초록 ▼

Ⅳ. 연구개발결과
MEP 경로 8개, PTase 9개, 대사체 조절관련 후보 유전자 17종에 대한 용 데이터베이스의 전사체 데이터를 이용하여 조직/발달시기별 발현양상 및 각종 호르몬과 biotic/abiotic 스트레스에 따른 각 유전자들의 발현양상을 상관 분석하여 프로파일링을 실시 하였고, 컬러쌀을 이용한 MEP 유전자 과발현체들의 플랫폼 식물체로서의 유용성 분석한 결과, OsDXS 과발현 형질전환체에서 기존에 구축된 stPAC 단일카세트 형질전환체보다 차별적으로 증진된 카로티노이드 생산됨을 확인하였다. 또한, CCD 유전자

Ⅳ. 연구개발결과
MEP 경로 8개, PTase 9개, 대사체 조절관련 후보 유전자 17종에 대한 용 데이터베이스의 전사체 데이터를 이용하여 조직/발달시기별 발현양상 및 각종 호르몬과 biotic/abiotic 스트레스에 따른 각 유전자들의 발현양상을 상관 분석하여 프로파일링을 실시 하였고, 컬러쌀을 이용한 MEP 유전자 과발현체들의 플랫폼 식물체로서의 유용성 분석한 결과, OsDXS 과발현 형질전환체에서 기존에 구축된 stPAC 단일카세트 형질전환체보다 차별적으로 증진된 카로티노이드 생산됨을 확인하였다. 또한, CCD 유전자 4종 도입벼 형질전환체에서 향기 및 방충 효과를 지닌 β-ionone과 β-cyclocitral 생성을 확인함으로서 새로운 CCD 관련 기작을 동정하였고 시스템 생물학적 접근을 통한 터펜 대사 경로간의 상호네트워크를 분석 하였다. 터페노이드와 페닐프로파노이드 동반조절관련 주요 후보유전자 선발하여, in planta 시스템 기반 동반조절관련 유전자의 기능을 확인 하였고, 페닐프로파노이드계 물질생성 시스템 구축하였다. 미생물 대사공학을 이용하여 최종산물인 레티노이드의 발효공정상에서의 in-situ 추출을 위한 2-상배양 시스템을 개발하고 특허를 출원 및 등록하였다. 본 발명의 레티노이드의 in-situ 추출에는 세포벽 분해를 위한 리소자임이 필요하지 않다. 레티노이드(C20, 이소프레노이드 분자)는 세포벽의 손실 없이 세포로부터 효과적으로 방출될 수 있다. 레티노이드 생산의 2-상 배양에서, β-카로틴은 레티노이드의 직접적인 전구체이기 때문에 세포 내에 계속 유지되어야 한다. β-카로틴이 도데칸 상에서 추출되는 경우, 이는 세포내에 위치한 BCD(M)O에 의해 절단될 수 없다는 사실을 분석 하였고, 피부 친화적 친유성 물질을 포함하는 배지에서 레티노이드를 생산하였다.
벼 잎에서 보고된 터페노이드 standard를 UPLC-Q-TOF-MS를 이용하여 분석하였고, 결과 총 23개 터페노이드 standard 중 UPLC-Q-TOF-MS로 8개 터페노이드 검출한계, 정량한계 측정하였다. 또한, 터페노이드 standard를 GC-TOF-MS를 이용한 용매별 (dichloromethane, chloroform, methanol+chloroform) 분석을 실시한 결과 총 23개의 터페노이드 standard 중 GC-TOF-MS로 19개 터페노이드 검출한계, 정량 한계 측정되었고, α-pinene, limonene, α-cardinol 등의 물질이 분석되어 벼 2차대사산물의 활용가능성을 타진하였다. 한편으로, LC-IT-MS를 이용하여 아스퍼질러스 8개종의 2차대사산물 분석하여, 아스퍼질러스 렌툴루스의 종 특이적 대사산물인 네오사토린은 스타필로코커스에 활성을 보이는 것을 확인 하였고, UPLC-Q-TOF-MS를 이용하여 LED 조사 rice leaves의 2차 대사산물 분석 결과, Rice leaves 2차 대사산물 11개를 확인(Luteolin-6-C-glucosyl-8-C-arabinoside, isoorientin glucoside 계열 2종, apigenin-6-C-glucosyl-8-C-arabinoside, isovitexin glucoside 계열 2종, isoscoparin glucoside 계열 2종, Tricin, Tricin glucoside 계열 2종) 하였고, 항산화와 양의 상관관계가 있는 7개 물질을 확인(Luteolin-6-C-glucosyl-8–C- arabino side, isoorientin glucoside 계열 2종, isovitexin-2-O-glucoside, isoscoparin glucoside 계열 2종, tricin-O-glucoside)하였다. 