Improvement of 1,2-propanediol production from methanol in metabolically engineered Methylorubrum extorquens AM1 by optimization of culture conditions

국문 초록 (Abstract)

메탄올은 온실가스의 주성분인 이산화탄소와 메탄으로부터 생산 가능하며 다양한 분야에 이용될 수 있어 화석연료 대체제로서 주목받고 있다. 또한 메탄올은 미생물 배양에 있어서 저렴하고 풍부하며 비식용 자원이라는 점, 배양 중 오염가능성을 줄이며 정제 공정이 용이하다는 점, 흔히 사용되는 당 기반 원료보다 환원도가 높아 생산 수율이 높다는 점에서 산업적으로 매우 유망한 원료이다. Methylorubrum extorquens AM1은 메탄올을 단일 탄소원으로 이용할 수 있는 메틸영양균으로, 이전 연구에서 메탄올로부터 1,2-프로판디올을 생산하도록 대사공학적으로 조작되었다. 본 논문에서는 개량된 M. extorquens AM1의 배양 조건을 최적화하여 1,2-프로판디올 생산을 향상시키고자 하였다. 첫번째로, 최적의 배양 온도와 교반 속도를 확립하였다. 두번째로, 질소원에 따른 세포 생장과 1,2-프로판디올 생산을 비교하여 요소가 최적의 질소원임을 확인하였다. 세번째로, 인산염 농도에 따른 pH 변화와 1,2-프로판디올 생산을 비교하여 30 mM의 인산염이 최적임을 확인하였다. 마지막으로, 과발현된 formate dehydrogenase를 통한 세포내 NADH의 재공급을 위해 배양 중 sodium formate를 첨가하였고, 그 결과, 10 mM의 sodium formate를 첨가했을 때 1,2-프로판디올 생산이 소량 증가하는 것을 확인하였다. 결론적으로, 본 논문에서는 개량된 M. extorquens AM1의 배양 조건을 최적화함으로써 4 g/L의 메탄올로부터 475 mg/L의 1,2-프로판디올을 생산하여 기존 배양 조건보다 1,2-프로판디올 생산이 52% 향상된 것을 확인하였다.

다국어 초록 (Multilingual Abstract)

Methanol can be produced from carbon dioxide and methane, the main components of greenhouse gases, and can be used in various industrial fields, so it is a promising fossil fuel substitute. Methanol is also an attractive feedstock for biotechnology such as microbial culture because it is abundant, relatively inexpensive, and not food-competitive. Moreover, it has the advantage of reducing the possibility of medium contamination and facilitating the downstream process and achieving high production yield because of a high degree of reduction per carbon compared to commonly used sugar-based feedstocks. Methylorubrum extorquens AM1 is a methylotroph that can utilize methanol as a sole carbon source and has been metabolically engineered to produce 1,2-propanediol from methanol in a previous study. In this study, the culture conditions of this strain were optimized to improve the production of 1,2-propanediol. First, the culture temperature and the agitation rate were optimized. Second, cell growth and 1,2-propanediol production according to the nitrogen source of the culture medium were compared, and it was confirmed that urea was the optimal nitrogen source. Third, the pH change and 1,2-propanediol production according to the phosphate concentration were compared, and the phosphate concentration was optimized to 30 mM. Finally, sodium formate was added to the medium during culture for regeneration of intracellular NADH through overexpressed formate dehydrogenase, and as a result, it was confirmed that 1,2-propanediol production was slightly increased when 10 mM of sodium formate was added. In conclusion, in this study, it was confirmed that 475 mg/L of 1,2-propanediol was produced from 4 g/L of methanol by optimizing the culture conditions of metabolically engineered M. extorquens AM1, which was improved by 52% compared to the culture conditions before optimization.