초록 ▼

1. 국내의 환경오염 토양의 메타게놈 추출 및 라이브러리 작성 : 총 4,900 메가 염기쌍 (123,900 클론)의 라이브러리를 작성하여 26,200 클론 (1,048 Mb 염기쌍)을 사업단에 기탁 (평균 삽입 DNA 35-40 kb)
2. Quorum sensing 조절 클론 확보 : AHL 분해 유전자원 2종 (ELP104, EST33), 3-OH PAME 분해 유전자원 4종 (LP86, LP96, ELP104, EST33), chloramphenicol 재활성과 분해 관련 유전자원 2종 (DL26, DL136), 총 8

1. 국내의 환경오염 토양의 메타게놈 추출 및 라이브러리 작성 : 총 4,900 메가 염기쌍 (123,900 클론)의 라이브러리를 작성하여 26,200 클론 (1,048 Mb 염기쌍)을 사업단에 기탁 (평균 삽입 DNA 35-40 kb)
2. Quorum sensing 조절 클론 확보 : AHL 분해 유전자원 2종 (ELP104, EST33), 3-OH PAME 분해 유전자원 4종 (LP86, LP96, ELP104, EST33), chloramphenicol 재활성과 분해 관련 유전자원 2종 (DL26, DL136), 총 8종의 quorum sensmg 조절 클론 확보
3. Quorum sensing 조절 클론의 유전자 분석 : 탐색 선발한 유전자원 중 3-OH PAME의 분해 효소는 2종 (ELP104, EST33)은 새로운 family에 속하는 지질 분해 효소, AHL 분해 관련 효소 EstD2 유전자는 신규 유전자로 논문 발표 (AMB, 2010)
4. 활성 유전자의 quorum sensing 조절 기능 결정 : 선발 8종 유전자원 중 7종을 단백질로 순수 정제하고 3-OH PAME 분해, AHL 분해, chloramphenicol 재활성 및 분해 관련기능 확인하고 3-OH PAME 분해 효소 3종 (LP86, ELP104, EST33)을 Ralstonia solnacearum에 형질전환하여 quorum sensmg 조절여부를 표현형과 유전자의 발현양상을 분석
5. 왁스합성 관련 WES 단백질 유전자 클론 확보 및 대장균 기능 결정 : 기대치 않은 성과로 왁스합성 관련 WES 단백질 유전자를 신규 유전자로 확보, 대장균과 애기장대 내에서 와스 합성 과정을 통한 유전자의 기능 검정
(출처 : 보고서 요약서 4p)

Abstract ▼

IV. Results
1. Construction of metagenomic library
Metagenomic DNA was isolated from alluvial soil of Eulsuk do Island at Nakdong River basin and industrial contaminated wastes oil nearby sasang industrial complex area. Isolated metagenome DNA was estimated as more than 20 kb through compariso

IV. Results
1. Construction of metagenomic library
Metagenomic DNA was isolated from alluvial soil of Eulsuk do Island at Nakdong River basin and industrial contaminated wastes oil nearby sasang industrial complex area. Isolated metagenome DNA was estimated as more than 20 kb through comparison with calculated standard and used for metagenomic library construction. Metagenomic library consisted of approximately 45,300 clones corresponding to 1,800 Mb of microbial genome from alluvial soil of Eulsuk do Island and approximately 78,600 clones corresponding to 3,100 Mb of microbial genome from industrial contaminated waste soil nearby sasang industrial complex area.

2. Screening of metagenomic clones with quorum sensing regulation
Metagenomic clones with quorum sensing regulation were not found using co-culture depending on violacein expression of Choromobacterium νiolaceum strain that produce limited chain length of AHL. Metagenomic clones with quorum sensing regulation were not found us ing co-culture with a reporter strain Agrobacterium tumefaciens NT1 producing variety chain length of AHL, either. The positive clones were selected using co-culture with metagenomic library clones depending on GFP expression of Escherichia coli JB525 by fluorescent activated cell sorter (FACS). But the positive clones could not be recovered on culture. Classical screening of lipolytic clones generated seventeen clones with lipid autoinducer removal using Ralstonia solanacearum producing 3-OH PAME as autoinducer. The unique four clones carrying hydrolyzing activity toward 3-OH PAME were selected by secondary library construction and subcloning. The lipolytic clones showed degradation activity towards AHL, designated EstD2 and ELP104, were also selected from the lipolytic clones which could hydrolyze the chemical structure of lactone ring.
Additionally, the two clones, DL26 and DL136, associated with chloramphenicol metabolism were also selected in the progress which is screening of active clones hydrolyzing zearalenone as mycotoxin producing in pathogenic Fusarium species.

