초록 ▼

Ⅳ. 연구개발결과
□ 1세부과제명 : 사이코스 대량 생산 세포공장 개발
- 코리네박테리움 글루타미쿰에서 사이코스 에피머라제(DPE) 유전자의 발현최적화
· 코리네박테리움용 단백질 고발현 셔틀벡터의 제작
· 프로모터 스크리닝을 통한 발현최적화
- 재조합 균체를 재사용한 연속반응성 시험
· 균체 재사용 4회 이상 가능 확인
- 발현벡터 내 DPE 유전자 양 증가
· 유전자량 증가시키기 위해 DPE를 두 개, 세 개 가지는 벡터 구축. 사이코스 생산속도 증가 확인
- 고온 안정 유전자 탐색

Ⅳ. 연구개발결과
□ 1세부과제명 : 사이코스 대량 생산 세포공장 개발
- 코리네박테리움 글루타미쿰에서 사이코스 에피머라제(DPE) 유전자의 발현최적화
· 코리네박테리움용 단백질 고발현 셔틀벡터의 제작
· 프로모터 스크리닝을 통한 발현최적화
- 재조합 균체를 재사용한 연속반응성 시험
· 균체 재사용 4회 이상 가능 확인
- 발현벡터 내 DPE 유전자 양 증가
· 유전자량 증가시키기 위해 DPE를 두 개, 세 개 가지는 벡터 구축. 사이코스 생산속도 증가 확인
- 고온 안정 유전자 탐색
· 고온 공정에서의 이점을 위한 고온 안정성 가지는 DPE 효소 자원 탐색으로 뛰어난 열 안정성을 가지는 Clostridium 유래의 DPE 발굴. 고온 공정에서 균체 재사용 횟수 증가시킴.
- GRAS 미생물 세포공장을 이용한 과당의 사이코스로의 생물전환반응 공정 최적화
· 최종 사이코스 생산량과 반응 속도에는 영향을 주지 않으면서, 고온에서 배지의 갈변 현상을 해결 할 수 있는 최소 배지의 조성 확립
· 반응온도를 바꾸어 전환반응 시킨 결과, 고온의 조건에서 전환 반응 속도가 현저히 증가함을 관찰 (70℃에서 3시간 만에 120g/L의 사이코스 생산)
· 고온에서 whole cell catalyst를 이용한 사이코스 전환 반응 기술 개발
□ 2세부과제명 : 사이코스 생산경로 효소들에 대한 분자모델링 연구
- 프럭토스 주위의 잔기들에 대한 점돌연변이시 에너지 변화량 계산을 바탕으로 활성의 증가가 기대되는 17개의 돌연변이 후보 선별
- 55℃에서 30ns 동안의 분자 동력학 모의실험 후, RMSD 분석을 통하여 계산 시간동안 WT 효소를 비롯한 각 돌연변이 구조들이 큰 구조적 변형 없이 안정적으로 유지됨을 확인
- DPE 효소 메커니즘에서 Glu244와 프럭토스 사이의 수소이동이 일어나기 적합한 환경을 유지하는지를 확인하기 위해 거리변화 측정, 이를 통해 WT과 비슷하거나 더욱 가까운 거리를 유지하는 7개의 돌연변이 (G65Q, A107K, E156R, G65L, L152K, H186R, - 선별된 7개의 돌연변이 후보들에 대하여 DPE와 프럭토스간의 결합에너지를 측정 결과, 3개의 돌연변이 (G65Q, H186R, Y6R/G65E) 들이 WT보다 더 낮은 에너지를 가지는 것으로 확인
□ 1협동과제명 : 사이코스 생산용 당 대사 재설계 코리네박테리움 개발
- IPTG를 이용하는 고발현 벡터 (총 3종) 및 C. glutamicum 유래의 strong promoter인 SOD promoter와 ilvc promoter를 이용한 항시적 고발현 벡터 시스템 (총 2종) 구축
- IPTG를 이용하는 고발현 벡터로 DPEase 고발현시 48 g/L의 psicose를 생산하였으며 (30℃, 48hr), 코리네 세균 유래의 항시적 고발현 벡터를 사용한 경우 27 g/L의 사이코스를 생산
- C. glutamicum의 ptsF 변이주에서 fructokinase 유전자의 발현 실험을 통한 myo-inositol facilitator 유전자(iolT1, iolT2)의 non-PTS 과당 수송 기능 규명
- ptsF 변이주를 숙주로 이용하여 DPEase 유전자와 과당 수송 막단백질 유전자 (iolT)의 동시 발현 시스템 구축 (총 3종)
- 최종 구축한 동시 발현 시스템을 이용하여 30℃에서는 85 g/L psicose (48hr)를 생산하였으며, 50℃에서는 105 g/L psicose (24hr)를 생산
□ 2협동과제명 : 사이코스 생합성 및 대사관련 대사체분석 플랫폼 구축
- 세포내 대사체 분석을 위한 시료 전처리방법 확립
· 사이코스 생합성 및 대사관련 대사체: 당 및 당 인산화물
· 시료내 당 분석을 위한 추출용매: CH3CN/i-PrOH/H2O(3/3/2)
· 시료전처리 조건 확립: 용매 추출후 원심분리하여 기기분석 수행
- 사이코스 및 전구체/대사산물 분석을 위한 LC-MS/MS 분석조건 확립
· 검출기: mass detector
· 당 분석 컬럼: Luna amine column (150 × 2 mm, 3 μm, 100Å)
· 당 인산화물 분석 컬럼: Reprosil-Pur Basic column (150 × 2 mm, 2.5 μm)
· 이동상: CH3CN/Water gradient condition
· SRM: 179 > 89 (fructose, glucose, psicose, mannose, altrose) 341 > 179 (sucrose), 259>97 (G-6-P, F-1-P, F-6-P), 421>241 (Sucrose-6-P)
- 사이코스 및 전구체/대사산물 동시 다중 분석법 확립 및 검증
· 공정분석법: GC-MS를 이용한 사이코스 및 전구체/대사산물 분석법 개발
· 유도체 시약: MSTFA
· 컬럼: Rxi-5Sil MS column (30 m × 0.25 mm, 0.1 μm)
· SIM: 73 (mannose), 217 (psicose, fructose), 319 (glucose), 341 (sucrose)
· 분석법 검증: 특이성, 직선성, 회수율, 안전성 평가 완료
- 동시 분석법을 이용한 사이코스 생산시스템 최적화 지원