또한, UPLC-Q-TOF-MS와 GC-TOF-MS를 이용하여 품종별 벼의 1차, 2차 대사체 동정한 결과, 벼의 2차 대사산물 12개(Cyanidin-3-glucoside, peonidin-3-glucoside, proanthocyanidin trimer, pronathocyanidin dimer, catechin, apigenin-6-C-glucosyl-8-C-arabinoside, tricin-O-rhamnoside- O-hexoside, pinellic acid, LysoPC 14:0, LysoPC 18:2, LysoPC 16:0, LysoPC 18:1) 와 1차 대사산물 26개(Valine, proline, serine, threonin, aspartic acid, GABA, glutamic acid, phenylalnine, asparagine, tryptophan, adenosine 아미노산 계열 11종, pyruvate, nicotinic acid, succinic acid, maleic acid, malonic acid, salicylic acid, citric acid 유기산 계열 7종, xylose, xylitol, fructose, glucose, gluconic acid, myo-inositol, sucrose, maltose 당 계열 8종) 이 확인되었고, 항산화와 양의 상관관계가 있는 4개 물질이(Cyanidin-3-glucoside,peonidin-3-glucoside, proanthocyanidin dimer, proanthocyanidin trimer) 확인 되었다. 한편으로, 대사공학으로 설계가 완성된 애기장대 식물(carotenoid cleavage product형)의 카로티노이드 분석을 통해 카로티노이드의 증감을 측정하였으며 의도한 효과를 스크리닝함으로서, 식물 시료의 HPLC 분석에 의한 카로티노이드 프로파일링 기술 확립하였고, GC-TOFMS 및 chemometrics tool을 이용하여 1차 및 2차 대사 경로 연결 부분 가시화 기술 체계 구축하였다.
또한, 이러한 식물/미생물 대사공학에 관한 연구를 바탕으로 다양한 조건에서 벼세포 대사흐름 예측 : 혐기/호기 조건하의 벼종자 발아, 정상 및 가뭄조건하의 광호흡 벼엽육 조직에서의 벼세포에 대한 대사/조절 네트워크 및 게놈수준의 인실리코 모델 개발하였고, 벼세포 인실리코 모델과 오믹스 분석을 통해 여러 가지 광원에서의 세포내 대사과정을 분석 하였으며, 인공유전자 합성을 위한 인실리코 디자인용 웹기반 툴을 개발하였다.

Abstract ▼

Terpenoid technology is powerful and extensible which can produce a number of high value compounds by combining stem technology producing IPP and branch technology consisting of target product pathway. Terpenoids has bulky market size (medical isoprenoids, carotenoids and its derivatives, US$ 2 bill

Terpenoid technology is powerful and extensible which can produce a number of high value compounds by combining stem technology producing IPP and branch technology consisting of target product pathway. Terpenoids has bulky market size (medical isoprenoids, carotenoids and its derivatives, US$ 2 billions; US$ 12 billions; isoprene, US$ 2 billions). Terpenoids are composed of IPP as building block is the largest compound class in nature, which have been known above 50,000. To improve terpenoid production based on in planta, it needs to understand interaction between other secondary metabolic pathway. Genes related in two different secondary metabolic pathway in plant were isolated by microarray analysis with rice grown under different light. Expression of transcriptional factor showed the positively correlation with structural genes involved in metabolism. Expression of putative transcriptional genes in plant will be able to give the knowledge to understand interaction between two different metabolic pathways.
