시스템 대사공학 기반 트립토판 대사경로 최적화 코리네박테리움 개발 = Development of Corynebacterium for optimized tryptophan metabolic pathway Based on Systems Metabolic Engineering

다국어 초록 (Multilingual Abstract)

Abstract
Tryptophan is one of essential amino acids, and used as feed and food additives. In this study, tryptophan-overproducing Corynebacterium glutamicum strains were developed from TP679 as a mother strain, which has following genotype; Δcsm ΔtrpL::Pilvc-M1 trpEm Δvdh::PilvcaroGm. Since anthranilate phosphoribosyltransferase in tryptophan biosynthesis is subjected to feedback inhibition by tryptophan, a mutation at 162 th amino acid of TrpD was introduced by site-directed mutagenesis, leading to construction of A162S, A162T, A162D and A162E mutants. According to TrpD enzyme assay, A162D mutant showed a strong feedback-resistance to high concentrations of tryptophan up to 5 mM. In addition, TP679 harboring a plasmid expressing trpDm (A162D) produced 2.5 g/l tryptophan but not anthranilate. To increase serine pool necessary for tryptophan biosynthesis, a truncated form of serA encoding mutant SerA with deletion of 197 amino acids at C-terminal as well as a deletion of sdaA encoding serine deaminase were introduced into genome of TP793, respectively. As a result, TP823 with a deletion of sdaA yielded 10% increase of tryptophan titer compared to mother strain. Finally, the effect of tryptophan excretion on tryptophan production was assessed by expression of yddG encoding tryptophan exporter from E. coli, yielding 3.19 g/L tryptophan in flask culture and 11.4 g/l tryptophan in a 5-L bioreactor.

목차 (Table of Contents)

• 목 차
• LIST OF FIGURES v
• LIST OF TABLES vii
• I. 서 론 2
• II. 실험재료 및 방법 9
• 1.1. 균주, 플라스미드 및 프라이머(primer) 9
• 1.2. 배지조성 9
• 1.3. 효소와 시약 14
• 2.1. C. glutamicum competent cell 제조 및 형질전환법 15
• 2.1.1. Competent cell 제조 15
• 2.1.2. Electrophoration에 의한 형질전환 15
• 2.2. 유전자 DNA 클로닝 방법 16
• 2.2.1. Overlap cloning 16
• 2.2.2. Gene fusion 16
• 2.2.3. DNA Ligation 16
• 2.3. E. coli 유래 trpD 삽입용 벡터 및 NCgl1112 결손 trpD 삽입균주 제작17
• 2.4. C. glutamicum 유래 wild 및 mutant trpD 발현 벡터 제작 20
• 2.5. 염색체 내 trpDA162D 변이용 벡터 및 변이균주 제작 22
• 2.6. serA 변이벡터 및 염색체 내 serA△591 결손균주 제작 24
• 2.7. sdaA 결손용 벡터 및 염색체 내 sdaA 결손균주 제작 26
• 2.8. E. coli 유래 yddG 유전자 발현 벡터 제작 28
• 2.9. 재조합 C. glutamicum 발효 30
• 2.9.1. 삼각플라스크를 이용한 트립토판 생산균 발효 30
• 2.9.2. 5-L Bioreactor를 이용한 트립토판 생산균 발효 30
• 2.10. 분석 방법 34
• 2.10.1. 세포파쇄, 단백질 정량 및 SDS-PAGE 34
• 2.10.2. TrpD 효소활성 분석 34
• 2.10.3. 발효산물 분석 35
• III. 결과 및 고찰 36
• 1. 최적 TrpD 발현에 의한 tryptophan 생산 36
• 1.1. 염색체 내 E. coli 유래 trpD 삽입균주 개발 및 tryptophan 생산 36
• 1.2. C. glutamicum 유래 feedback-resistant TrpD 개발 및 tryptophan 생산 40
• 1.2.1. A162 변이 TrpD 개발 및 발현 40
• 1.2.2. A162 변이 TrpD 효소활성 42
• 1.2.3. Plasmid 내 변이 trpD 발현에 의한 trytpophan 생산 45
• 1.2.4. 염색체 내 trpDA162D 변이에 의한 tryptophan 생산 47
• 2. Serine pool 공급 향상을 통한 tryptophan 생산 50
• 2.1. 염색체 내 serAΔ591 결손균주 개발 50
• 2.2. 염색체 내 sdaA 결손균주 개발 51
• 2.3. 개발균주 평가 51
• 3. 세포 외 tryptophan 배출능 향상을 통한 tryptophan 생산 54
• IV. 결론 57
• V. 참고문헌 58
• Abstract 64