초록 ▼

Ⅳ. 연구개발결과
1. 당사슬 생합성 회로의 부품화와 위치 조립을 통한 인체형 당단백질 생산 숙주 개발
○ 만노스 인산화 효소 YlMpo1을 발굴하였으며, 이를 이용하여 당사슬 생합성 회로를 조작하여 효소 치료제의 라이소좀 타겟팅에 유용한 만노스-6-인산 부가 능력이 증대된 효모를 개발함
○ 재조합 발현한 YlMpo1을 이용해서 분자 수준에서 특성을 규명하고 이를 이용한 효과적인 in vitro 효소 반응공정을 개발함
○ 세포벽 형성에 중요한 Gas1 단백질과 GAS1 유전자가 결손된 효모 균주를 이용하여 분비

Ⅳ. 연구개발결과
1. 당사슬 생합성 회로의 부품화와 위치 조립을 통한 인체형 당단백질 생산 숙주 개발
○ 만노스 인산화 효소 YlMpo1을 발굴하였으며, 이를 이용하여 당사슬 생합성 회로를 조작하여 효소 치료제의 라이소좀 타겟팅에 유용한 만노스-6-인산 부가 능력이 증대된 효모를 개발함
○ 재조합 발현한 YlMpo1을 이용해서 분자 수준에서 특성을 규명하고 이를 이용한 효과적인 in vitro 효소 반응공정을 개발함
○ 세포벽 형성에 중요한 Gas1 단백질과 GAS1 유전자가 결손된 효모 균주를 이용하여 분비 능이 우수한 효모를 선별하기 위한 스크리닝 방법을 개발함
2. 식물유래 폴리케타이드계 화합물 생합성 인공모듈 구현 최적화 플랫폼 대장균 개발
○ 타이로신 고생산 생합성 모듈의 대장균 내 삽입을 통한 발효공정에 적합한 타이로신 생산 균주 (ΔCOS1)개발
○ Malonyl-CoA로 전환수율을 향상하기 위해 acetyl-CoA에서 acetate로의 전환을 차단: pta 와 ackA 유전자 제거 균주(ΔAP), eutI 유전자 제거 균주 (ΔEutI), 그리고 bioC 유전자 제거 균주(ΔBioC) 3종 개발.
○ 타이로신 생산 균주(ΔCOS1)에서 5종의 인공대사 경로 구현을 통해 최고 17배의 쿠마린산 생산 증가확인
○ 타이로신 생산 균주(ΔCOS1)을 바탕으로 acetyl-CoA 생합성 경로 유전자(pta & ackA, eutI, bioC)가 제거된 다중회로 조작 균주 3종 개발 (ΔCOS2, ΔCOS3, ΔCOS4)
○ 개발된 ΔCOS1~4균주에서 폴리케타이드계 화합물(레스베라트롤) 생합성 인공대사 경로(pET-opTLvS) 발현을 통한 레스베라트롤 생산이 최고 24.2mg/L 확인 및 기존 대장균 대비 730% 생산성 증가 확인.
3. 아이소프레노이드 생합성 생물부품 발굴 및 조립을 통한 유용 터펜의 합성
○ 생물부품으로서 (-)-a-bisabolol synthase (German chamomile), caryophyllene synthase (Piper nigrum), drimenol synthase (Valeriana officinalis), IDS promter (Ginkgo biloba)를 동정하고 특성을 확인함
○ 담배에 (-)-a-bisabolol synthase와 함께 HMGR, 표피세포 고유 promoter pCer6 를 이용하여 발현시켰을 때 15 mg kg^-1의 생산을 나타내었으며 acetyl CoA carboxylase의 BCCP subunit를 과발현하였을 때 생산성은 약 30% 증가함.
4. 바이오네트워크 재조립을 통한 바이오센서 및 GABA생산 숙주 개발
○ 글루탐산인식 chimera two-component system제작
- Pseudomonas putida의 AauS의 sensing domain과 대장균의 EnvZ의 catalytic domain융합
- 외부의 글루탐산을 인식하여 ompC 유전자를 발현 (0.1 mM 글루탐산 인식)
○ 형광기반 리포터 플라스미드 제작
- ompC 유전자의 프로모터와 형광단백질인 gfp를 융합
- 글루탐산에 의하여 형광이 발현되는 시스템
○ 글루탐산인식 바이오센서 제작
- Chimera two-component system과 형광리포터 도입
- 외부의 글루탐산 인식하여 형광발현 (0.1 mM 글루탐산 인식)
5. 바이오부품의 기능 분석 및 HTS를 위한 미세유체기술 개발
○ 미생물 대사산물 과량발현 균주선별을 위한 유체패터닝 기술 개발
- 유체패터닝 (Fluid patterning) 기술 개발.
- 물-기름의 안 섞이는 자연현상을 응용, 유체를 특정 공간에 패터닝하는 기술 개발.
- 국제학술지 논문 준비 중
○ 자가조립멤브레인(SAPM) 기반 미생물 집적 장치 개발
- 미세유체 기술을 기반으로 자가조립멤브레인 기술을 개발.
- 인공적으로 합성된 E. coli를 고농도 집적화에 성공.
- 특허출원 (10-2014-0187537) 자가조립멤브레인 기반 미생물 집적 장치
○ 미생물 중금속 검출용 바이오센서의 민감도를 극대화 시키는 바이오칩 개발
- 중금속 검출을 위한 미생물 바이오센서의 민감도를 103배 이상 증가시킴
- 특허출원 (10-2014-0169737) 미생물을 이용한 중금속 탐지 바이오센서
- 국제학술지 논문 게제 (Biosensors and Bioelectronics 65, (2015) 257-264)