Plants produce various isoprenoids with essential functions through the MEP pathway, including the major photosynthetic pigments (chlorophylls and carotenoids) as well as other key compounds related to photosynthesis (plastoquinones, phylloquinones and tocopherols), phytohormones (gibberellins and abscisic acid) and monoterpenes. Carotenoids, including in β -carotene, are essential components for human health as precursor of vitamin A. carotenoid degradation pathway involving carotenoid cleave dioxygenase (CCD) would also be promising targets for stabilizing and increasing the level of β-carotene in seeds using a combination of linkage mapping and a genome-wide association study. It identified the AtCCD4 gene as a major locus negatively regulating carotenoid levels in Arabidopsis seeds. To apply this concept to major food crops, we are investigating the functions of four CCD genes (AtCCD4, OsCCD1, 4a, 4b) in transgenic rice plants. Over-expression of four CCD genes resulted in the decrease of carotenoids and the increase/de novo biosynthesis of apocarotenoids as carotenoid cleavage products. Their transcriptomic and metabolomic DATA are being integrated to understand the in planta function of CCD genes. Furthermore, three RNAi-mediated CCD suppression lines (OsCCD1-Ri, 4a-Ri, 4b-Ri) were obtained and then crossed with β-carotene rice. Their enhancing effects on seed carotenoid levels via genetic manipulation of carotenoid catabolism will be analyzed with homozygous transgenic seeds soon. This study might be helpful to get the answer whether carotenoid degradation pathway can substantially influence to biofortification programs for seed crops or not. The MEP pathway is composed of eight essential enzymatic steps by DXS, DXR, IspD, IspE, IspF, IspG, IspH and IPI to produce sopentenyl diphosphate (IPP) and dimethyl allyl diphosphate (DMAPP) that a role as building blocks for the biosynthesis of diverse isoprenoid compounds, from pyruvate and D-glyceraldehyde 3-phosphate. Among 11 genes that have been collected to be responsible for the eight key enzymes based on GenBank database, 8 genes were isolated for functional studies of their major roles in rice. Terpenoids are synthesized from the C5-isoprene units, IPP and DMAPP, and classified into hemiterpenes (C5), monoterpenes (C10), sesquiterpene (C15) and diterpenes (C20), based on the number of C5 units. The first precursors of them are geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), respectively, and the synthesis of them is accomplished by basically condensing allylic pyrophosphate between IPP and DMAPP, and adding further IPP units on to the growing polymer. This basic polymerization reaction of isoprene units is catalyzed by prenyltransferases (PTase). In this study, 8 genes of PTases were identified based on GenBank database. For all of MEP–and PTase-genes, their expressed gene sequences expression patterns on various developmental stages and rice organs were characterized using Real-Time PCR and transcriptome database, and t e subcellular localization of those genes were characterized using GFP-fusion constructions. For the functional analysis, transgenic rice plants to over-express the genes were constructed and the T-DNA knock-out rice plants of them were analyzed. At the same time, the overexpression cassettes of the genes were cloned with the expression cassette for producing β-carotene in rice endosperm, and then the transgenic rice plants were also constructed and analyzed, in order to analyze which one was a determinant gene for the production of β-carotene. Based on the intensity of yellow color on endosperm, a rice DXS was shown to play an important role for enhancing the production of β-carotene, compared with other MEP genes. These results proposed that transgenic rice plants harboring the key enzymes of MEP pathway could be provided as the useful platform for metabolic engineering to produce or enhance the levels of functional isoprenoid-derived compounds in rice.
Terpenoids is very useful metabolites in terms of plants and animals. To improve terpenoid production based on in planta, it needs to understand interaction between other secondary metabolic pathway. From previous study, we had been isolated several transcription factor genes from rice including AP2, NAC, WRKY, and bZIP etc. To confirm the interaction between the transcription factors related in regulating two different metabolic pathway, Y2H assay was performed. From transient assay with protoplast, we selected the transcription factor that regulated terpenoids and phenypropanoids and carried out Arabidopsis transformation for identification of gene function. For identification of gene function in planta, transgenic plant overexpressing TF3 and TF13 gene showed different phenotype compared to wild type plant. From qRT-PCR analysis, TF3 plant displayed the different expression of genes related in GA biosynthesis (GA precusors and inactive GA synthesis). In TF13 plant, gene expression of GGPS, PSY and ZDS was remarkly upregulated. In the future, we will analyze the metabolites for GAs and terpenoids to confirm the TF function. To investigate cross-interaction between different metabolites, we developed phenylpropnoid derived anthocyanins accumulating plant. Transgenic Arabidopsis plants showed strong pigmentation in whole plant body and had high level of gene expression related in structural and regulatory genes of anthocyanins. In the future, we will conduct the transcriptome analysis to obtain the knowledge about the terpenoids biosynthetic mechanism based on phenylpropanoid production system.
Of terpenoids, retinoids can be produced from E. coli when introduced with the β-carotene biosynthesis pathway and the BCMO gene. E. coli has no inherent metabolic pathways related to retinoids, therefore only retinal should be produced from the cleavage of β-carotene by BCMO. However, retinol and retinyl acetate were also produced in significant amounts, by the non-specific activity of inherent promiscuous enzymes or the antibiotic resistance marker of the retinal-producing plasmids. Retinol was produced by the ybbO gene of E. coli which encodes oxidoreductase and retinyl acetate was produced by the chloramphenicol resistance gene, called cat gene which encodes chloramphenicol acetyltransferase, present within the pS-NA plasmid that also contains the mevalonate pathway. The composition of retinoids could be modulated by manipulating the relevant genes. The composition of retinol, a commercially important retinoid, was significantly increased by the overexpression of ybbO gene and the removal of cat gene in the recombinant E. coli, which suggests the possibility of selective retinoid production in the future. In order to prevent degradation of the intracellular retinoids through in-situ extraction from the cells, a two-phase culture system was performed for retinoids production. Several organic solvents, including n-alkanes, mineral oils and cosmetic raw materials, were applied as the extraction phase. Out of the n-alkanes, n-decane showed the highest retinoid production as 134.2 mg/l after 72 hours. For mineral oil, light and heavy mineral oil showed retinoid production of 158 mg/l and 174 mg/l after 96 hours, respectively. Out of the cosmetic raw materials, isopropyl myristate showed the highest retinoid production of 181 mg/l. These results indicate that many kinds of oils can be applied for retinoid production, and optimization of the in-situ extraction process will lead to further improve of economical production for the industrial purpose.
