학술논문
Microbial production of squalene using CRISPR/Cas system-engineered Corynebacterium glutamicum / 크리스퍼-카스 시스템을 이용한 코리네박테리움 글루타미쿰 균주의 스쿠알렌 생산
Document Type
Dissertation/ Thesis
Author
Source
Subject
Language
English
Abstract
스쿠알렌은 탄소 30개로 이루어진 불포화 탄화수소로 상어 간유, 올리브 등에 많이 함유되어 있다고 알려져 있다. 스쿠알렌은 환원력이 강력한 물질로 체내 활성산소와 결합하여 세포의 산화 및 손상을 예방해주는 효능이 있어 미용보조제, 건강보조식품, 면역 및 백신보조제등으로 이용되고 있다. 이러한 기능성으로 스쿠알렌의 세계시장은 지속적으로 증가하고 있다. 증가하는 수요에 따른 공급이 필요하며 스쿠알렌은 상어 간유로부터 높은 수율과 순도로 얻을 수 있지만 상어의 포획이 제한됨에 따라 상어 간유로부터의 스쿠알렌 추출이 어려워지게 되었고, 식물소재의 스쿠알렌은 추출법에 따른 수율과 순도가 낮다고 보고되어왔다. 따라서, 본 연구에서는 스쿠알렌의 효율적이며 지속적인 생산을 위해 산업적으로 아미노산을 대량 생산하는 미생물 균주로 알려진 코리네박테리움 글루타미쿰 균주를 이용하여 최신유전자 편집 및 응용기술인 크리스퍼/카스 시스템을 본 균주에 접목시켜 고효율의 스쿠알렌 생산을 목적으로하였다. 먼저, 비메발론산 경로(MEP pathway)의 과발현 및 스쿠알렌 생산효소 도입을 통해 스쿠알렌 생성능을 가진 코리네박테리움 글루타미쿰 기본 균주를 최초로 구축하여 23.9 ± 2.29 mg/L의 스쿠알렌 생산량을 확인하였다. 또한, 유전자 편집 응용기술인 CRISPR/dCas9 기술을 적용한 균주의 고효율 배양을 통해 스쿠알렌 생성능이 향상된 균주를 스크리닝 하였다. 스쿠알렌 생성능이 가장 향상된 균주는 스쿠알렌 생산 경로와 경쟁하는 경로를 블로킹하기 위해 idsA 유전자를 저해한 균주로 확인하였다. 해당 균주는 105.3 ± 3.0 mg/L의 스쿠알렌을 생산하였으며 기본 균주 대비 약 5.2배 스쿠알렌 생산량이 증가하는 결과를 확인하였다. 추가적인 스쿠알렌 생산 우량균주를 제작하기 위하여 스쿠알렌 합성효소의 탐색 및 단백질 공학, 발현량 최적화를 진행하였고, 유전자 편집 기술인 CRISPR/Cas12a 기술을 적용하여 스쿠알렌 생산 대사경로를 최적화하는 연구를 진행하였다. 이를 통해, 고효율의 스쿠알렌 생성능을 가지는 우량균주를 구축하였고, 해당 균주의 5L lab-scale 유가배양을 통해 1406 ± 16 mg/L의 스쿠알렌 생산량을 확인하였다. 마지막으로,효율적인 바이오프로세스를 위해 소수성 물질인 스쿠알렌의 특성을 고려한 오일-오버레이 발효공정법을 설계하여 발효액으로부터 효과적인 스쿠알렌 추출 및 분리 방법을 제시하였다. 또한, 추출된 미생물유래 바이오스쿠알렌의 나노에멀젼 형성 및 그 성능을 평가하였고, 아미노산 대량생산 균주인 코리네박테리움 글루타미쿰의 라이신과의 코-프로덕션 전략을 도입하면서 스쿠알렌을 생산하는 코리네박테리움 글루타미쿰 균주의 산업적인 가능성을 보여주었다.