Abstract ▼

(제1세부과제)
More than 60% of protein therapeutics is glycoproteins attached with glycans which are important for folding, therapeutic efficacy, in vivo half-life and immunogenecity. Although protein productions in yeast have been widely used with the advantages of low cost, easy handling and safety,

(제1세부과제)
More than 60% of protein therapeutics is glycoproteins attached with glycans which are important for folding, therapeutic efficacy, in vivo half-life and immunogenecity. Although protein productions in yeast have been widely used with the advantages of low cost, easy handling and safety, it has a great problem to produce glycoproteins attached with non-human high-mannose type glycans. Because these non-human glycan structures often provoke immune reaction and lead to rapid clearance, glycosylation pathway engineering of yeast is essentially required for its therapeutic use. Yeast-specific mannose chain synthesis was blocked by disrupting the gene encoding the first glycosyltransferase of that pathway. We are further reconstructing and optimizing the glycosylation pathway for production of therapeutic enzymes. For recombinant glcucocerebrosidase (Cerezyme), the strain producing glycoproteins homogeneously glycosylated with human-compatible mannose type glycan (Man₅GlcNAc₂) was generated through optimized localization of alpha(1,2)-mannosidase especially on the cell wall surface. On the other hand, other therapeutic enzymes including alpha-galactosidase (Fabrazyme and Myozyme) require the addition of N-glycans containing mannose-6-phosphates (M-6-Ps), which is recognized by M-6-P receptors on plasma membrane for cellular uptake and targeting to lysosomes. Although M-6-P-containing glycans are found only in mammalian cells, the mannosylphosphorylated mannose structure (mannose-1-phosphate-6-O-mannose) of N-glcyans in yeast can be converted to M-6-P structure (phosphate-6-O-mannose) by uncapping the outer mannose residue. In the traditional yeast Saccharomyces cerevisiae, both ScMNN4 and ScMNN6 genes are required for efficient mannosylphosphorylation. ScMnn4 protein has been known to be a positive regulator of ScMnn6p, a real enzyme for mannosylphosphorylation. On the other hand, YlMpo1p, a ScMnn4p homologue, mediates mannosylphosphorylation in Yarrowia lypolytica without the involvement of ScMnn6p homologues. We show that heterologous expression of YlMpo1p can perform mannosylphosphorylation in S. cerevisiae in the absence of ScMnn4p and ScMnn6p. Moreover, mannosylphosphorylation of N-glycans enhanced by YlMpo1p overexpression is much higher than that with ScMnn4p overexpression, and this is highlighted further in Scmnn4- and Scmnn6-disrupted mutants (S. cerevisiae mnn4D, mnn6D, and mnn4Dmnn6D strains). We applied the strategy of mannosylphosphorylation enhancement by YlMpo1p overexpression to a glyco-engineered S. cerevisiae (Scoch1Dmnn1D strain) in which the synthesis of yeast-specific immunogenic glycans is blocked. When compared to ScMnn4p overexpression, a great increase of bi-mannosylphosphorylated glycan is observed. Through an in vitro process involving the uncapping of the outer mannose residue, this bi-mannosylphosphorylated structure is changed to a bi-phosphorylated structure with high affinity for mannose-6-phosphate receptor. The superior ability of YlMpo1p to increase bi-mannosylphosphorylated glycan in y ast shows promise for the production of therapeutic enzymes with improved lysosomal targeting capability. However, since the production yields of therapeutic enzymes in yeast were very low, we also developed the screening method to select a yeast with improved secretion.
Gas1 protein is a beta(1,3)-glucanosyltransglycosylase playing an essential role in the assembly of cell wall as localized on the yeast surface through a glycosylphsphatidylinositol (GPI) anchor. When GAS1 gene was disrupted in yeasts, the resulting mutant strain were reported to exhibit hypersensitivity to cell wall-perturbing reagents together with increased capability of protein secretion due to the loosed cell wall structure. Functional complementation of cell wall-defective phenotype of Gas1-deletion mutant using recombinant expression of Gas1 protein was employed to generate a screening system for a strain with improved capability of protein secretion. We constructed the expression vectors encoding fusion proteins with N-terminal secretory protein of interest linked to Gas1p without signal sequence. After these vectors were transformed into Gas1-deletion mutant, the growths of the resulting transformants were tested on the agar plates containing cell wall-perturbing reagents. Only the strains expressing Gas1p fused to well secreted proteins showed restored growth phenotype under cell-wall stress condition. This system can be used to enrich yeasts with improved secretion capability after genome-wide random mutagenesis, which would contribute to the development of super secretory yeasts. All these efforts would contribute to generating ‘smart super secretory yeast’ producing therapeutic enzymes attached with the glycans optimized for lysosomal targeting.

