants, we constructed and introduced into the high producers the respective expression vector with Vitreoscilla hemoglobin(VHb) gene. Notably, the resulting transformant, SELI8-113/VHb+, harboring the wild VHb gene showed approximately 2.5 fold higher lovastatin productivity than the parallel nontransformed strains in 5L-bioreactor fermentations performed under the relatively low levels of dissolved oxygen environments. Furthermore, production stabilities of most of the strains screened from the SELI8-113/VHb+ were excellent, exhibiting sharp contrast to the results obtained from the nontransformants. Therefore, it was concluded that optimal supply of oxygen was prerequisite for the enhanced biosynthesis of lovastatin as well as production stability of the high-yielding producers.
      In addition, we tested fill and draw mode of fermenter operation in order to maximally utilize the fermentation characteristics of the high-yielding transformants, as observed in the previous experiments. In the fill and draw cultures performed by exchanging 30%, 50%, and 70%, respectively, of the fermention broth with a fresh production medium, notable results were observed that the transformant(SELI8-113/VHb+) maintatined its higher lovastatin productivity as well as cell growth rate until the third round of repeated batch operations. In contrast to these results, the nontransformed strain(SELI8-113) showed significantly reduced level in terms of biosynthetic capability of lovastatin and cell proliferation in the comparative repeated batch fermentations (i.e., fill and draw culture) performed under the identical culture conditions. Also notable was the positive role of glycerol added in the production medium, since it contributed to remarkable enhancement in the lovastatin-biosynthetic capability of the transformant. The optimum concentration of glycerol supplemented to the previously utilized production medium, which did not cause catabolite repression/inhibition phenomenon to the higher producer was determined to be 12 g/L through statistical medium optimization studies. (Response surface method(RSM) based on central composite design(CCD) was adopted.). In another set of fill and draw fermentations performed with medium exchange of 70%, 80% and 90% (v/v) respectively, optimum amount of exchanged medium producing maximum amount of lovastatin was observed to be 70% (v/v), (In theses fermentations, the newly modified medium was adopted, as explained above.).

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      목차 (Table of Contents)

      • Ⅰ. 서론
      • 1. Lovastatin = 1
      • 2. Vitreoscilla hemoglobin = 4
      • 3. Efflux pump (lovI) = 6
      • 4. Fill & Draw culture = 8
      • Ⅰ. 서론
      • 1. Lovastatin = 1
      • 2. Vitreoscilla hemoglobin = 4
      • 3. Efflux pump (lovI) = 6
      • 4. Fill & Draw culture = 8
      • 5. 연구 목적 = 10
      • Ⅱ. 실험 재료 및 방법 = 10
      • 가. 실험 재료 = 10
      • 1. 균주 및 균주 보관 = 13
      • 2. 배지 및 배양 조건 = 13
      • 나. 실험 방법 = 15
      • 1. 균체농도 분석 = 15