Microbial production of squalene has driven caused by the illegal harvesting from shark liver and the limitation of low-yielding bioprocesses from plants. To broaden its utilization of metabolic engineering in C. glutamicum, CRISPR/Cas technologies including CRISPRi and CRISPR/Cas12a system have been applied to microbial cell for rapid development of metabolic engineering in microbial cell factories. C. glutamicum were metabolically engineered to produce squalene from glucose by expressing truncated squalene synthase and over-expressing key enzymes (dxs, idi, and ispA) of MEP pathway. To enhance squalene production, combinatorial target genes were repressed by CRISPR-dCas9 for precursor rebalancing, redox balancing, and blocking the competing pathway. In addition, the idsA gene was deleted using the CRISPR-Cas12a assisted recombineering. As a result, the best squalene production strain was found with blocking competing pathway by repressing the idsA gene using high-throughput cultivation. Using this strain, 105.3 ± 3.0 mg/L squalene were analyzed, which was a 5.2-fold increase over the parental squalene production strain.To improve the production of squalene, protein engineering of the key enzymes was resulted in a 4.6-fold improvement of squalene production. To increase the total squalene production, a lactate-bypass was designed to increase the pyruvate level that can be reloaded from lactate utilization, by inactivating the pyruvate carboxylase. Fed-batch fermentation resulted in 1406 ± 16 mg/L of total squalene production (32% of tSQ in the medium). In addition, a biocompatible dodecane overlay were used to mitigation of intracellular squalene to the cell/dodecane-mixed layer, which occupied 87.7 % of tSQ in 10% of the culture volume. For its cosmetic applications, bio-squalene/dodecane nanoemulsions were formulated and analyzed. Moreover, for simultaneous production of L-lysine with squalene, L-lysine producing C. glutamicum strain was engineered to produce squalene by generating mutant using CRISPR/Cas12a system and repressing targeted gene using CRISPR/dCas9 system. Thus, this study could be useful for metabolic engineering of C. glutamicum to produce hydrophobic chemicals and other high valued chemicals.
Microbial production of squalene has driven caused by the illegal harvesting from shark liver and the limitation of low-yielding bioprocesses from plants. To broaden its utilization of metabolic engineering in C. glutamicum, CRISPR/Cas technologies including CRISPRi and CRISPR/Cas12a system have been applied to microbial cell for rapid development of metabolic engineering in microbial cell factories. C. glutamicum were metabolically engineered to produce squalene from glucose by expressing truncated squalene synthase and over-expressing key enzymes (dxs, idi, and ispA) of MEP pathway. To enhance squalene production, combinatorial target genes were repressed by CRISPR-dCas9 for precursor rebalancing, redox balancing, and blocking the competing pathway. In addition, the idsA gene was deleted using the CRISPR-Cas12a assisted recombineering. As a result, the best squalene production strain was found with blocking competing pathway by repressing the idsA gene using high-throughput cultivation. Using this strain, 105.3 ± 3.0 mg/L squalene were analyzed, which was a 5.2-fold increase over the parental squalene production strain.To improve the production of squalene, protein engineering of the key enzymes was resulted in a 4.6-fold improvement of squalene production. To increase the total squalene production, a lactate-bypass was designed to increase the pyruvate level that can be reloaded from lactate utilization, by inactivating the pyruvate carboxylase. Fed-batch fermentation resulted in 1406 ± 16 mg/L of total squalene production (32% of tSQ in the medium). In addition, a biocompatible dodecane overlay were used to mitigation of intracellular squalene to the cell/dodecane-mixed layer, which occupied 87.7 % of tSQ in 10% of the culture volume. For its cosmetic applications, bio-squalene/dodecane nanoemulsions were formulated and analyzed. Moreover, for simultaneous production of L-lysine with squalene, L-lysine producing C. glutamicum strain was engineered to produce squalene by generating mutant using CRISPR/Cas12a system and repressing targeted gene using CRISPR/dCas9 system. Thus, this study could be useful for metabolic engineering of C. glutamicum to produce hydrophobic chemicals and other high valued chemicals.