초록 ▼

본 연구개발은 지구온난화의 주범이며 산업화에 의한 화석연료 사용량의 증가에 따라 배출량이 증가하고 있는 CO2를 고부가가치 화합물로 전환하는 생물학적 공정개발을 목표로 함. 본 단계에서 Moorella thermoacetica 균주를 활용하여 배지 조성 및 배양 조건 최적화를 통하여 이산화탄소 대사 균주를 개발함. 더 나아가, 이산화탄소 전환 균주의 보다 심도깊은 이해를 위하여, 시스템 level의 분석을 진행하였음. 유전자 서열(whole genome)과 기존 보고된 서열의 차이점 존재 유무를 판단하기 위하여 resequencin

본 연구개발은 지구온난화의 주범이며 산업화에 의한 화석연료 사용량의 증가에 따라 배출량이 증가하고 있는 CO2를 고부가가치 화합물로 전환하는 생물학적 공정개발을 목표로 함. 본 단계에서 Moorella thermoacetica 균주를 활용하여 배지 조성 및 배양 조건 최적화를 통하여 이산화탄소 대사 균주를 개발함. 더 나아가, 이산화탄소 전환 균주의 보다 심도깊은 이해를 위하여, 시스템 level의 분석을 진행하였음. 유전자 서열(whole genome)과 기존 보고된 서열의 차이점 존재 유무를 판단하기 위하여 resequencing을 거쳤으며, 1곳의 특이서열을 발굴함. 또한, Flavonoid 및 GAG 고생산 균주의 스크리닝을 위하여 목적화합물 특이적 바이오센서를 개발 및 개량하였음. 본 연구에서 최종적으로 연구팀이 구축한 시스템으로부터 아세트산을 기반으로 Flavonoid 및 GAG의 성공적으로 생합성되는 것을 확인하였으며, 대사공학, 합성생물학 전략을 통해 생산성을 최적화 하였음.

(출처 : 보고서 요약서 3p)

Abstract ▼

□ Purpose
CO₂ is considered as one of major factors of global warming and whose concentration in atmosphere is currently increasing. Especially, industrial waste gas and thermal power generation contributes significant proportion of anthropogenic CO₂ emission. In this research, we are going to de

□ Purpose
CO₂ is considered as one of major factors of global warming and whose concentration in atmosphere is currently increasing. Especially, industrial waste gas and thermal power generation contributes significant proportion of anthropogenic CO₂ emission. In this research, we are going to develop a biological process that convert CO₂ to value-added chemical products. First, whole bioprocess will be designed as two-stage conversion. In the first stage, CO₂ will be converted to acetate which will be further converted to various value-added compounds in the second stage. Specialized microbial strains will be developed for each stage. In particular, we are going to develop a strain that can efficiently utilize CO₂ as a sole carbon source and produce a large amount of acetate. Also, microbial strains that converts acetate into diverse flavonoids and glycosaminoglycans will be constructed. Then, two-stage bioprocess will be optimized to improve the productivity and yield of the process.

□ contents
ㅇ Microbial strain for efficient conversion of CO₂
We are going to develop biological process which converts CO₂ in industrial waste gas into soluble, multi-carbon substrate that can be utilized by Escherichia coli. First, natural CO₂-fixing microorganisms will be screened for their conversion rate. Then, we will apply adaptive evolution to highly efficient parental strains to further improve their growth rate and acetate production. Also, we will devise synthetic biology tools which enable manipulation of CO₂-fixing microbes.
ㅇ High-throughput screening tool and dynamic regulation system
Genetic biosensors will be constructed that specifically detects the target value-added products. To accomplish the goal, we will explore natural biosensors including riboswitches and transcription factors. Also, artificial sensors will be developed through SELEX. The sensors will be introduced to producer E. coli strains for high-throughput screening and dynamic regulation of metabolic pathways.
ㅇ Microbial strains that produce various value-added products from acetate
Metabolic pathways will be redesigned including acetate assimilation pathway and biosynthetic pathways for the target metabolites. Moreover, high-throughput screening device will be applied to optimize strain performance and enhance yield and productivity. Finally, we will introduce dynamic modulation system to further improve the producer strains.
ㅇ Efficient process for conversion of CO₂ and biosynthesis of value-added products
With systems biological analysis of bacteria in bioreactor, candidate metabolites affecting chemical production will be identified. This information could be utilized in another round of engineering of CO₂ fixation/biochemical production strain. Finally, parameter study will be conducted to develop a stable production process through continuous monitoring.