      • 2. Lovastatin 추출 및 정량분석 = 15
      • 3. Glucose, Glycerol 정량 분석 = 16
      • 4. Vitreoscilla hemoglobin (VHB) 발현 벡터 제조 = 17
      • 5. Efflux pump (lovI) 유전자 발현을 위한 alcA promoter system 개발 = 17
      • 5-1. 균체 chromosomal DNA 분리 = 22
      • 5-2. A. nidulans로부터 alcA promoter 및 alcR 유전자 클로닝 = 22
      • 5-3. A. terreus로부터 efflux pump 유전자 클로닝 = 23
      • 5-4. trpC promoter와 hygromycin 저항성 유전자 클로닝 = 23
      • 5-5. Ligation = 25
      • 5-5-1. pBARALC1 벡터제조 = 25
      • 5-5-2. pBARALCLOVI-1 벡터제조 = 25
      • 5-5-3. pHPHALCLOVI-1 벡터제조 = 25
      • 5-5-4. alcR 유전자 발현 벡터 제조 = 29
      • 6. 형질전환 = 29
      • 6-1. 원형질체 준비 = 29
      • 6-2. 원형질체 재생 = 31
      • 6-3. Phosphionthricin 및 Hygromycin MIC 측정 = 33
      • 6-3-1. Phosphinothricin MIC 측정 = 33
      • 6-3-2. Hygromycin MIC 측정 = 33
      • 6-4. PEG를 이용한 형질전환 = 33
      • 7. 형질전환체 서별 및 분석 = 34
      • 7-1. PCR에의한 host DNA에 VHb 유전자 삽입확인 = 34
      • 7-2. RT-PCR에의한 VHb 유전자 발현 확인 = 35
      • 7-3. CO-difference spectra를 이용한 VHb 활성 측정 = 36
      • 8. 반응표면분석 방법을 이용한 생산 배지 최적화 = 36
      • 9. 5L 생물배양기 배양을 통한 생산균주의 배양 생리학적 특성 연구 = 37
      • 9-1. 회분식 배양 = 37
      • 9-2. Fill & Draw 배양 = 39
      • Ⅲ. 결과 = 40
      • 가. VHb 유전자 도입을 통한 lovatatin 고생산 형질전환체 개발 = 40
      • 1. VHb 발현 벡터 제조 = 40
      • 2. VHb 발현 형질전환체 제조 = 40
      • 2-1. Phosphinothricin MIC 측정 = 41
      • 2-2. PEG를 이용한 형질전환 = 41
      • 3. VHb 발현 형질전환체 선별 및 분석 = 44
      • 3-1. PCR을 통한 VHb 유전자 삽입 확인 및 플라스크 배양 = 44
      • 3-2. RT-PCR을 통한 VHb 유전자 발현 확인 = 48
      • 3-3. CO-difference spectra를 이용한 VHb 활성 확인 = 48
      • 3-4. random screening을 통한 모균주와 형질전환체 생산성 및 생산 안정성 확인 = 48
      • 나. 회분식 배양 연구 = 52
      • 1. 모균주와 형질전환체의 lovastatin 생산성 및 배양 생리학적 특성조사 = 52
      • 2. 용존산소 제한 조건에서의 목균주와 형질전환체의 특성비교 = 61
      • 다. Fill & Draw 배양연구 = 65
      • 1.형질전환체를 이용한 회분식 배양과 fill & draw 배양 비교 = 65
      • 2. 모균주와 형질전환체의 50% fill & draw 배양 = 71
      • 3. lovastatin 생합성을 위한 최적 glycerol 농도 조사 = 78
      • 3-1. 다양한 glycerol 농도에 대한 lovastatin 생산성 비교 = 78
      • 3-2. 생물반응기 배양을 통한 glycerol 첨가 효과 조사 = 80
      • 3-2-1. 형질전환체의 50% fill & draw배양에서 lovastatin 생산에 대한 glycerol 효과조사 = 80
      • 3-2-2. 70% fill & draw 배양에서 모균주와 형질전환체의 특성 비교 = 84
      • 3-3. 반응표면분석 방법을 이용한 최적 glycerol 농도 조사 = 90
      • 4. 형질전환체를 이용한 fill & draw 배양에서 교체가능한 배지의 최적 부피 조사 = 93
      • 5. Fill & Draw 배양에서 생산 배지의 feefing strategy 연구 = 98
      • 라. Efflux pump (lovI) 유전자 발현 형질전환체 개발 = 98
      • 1. alcA promoter system을 이용한 lovI 발현 벡터 제조 = 98
      • 1-1. A.nidulans로부터 alcA promoter 및 alcR 유전자 클로닝 = 98
      • 1-2. A. terreus로부터 lovI 유전자 클로닝 = 99
      • 1-3. trpC promoter와 hygromycin 저항성 유전자 클로닝 = 99
      • 1-4. 제한효서 처리 및 ligation = 101
      • 1-4-1. PHPHALCLOVI-1 벡터 제조 = 101
      • 1-4-2. pBARALCR1 벡터 제조 = 102
      • 2. Efflux pump(lovI) 유전자 과발현 형질전체 제조
      • 2-1. Hygromycin MIC 측정 = 102
      • 2-2. PEG를 이용한 형질전환 = 105
      • 3. Efflux pump 유전자 과발현 형질전환체 선별 및 분석 = 105
      • Ⅳ. 결론 = 107
      • Ⅴ. 참고문헌 = 111
      • Ⅵ. 영문요약 = 115
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