1) 지의류 형성 곰팡이의 whole genome sequencing ○ 차세대 염기서열 분석방법을 이용하여 전체 유전체 서열 확보 ○ 유전체 염기서열의 생물정보학적 분석을 통한 scaffold 생성, gene annotation(유전자 부위 결정) 및 지의류 유전체 특성 분석 ○ 생리활성물질 생합성 유전자(polyketide synthase 포함) annotation 및 다른 진균 유래 유전자와의 비교 분석 2) 유용 생리활성 물질 생합성 유전자의 과발현 시스템 확립 ○ 생합성 유전자의 발달단계 및 환경
1) 지의류 형성 곰팡이의 whole genome sequencing ○ 차세대 염기서열 분석방법을 이용하여 전체 유전체 서열 확보 ○ 유전체 염기서열의 생물정보학적 분석을 통한 scaffold 생성, gene annotation(유전자 부위 결정) 및 지의류 유전체 특성 분석 ○ 생리활성물질 생합성 유전자(polyketide synthase 포함) annotation 및 다른 진균 유래 유전자와의 비교 분석 2) 유용 생리활성 물질 생합성 유전자의 과발현 시스템 확립 ○ 생합성 유전자의 발달단계 및 환경조건에 따른 발현양상 정량 분석 (real-time RT-PCR) ○ 이형질전환체 생산을 위해 생합성 유전자 cDNA 과발현 벡터 제작 (5개 이상) ○ 모델 사상성 진균(Aspergillus sp.)을 이용하여 과발현 형질전환체 제작 및 생리 활성능 검정 후 선발(유전자 1-2개 이용) ○ 선발된 과발현 형질전환체에서의 생리활성물질 추출 및 동정
Abstract▼
Lichens are symbiotic organisms, principally composed of lichen-forming fungi (mycobiont) and algae or cyanobacteria (photobionts). Moreover, there are a variety of fungi that inhabit lichens, including the lichenicolous and endolichenic fungi. Lichen-forming fungi synthesize a broad variety of seco
Lichens are symbiotic organisms, principally composed of lichen-forming fungi (mycobiont) and algae or cyanobacteria (photobionts). Moreover, there are a variety of fungi that inhabit lichens, including the lichenicolous and endolichenic fungi. Lichen-forming fungi synthesize a broad variety of secondary metabolites that exert biological effects including antimicrobial, anti-inflammatory, analgesic, antipyretic, and antiproliferative and cytotoxic effects. Recently, interest has increased in the pharmaceutical properties of compounds derived from lichens. However, a major obstacle to the large-scale use of lichens is their limited availability, which is attributable to their slow growth and specific ecological requirements. Lichens produce unique polyketide secondary metabolites including depsides, depsidones, dibenzofurans, and depsones. The biosynthesis of these compounds is governed by polyketide synthase (PKS), but the mechanism via which they are produced has remained unclear until now. A cultured mycobiont of Cladonia metacorallifera was employed in the isolation and characterization of polyketide synthase genes. Whole genome sequence of the mycobiont was conducted for the first time to screen PKS genes. Total genome size was 34.7 Mb, and 10,815 genes were predicted from the mycobiont, which was quite different from the model filamentous fungus, Aspergillus nidulans. Total 34 PKS genes were annotated from the mycobiont. Among them, 8 reducing PKSs (CmPKS1, 3, 4, 6, 7, 9, 11, 14) and 2 non-reducing PKSs (CmPKS18, 36) were unique in lichen-forming fungi. We reported the 6-methylsalicylic acid synthase (6-MSAS) type of PKS gene, which is a member of the fungal-reducing PKSs. The domain organization of CmPKS1 (KS-AT-DH-KR-PP) is a typical 6-MSAS-type PKS and the results of phylogenetic analysis showed that CmPKS1 grouped with other fungal-reducing PKSs. Fungal reducing PKSs having 6-MSAS type architecture not only CmPKS1 but also has been found another fungal reducing PKS. Quantitative real-time RT-PCR analyses showed that CmPKS1 was expressed preferentially in the early growth stage of the axenically cultured mycobiont. Furthermore, CmPKS1 expression was found to be dependent on the carbon sources and concentrations in the medium. Full length sequences of cDNA of 5 CmPKS genes (1, 3, 7, 35, 36) was successfully cloned to develop transformants into Aspergillus nidulans. Heterologus expression of CmPKS1 and CmPKS35 in A. nidulans was confirmed by detecting unique picks of HPLC profile using cultural extracts of the transformants.
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