(제2세부과제)
Polyketides represent a diverse group of natural products that have a variety of biologically active characteristics, such as anticancer, anti-cholesterol, and antimicrobial properties. Among these widespread synthases are type III polyketide synthases (PKSs) hich can produce a broad class of natural products that ave applications in medicine, agriculture, and other fields, due to their notable structural diversity and biological activities. Plant polyketides are the precursors to a range of important plant metabolites such as the secondary metabolites belonging to the flavonoid/stilbenoid class of compounds. Although these compounds are widely used in human health care and industrial material, at present they are mainly obtained by extraction from plants, and extraction yields are low because most of these metabolites accumulate at low levels in plant cells. Furthermore, their isolation from plant material, especially as pure compounds, remains a challenge.
In our study, several genetic strategies were used to modify the bottleneck of high-level production in E. coli comes from the insufficient supply of intracellular precursors, tyrosine and malonyl-CoA. We outline the construction and evaluation of a series of strains capable of circumventing both of the critical limitations. Our artificial plant polyketides (resveratrol) biosynthetic pathway was assembled with in engineered strain for high L-tyrosine production in order to able the production of resveratrol directly from glucose. Also, when combined with the overexpression of malonyl-CoA, the enhancement of resveratrol in pathways will be increased the production level.

(제1협동과제)
-Title: Production of value-added terpenes through discovery and assembly of biocomponents for isoprenoid biosynthesis in noble plant epidermal cell platform.
- Aim of the Research: The purpose of this research is to discover biocomponents necessary for value-added sesquiterpene synthesis and to establish plant epidermal cell platform by using synthetic biology approach.
- Rational of the Research: Sesquiterpenoids have interesting bioactivity useful for human use. However, the terpenoids have diverse carbon skeleton with stereochemical centers, which render synthetic approach for the stuructures unpractical. Here, we tried to isolate biocomponents for terpene synthesis and to apply synthetic biology for production of terpenoids in plant epidermal cells to achieve the efficient synthesis of those complex structures.
- Achievements
1. (-)-a-Bisabolol synthase from German chamomile and drimenol synthase from valerian were cloned based on publicly available database. The genes were expressed in yeast platform and the function of the cloned terpene synthase was identified by elucidating the product of the yeast fermentation.
2. Plant epidermal cells were engineered to effectively produce sesquiterpenes by overexpressing HMGR and partially inhibiting ACC by overexpression of BCCP subunit.
3. Characteristics of GbIDS promoter was studied. The promoter was specific for root and developing young leaves.
4. Caryophyllene synthase was identified from black pepper.
5. Genes involved in protoanthocyanin in pea were identified.
6. Theses researches were published as 6 articles in high impact factor journals such as Biochemical Journal.
- Further Plan: Discovery of terpene genes, especially terpene oxidases, with emphasis on rotundone biosynthesis in black pepper will be highlighted in the next step of theresearch. Optimization of epidermal cell system as terpene synthesis platform will be further pursued.

(제2협동과제)
In this study, amino acid sensing recombinant bacteria was constructed via chimera two-component system strategy. Sensing domain of Pseudomonas putida AauS and catalytic domain of Escherichia coli EnvZ was integrated. Constructed bacteria can sense amino acid and express green fluorescence prtein. To construct gamma-aminobutyric acid producingrecombinant E. coli, glutamate decarboxylase and glutamate/GABA antiporter was physically connected via synthetic protein scaffold.

(제3협동과제)
The directed evolution method for a synthetic microbe has turned out to be highly useful for both protein engineering and industrial purposes such as the over-production of various biochemicals. However, conventional high-throughput screening techniques still have many difficulties such as complex fabrication process for fully compartmentalized environments.
Therefore, microfluidic methods that provides miniaturization, easy manipulation, and fabrication process have attracted many interest from several biological fields. In this project, we developed several high-throughput screening technologies to overcome conventional limitations. First, fluid patterning technology was proposed to address problems of both sample selectability and throughput by applying the concept 'immiscible' nature phenomena between oil and aqueous phase. Second, we propose a multi-purpose, versatile self-assembled particle membrane (SAPM) microbioreactor array device that can control chemical culture environment simultaneously. Finally, we propose a high-throughput,chemostat-like microfluidic platform that can continuously supply both nutrients and inducers using microfabricated ratchet structures and a mixing microchannel network. Using this device, we improved the sensitivity of microbial biosensor with 10³ times enhancement.
In summary, high-throughput screening technologies are considered to be a critical process for the advancement of synthetic biology, microbiology, and related pure biologies but appear to be still challenging. However, the technologies we developed in the last stage and in the following stage shows a remarkable potential to address the conventional limitations in high-throughput screening.