Effect of nucleoside diphosphate kinase expression on 2’-fucosyllactose production in recombinant Corynebacterium glutamicum

다국어 초록 (Multilingual Abstract)

The purpose of this study was to obtain 2’-fucosyllactose of high yield from recombinant Corynebacterium glutamicum, overexpressing nucleoside diphosphate kinase. Nucleoside diphosphate kinase (NDK) is a protein that catalyzes the reaction of producing GTP using ATP as a substrate of GDP. It plays an important role in the synthesis of nucleoside triphosphates (NTPs) other than ATP, and ATP gamma phosphate is passed to NDP beta phosphate through the ping-pong mechanism using phosphatized active site intermediates. In this experiment, the NDK gene was introduced and overexpressed in Corynebacterium glutamicum to increase the production of 2’-fucosyllactose. In batch and fed-batch fermentation of strains, the result of flask fermentation of NDK gene from C. glutamicum showed 843mg/L 2’-FL which was improved by 45 % compared to that of the pEGWTT(CO)/pVBCL strain without the overexpression of the NDK gene,
Also, I tried to verify that the overexpression of the NDK gene might positively affect throughout the fed-batch fermentation has effects on efficient mass production of 2’-fucosyllactose in Corynebacterium glutamicum throughout the fed-batch fermentation. I selected the pEGWTT(CO) /pVBCLN (CG) strains, which gave the best results in flask fermentation, and performed fed-batch fermentation in 2.5L bioreactors. As a result, the result of fed-batch fermentation of the pEGWTT(CO)/pVBCLN(CG) strain which was overexpressed NDK gene from C. glutamicum showed 10g/L 2’-FL. Productivity was improved by 53 % compared to that of the pEGWTT(CO)/pVBCL strain without the overexpression of the NDK gene. Through this result, I demonstrated that overexpression of NDK gene could increase the production of any bioreactors adopting GTP as a cofactor.

국문 초록 (Abstract)

Nucleotide diphosphate kinase(NDK) 효소는 GDP를 기질로 ATP를 사용하여 GTP를 생산하는 반응을 촉매하는 단백질이다. ATP이외의 Nucleoside trophosphates의 합성에 중요한 역할을 하고, ATP 감마 인산염은 인산화된 활성부위 중간체를 사용하여 핑퐁 메커니즘을 통해 NDP 베타 인산염으로 전달된다.
이전 실험에서는 2’-푸코실락토오즈의 생산을 높이기 위해 플라스미드에 Nucleoside diphosphate kinase (NDK) 유전자를 도입하고 과발현 시켰다. 본 실험에서는 이를 코리네박테리움 균주에 도입하고 여러 발효을 통해 2’-푸코실락토오스 생산성 향상 효과를 검증하였다. 플라스크 발효 결과 C. glutamicum 유래 NDK는 대조군 대비 O.D의 증가를 보였으며 843mg/l의 2’-FL을 생산하였다. 또한 2’-푸코실락토오스의 생산성은 45% 향상되었다.
또한 Fed-batch를 통해 NDK 유전자의 과발현이 대량생산에도 도움을 준다는 것을 검증하려고 하였다. Flask 발효에서 효과가 좋았던 pEGWTT/pVBCLN(CG) 균주를 선택하여 대조군과 함께 2.5L 바이오리액터에 발효를 진행하였다. 그 결과, NDK유전자를 과발현시키지 않은pEGWTT(CO)/pVBCL와 비교하여 코리네박테리움 글루타미쿰 유래의 NDK유전자가 과발현된 균주의 2’-‘FL생산량은 약 10g/L로 생산성이 53% 향상되었다. 또한 NDK효소활성 측정을 통해, NDK유전자의 발현도가 높을수록 2’-푸코실락토오즈의 생산성도 높아진다는 것을 증명하였다. 이 결과를 통해, 대량 발효에서도 NDK가 생산량에 영향을 주는 것을 증명하였고, 산업에서도 도움이 될 것이라고 생각한다.

목차 (Table of Contents)

• Ⅰ. INTRODUCTION 1
• Ⅱ. MATERIALS AND METHODS 6
• 1. Strains and plasmids 6
• 2. Primers 6
• 3. Culture media and fermentation conditions 13
• 4. Enzyme activity assay 16
• 5. Instrumental analysis 18
• Ⅲ. RESULTS AND DISCUSSIONS 20
• 1. Effect of nucleoside diphosphate kinase gene on 2'-fucosyllactose production in Corynebacterium glutamicum 20
• 1.1 Batch culture 20
• 1.2 mRNA expression of NDK gene 26
• 1.3 Quantity measurement of intracellular and extracellular 2’-fucosyllactose 28
• 1.4 ATP activity assay for proof of correlation ATP with NDK 30
• 1.5 Results of nucleoside diphosphate kinase assay 33
• 2. Fed-batch fermentation for improving effect of NDK gene on 2’-FL production in recombinant Corynebacterium glutamicum 35
• IV. CONCLUSIONS AND DISCUSSIONS 42
• 국문요약 44
• REFERENCES 46