□ Developement results
ㅇ Media and culture conditions were optimized for cultivation of Moorella thermoacetica. Specifically, the initial carbon source, the buffer solution, and the cultivation methods were optimized. As a result, 15.6 g/L acetate was produced from 112 hr cultivation using CO₂ as a sole carbon source. Using this optimized conditions, adaptative evolution is now underway to improve CO₂ fixation rate.
ㅇ Systems level analysis was conducted to attain a deeper understanding of gene regulation pattern in M. thermoacetica. Whole genome of the current strain was resequenced to check whether there are differences between the genome sequence of the strain and that of the previously reported strain. We identified 1 different sequence between them. Additionally, RNA transcripts were sequenced to understand metabolic fluxes required for CO₂ fixation.
ㅇ Biosensors for the traget products were developed and improved. First, specificity of the RNA-based naringenin biosensor was studied, and its application to the producer strain was validated. Furthermore, transcription factor-based biosensors for flavonoids were also developed. For the detection of GAG, a UDP-glucose-dependent biosensor module was found from literature search. Finally, an acetate-specific biosensor was developed.
ㅇ Flavonoid and GAG were produced from engineered E. coli strains using acetate as a carbon source. We first engineered a L-tyrosine producer strain that utilizes acetate. L-Tyrosine is an important precursor for synthesis of flavonoids. This strain produced 320 mg/L L-tyrosine using acetate as a sole carbon source. Furthermore, it was confirmed that p-coumaric acid. This strain produced 91.98 mg/L of naringenin. For GAG, it was confirmed that more than 20 mg/L of condroitin could be produced using acetate as a sole carbon source. Furthermore, E.coli with enhanced availability of PAPS was developed for the biosynthesis of highly sulfated GAGs.

□ Expected Contribution
This research will provide genuine technologies which enable economic conversion of CO₂ to diverse value-added products. The outcome of this research will complement existing technologies in which CO₂-fixing microbes are directly engineered, and thus repertoire of products was limited. Two-stage bioconversion process will be established based on specialized microbial strains. The diversity of products from CO₂ will easily be increased depending on the microbial strain in the second stage. Additionally, biosensors and dynamic regulators will be applied to many different bioprocesses for chemical production, increasing proportion of bio-based production in contemporary chemical industry.

(출처 : SUMMARY 6p)

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 보고서 요약서 ... 3
• 요 약 문 ... 4
• SUMMARY ... 6
• 목차 ... 8
• 1. 연구개발 목표 및 내용 ... 9
• 가. 최종 목표 ... 9
• 나. 단계목표(당초 목표 및 수정․보완 목표) ... 9
• 다. 당초 목표의 수정․보완(중요 연구변경) 사유 ... 9
• 라. 1단계 연차별 연구목표 및 내용 ... 9
• 2. 연구 추진전략 및 방법 ... 10
• 3. 주요 연구개발 결과 ... 10
• 가. 목표달성도 ... 10
• 나. 대표적 연구업적 ... 33
• 4. 연구결과의 활용계획 ... 38
• 5. 연구개발성과 현황 ... 39
• 6. 국가과학기술종합정보시스템에 등록한 연구시설․장비 현황 ... 67
• 7. Final report for 1st Phase of GRL ... 68
• I. Report ... 68
• II. Budget Report ... 78
• III. Attachments ... 78
• 끝페이지 ... 79