Biological production of C5-C8 medium chain fatty acids and in-vivo evaluation of key enzymes

Abstract

Depletion of fossil fuel and climate change are the largest issues in worldwide. These issues have been deteriorating since industrial revolution. As solutions to overcome these issues, alternatives like bioethanol have been being utilized in several developed and developing countries since 1990s. Despite of these efforts, it seems difficult to replace all part of fossil fuels. Recently, several researchers in field of environmental engineering are trying to produce C5-C8 carboxylic acids from chemicals containing lower carbon atoms like ethanol, acetate, lactate and propionate. C5-C8 carboxylic acids produced biologically would be converted into various liquid fuels and chemicals. In this thesis, bacteria producing C5-C8 carboxylic acids were isolated newly from cow rumen. The strain was characterized in physiological level and evaluated for production performance. Interestingly, the strain extended the carbons of external carboxylic acids. Addition of acetate increased production of hexanoic acid and octanoic acid. Addition of propionate increased production of pentanoic acid and heptanoic acid. Especially, medium composition for C6 carboxylic acid, hexanoic acid, among C5-C8 carboxylic acids was optimized using statistical methods. The isolated strain produced hexanoic acid to 10.6 g/L. When products removal system was used, the strain produced hexanoic acid to 140 g/L. In order to investigate reasons for carbon elongation of supplemental carboxylic acids in genomic level, their whole genomes were analyzed completely and key enzymes for hexanoic acid production were screened. RNA expression levels of each gene were compared through transcriptomic analysis, which indicated that selected genes played significant role during carbon elongation of supplemental carboxylic acids. To confirm metabolic pathway based on selected genes, selected genes expressed heterogeneously in Escherichia coli. Finally, metabolic pathway of C5-C8 carboxylic acids was constructed. The most important enzyme in the pathway, thiolase was compared with other thioloases(bktB, CacThl, and AtoB) related to acyl-CoA condensation reaction of fatty acid synthesis. When the thiolase from isolated strain were evaluated. The thiolase showed the best performance for hexanoic acid production. These results would be utilized for follow up studies and commercial production of C5-C8 fatty acid.|화석연료의 감소와 기후변화에 대한 전 지구적 문제는 가속화 되고 있다. 이러한 문제를 해결하기 위한 다양한 방법들이 제안되고 있고, 그 해결책으로서 재생 가능한 에너지자원들이 사용되고 있지만 화석연료로부터 공급되고 있는 모든 원료자원을 대체하기는 어려워 보인다. 이러한 원료자원을 대체하기 위하여 바이오 기반의 연료 및 원료 물질 생산에 대한 연구가 필요하다. 전세계적으로 C2-C4 유기산 및 알코올을 생산하는 연구는 많이 진행되었으며, 이중 C2 알코올인 에탄올은 가솔린과 함께 사용되고 있다. 하지만 이러한 원료물질들이 가지고 있는 적은 탄소수로 인해 화석연료 기반으로 만들어진 플랫폼을 일부 전환해야 하는 단점을 가지고 있다. 본 연구에서는 저등급 탄소 화합물을 이용하여 좀 더 연장된 C5-C8 유기산을 미생물로부터 생산하는 방법을 개발하였다. 본 학위 논문의 목표를 달성하기 위하여 혼합미생물로부터 C5-C8 유기산을 생산하는 균주를 분리하는 방법을 개발하였으며, 이로부터 C5-C8 유기산을 고농도로 생산하는 Megasphaera sp. MH 균주를 분리하였다. 이 균주의 C5-C8 유기산생산 성능을 평가함과 동시에 전 유전체와 RNA 수준에서의 발현양은 비교 평가되었다. 이러한 연구를 통해 핵심 유전자를 선택할 수 있었으며, C5-C8 유기산 생산에 대한 대사경로를 확보할 수 있었다. 선택된 유전자를 바탕으로 대장균에서 C6 유기산을 생산하는 대사경로를 재구축하여, 유기산 생산 플랫폼 미생물을 개발하였으며 이를 통해 유기산 생산 대사경로의 첫 번째 핵심 유전자인 Thiolase를 비교 평가할 수 있었다. 이 유전자의 단백질 구조를 다른 구조들과 비교하여 기질결합에 영향을 미치는 핵심 아미노산 잔기를 선택할 수 있었다. 이 핵심 아미노산 잔기는 기질이 결합하는 포켓의 크기에 영향을 주었으며, Megasphaera sp. MH 로부터 유래한 Thiolase의 포켓크기는 상대적으로 더 큰 것으로 나타났다. 이러한 유전자를 보유한 Megasphaera sp. MH 균주를 바탕으로 대표적 저등급 유기산인 acetate와 butyrate를 이용하여 C5-C8 유기산 생산을 시도하였으며, 추출 조건과 배양조건을 최적화하여 생산물을 C6 유기산인 hexanoic acid로 단순화 시킬 수 있었다. Megasphaera sp. MH에 추출발효를 적용하여 10일동안 140 g L-1의 hexanoic acid생산을 달성할 수 있었다. 이러한 배양 방식과 대사경로 및 핵심 유전자는 산업적 유기산 생산과 후속연구에 사용될 것으로 기대된다.; Depletion of fossil fuel and climate change are the largest issues in worldwide. These issues have been deteriorating since industrial revolution. As solutions to overcome these issues, alternatives like bioethanol have been being utilized in several developed and developing countries since 1990s. Despite of these efforts, it seems difficult to replace all part of fossil fuels. Recently, several researchers in field of environmental engineering are trying to produce C5-C8 carboxylic acids from chemicals containing lower carbon atoms like ethanol, acetate, lactate and propionate. C5-C8 carboxylic acids produced biologically would be converted into various liquid fuels and chemicals. In this thesis, bacteria producing C5-C8 carboxylic acids were isolated newly from cow rumen. The strain was characterized in physiological level and evaluated for production performance. Interestingly, the strain extended the carbons of external carboxylic acids. Addition of acetate increased production of hexanoic acid and octanoic acid. Addition of propionate increased production of pentanoic acid and heptanoic acid. Especially, medium composition for C6 carboxylic acid, hexanoic acid, among C5-C8 carboxylic acids was optimized using statistical methods. The isolated strain produced hexanoic acid to 10.6 g/L. When products removal system was used, the strain produced hexanoic acid to 140 g/L. In order to investigate reasons for carbon elongation of supplemental carboxylic acids in genomic level, their whole genomes were analyzed completely and key enzymes for hexanoic acid production were screened. RNA expression levels of each gene were compared through transcriptomic analysis, which indicated that selected genes played significant role during carbon elongation of supplemental carboxylic acids. To confirm metabolic pathway based on selected genes, selected genes expressed heterogeneously in Escherichia coli. Finally, metabolic pathway of C5-C8 carboxylic acids was constructed. The most important enzyme in the pathway, thiolase was compared with other thioloases(bktB, CacThl, and AtoB) related to acyl-CoA condensation reaction of fatty acid synthesis. When the thiolase from isolated strain were evaluated. The thiolase showed the best performance for hexanoic acid production. These results would be utilized for follow up studies and commercial production of C5-C8 fatty acid.