3. Characterization of metagenomic clones with alltoinducer degradation activity
Four lipolytic clones which showed degradation activity towards 3-OH PAME, were obtained throllgh gas chromatography. And then, four subclones containing only the liypolytic gene were selected depending on 3-OH PAME degradation activity. DNA nucleotide sequences of the selected four clones had low level identities to Published nucleotide sequences. Three proteins were over-expressed from three genes of selected clones after cloning into pET-30b. Over-expressed proteins were purified using Ni-column and FPLC for assay of 3-OH PAME hydrolysis by GC. Also, the active genes were transformed into R. solanacearum using pRK415 as a broad host range vector for estimation of qllorllm sensing regulation. Biofilm formation due to exopolysaccharide production in R. solanacearum strains carrying the active genes was dramatically reduced. Expression of the genes involved in EPS production was also dramatically down-regulated in R. solanacearum strains carrying the active genes. Two prote ins with AHL hydrolysis, LP2 and LP104, were also selected based on the decrease in GFP expression by co-culture with E. coli JB525 and C₆-HSL.

4. Characterization of metagenomic clones carrying chloramphenicol reactivation and degradation activity
We chose subclones, called DL26 and DL136, which showed chloramphenicol reactivation and degradation activity from metagenomic library. Deduced amino acid sequences of EstDL26 and EstDL136 revealed that they are novel esterases. Es tDL26 and EstDL136 showed similarity to many esterases of the microbial HSL family. Alignments of EstDL26 and EstDL136 with similar proteins located a conserved GxSxG motif at the active site. Over-expressed and purified EstDL26 and EstDL136 showed high activity toward short-chain p-NP esters, suggesting that both are esterases. Otherwise, EstDL26 and EstDL136 both exhibited mesophilic property. Thus, EstDL26 was incapable of deacetylating the C1 acetyl as it deacetylated 3-acetyl BCAM to BCAM, and 1,3-diacetyl BCAM to 1-acetyl BCAM, indicating a potential regioselectivity. On the other hand, EstDL136 showed strong CAE activities toward both C1 and C3 acetyls that completely hydrolyzed all BCAM acetates to BCAM. The results with LC-MS and NMR suggested that the structures of Cm hydrolysate and p-nitrophenylserinol are identical therefore, we concluded that the hydrolysate of Cm is p-nitrophenylserinol, which is generated from hydrolyzing the amide linkage of Cm. The hydrolysis of florfenicol by EstDL136 over time was also apparent through HPLC analysis. Both in vivo and in vitro, EstDL136 hydrolyzed Cm and Ff, and generated functionally inactive derivatives, suggesting that EstDL136 can cause amphenicol antibiotics to be ineffective. To investigate this hypothesis, a susceptibility test was performed with E. coli DH5 a carrying pUEst136 and we found that E. coli carrying pUEst136, was tolerant to the normally effective concentration (15μg/ml) of Cm and Ff. Although E. coli DH5 a carrying pUC119 was susceptible to Cm and Fffor the longest incubation period in this study, E. coli carrying pUEstl36 was recovered following antibiotic exposure (16-128μg/ml) of a variable duration. DNA sequence analysis of the 14.6 kb of pDL136 revealed the presence of 14 different putative orfs including estDL136, based on their DNA sequences and deduced amino acid sequence similarity to known genes in GenBank. Most orfs located near to estDL136 encoded proteins that were similar to many functional enzymes, including amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxygenase.

5. Characterization of me tag enomic clones carr ying wax sy nthase activity
A gene encoding wax ester synthase (WES) was also obtained during screening of metagenomic clones with activity for quorum sensing regulation. Deduced amino acid sequences of wes gene revealed that the gene encode novel wax synthase. The wes gene was transformed into E. coli for estimation of production of wax ester. We confirmed that E. coli carrying wes gene produced unique several wax esters by GC, when appropriate intermediates were supplemented in the culture. We introduced the gene wes into Arabidopsis plants to express wax ester synthase gene in plants. Six transgenic Arabidopsis lines were selected by RT-PCR. Four lines of selected transgenic Arabidopsis lines produced wax esters similar with wax ester produced in E. coli carrying wes gene. As results of the performing drought treatment and evapotranspiration calculation, transgenic Arabidopsis exhibited the stronger tolerance and lower transpiration metabolism than wild type Arabidopsis.
(출처 : SUMMARY 11p)

목차 Contents

• 표지 ... 1
• 제출문 ... 3
• 보고서 요약서 ... 4
• 요약문 ... 5
• SUMMARY ... 10
• Contents ... 15
• 목차 ... 16
• 제 1 장 연구개발과제의 개요 ... 17
• 제 1 절 연구개발의 필요성 ... 17
• 제 2 장 국내외 기술개발 현황 ... 22
• 제 1 절 국외 타 연구기관의 연구개발 실적 ... 23
• 제 2 절 국내 타 연구기관의 연구개발 실적 ... 24
• 제 3 장 연구개발수행 내용 및 결과 ... 25
• 제 1 절 메타게놈 라이브러리 구축 ... 26
• 제 2 절 Quorum sensing 조절활성 클론 확보 및 유전자의 분석 ... 28
• 제 3 절 Autoinducer 분해 활성 클론 분석 ... 32
• 제 4 절 Chloramphenicol reactivation 및 분해활성 클론 분석 ... 39
• 제 5 절 Wax 합성관련 메타게놈 클론 분석 ... 50
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 58
• 제 5 장 연구개발결과의 활용계획 ... 60
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 61
• 제 7 장 연구시설·장비 현황 ... 63
• 제 8 장 참고문헌 ... 64
• 끝페이지 ... 69