The suitable methods for analysis of various triterpenoids by comparing the sensitivity of detection of ultra-performance liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and gas chromatography (GC)‑TOF-MS was evaluated. Standard terpenoid compounds, including nine mono-, six sesqui-, four di-, and three triterpenoids, were used. To find the most suitable method for analysis of a wide range of various terpenoids using GC-TOF-MS and UPLC-Q-TOF-MS, we used three dissolvents and three mobile phases to compare the detection number of terpenoids. Using dichloromethane as the dissolvent for GC-TOF-MS analysis and 5mM ammonium acetate in water and methanol as the mobile phases for UPLC-Q-TOF-MS analysis were most suitable for analyzing diverse terpenoids. The limit of detection (LOD) and limit of quantification (LOQ) of each terpenoid compound were measured, and reasonable linearity (r² > 0.99)andprecision(RSD <20%)wereobtained.GC-TOF-MS was more sensitive in detecting volatile mono- and sesquiterpenoids (LOD 0.05‑10 μg/mL), whereas UPLC-Q-TOF-MS was more sensitive in detecting loganin, abscisic acid, gibberellic acid, aconitine, β-amyrin, α-amyrin, and lupeol (LOD 0.01‑3.8 μg/mL). Mono- and sesquiterpenoids in tangerine extracts were well detected and quantified with our method. Thus, we suggest that a combination of mass spectrometry techniques is needed to analyze various terpenoid compounds. Nine varieties of pigmented rice (Oryza sativa L.) seeds that were black, red, or white were used to perform metabolite profiling by using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and gas chromatography (GC) TOF-MS, and to measured antioxidant activities. Clear grouping patterns determined by the color of rice seeds were identified in principle component analysis (PCA) derived from UPLC-Q-TOF-MS. Cyanidin-3-glucoside, peonidin-3-glucoside, proanthocyanidin dimer, proanthocyanidin trimer, apigenin-6-C-glugosyl-8-C-arabiboside, tricin-O-rhamnoside-O-hexoside, and lipids were identified as significantly different secondary metabolites. In PCA score plot derived from GC-TOF-MS, Jakwangdo (JKD) and Ilpoom (IP) species were discriminated from the other rice seeds by PC1 and PC2. Valine, phenylalanine, adenosine, pyruvate, nicotinic acid, succinic acid, maleic acid, malonic acid, gluconic acid, xylose, fructose, glucose, maltose, and myo-inositol were significantly different primary metabolites in JKD species, while GABA, asparagine, xylitol, and sucrose were significantly distributed in IP species. Analysis of antioxidant activities revealed that black and red rice seeds had higher activity than white rice seeds. Cyanidin-3-glucoside, peonidin-3-glucoside, proanthocyanidin dimers, proanthocyanidin trimers, and catechin were highly correlated with antioxidant activities and were more contents in black and red rice seeds. These results are expected to provide valuable information that could help improve and develop rice-breeding techniques. Gas-chromatography coupled with time-of-flight mass spectrometry (GC-TOFMS) was used to analyze the relationships between primary metabolites and phenolic acids in rice (Oryza sativa L.). A total of 52 metabolites were identified, including 45 primary metabolites and seven phenolic acids from rice seeds. The individual phenolic acids were identified by GC-TOFMS including tert-butyldimethylsilyl (TBDMS) derivatization. Furthermore, we investigated the application of HPLC techniques to the screening of carotenoids in plants. The GC-TOFMS-based metabolic profiling and carotenoid profiling approach could be used as an alternative method to predict food quality and identify metabolic links in complex biological systems.
Also, we reconstructed metabolic network of model crop plant, Rice (Oryza sativa) cell, to understand their metabolic characteristics and applied it to identify essential genes/reactions of the rice photorespiration as well as metabolic and transcriptional regulatory mechanism underlying the anoxic adaptation of rice coleoptile. We also devised computational codon optimization method for de novo gene synthesis as key synthetic biological tools and developed the online tool. Finally, systems biotechnological strategy was presented for characterizing various cell factories by combined in silico modeling and omics analysis; its applicability was successfully demonstrated via microbial and mammalian cells. Thus, the current project allowed us to establish necessary components for building virtual systems and synthetic biology platform and tools for terpenoid production. As outcome of this project, seventeen papers were published in relevant biotechnology, systems biology and bioinformatics journals.
Taken all technologies together above, development of platform technology can be established for production of all kind of high value added terpenoids by using plant/microbe metabolic engineering.