Abstract ▼

D-Psicose, a carbon -3 epimer of D-fructose, known as a valuable rare sugar in sugar industry, is present in very small quantities in commercial carbohydrate or agricultural products. D-Psicose, which has 70% of the sweetness of sucrose but almost no calories, is approved as generally recognized as

D-Psicose, a carbon -3 epimer of D-fructose, known as a valuable rare sugar in sugar industry, is present in very small quantities in commercial carbohydrate or agricultural products. D-Psicose, which has 70% of the sweetness of sucrose but almost no calories, is approved as generally recognized as safe (GRAS) by the US Food and Drug Administration in 2011. It has several health benefits like as suppressing lipid synthesis in the liver to reduce abdominal obesity, preventing the development of diabetes and functioning as a medicine for arteriosclerosis. D-Psicose can be produced from fructose through bioconversion process. Bioconversion refers to the use of biocatalysts, often microorganisms or enzymes, to carry out a chemical reaction that is more costly or not feasible non-biologically. This study aimed to improve productivity of D-psicose in resting cell bioconversion study using a Gram-positive, facultative anaerobic, non-pathogenic soil bacterium, Corynebacterium glutamicum known as GRAS.
C. glutamicum was engineered to overexpress D-psicose 3-epimerase gene (dpe) cloned from Agrobacterium tumefaciens C58. D-Psicose 3-epimerase(DPE) can catalyze epimerization of free keto-sugars, including the conversion of D-fructose to D-psicose with yield of approximately 30%. In order to overexpress DPE, pSGT208 was constructed from E. coli - C. glutamicum shuttle vector pCES208 through modification of promoter region to be switchable with different promoters along with introduction of transcription terminator region. The dpe gene was cloned into pSGT208 containing lac promoter, which resulted in pS208-DPE, and the promoter was changed with a strong trc promoter to create pS208cT-DPE. For the resting cell conversion reaction producing D-psicose, the recombinant C. glutamicum transformed with pS208-DPE and pS208cT-DPE were harvested after 12 hours of culture in 2YT medium and resuspended in the cell concentration (OD) of 40 in a conversion reaction medium containing 40% (w/v) of D-fructose. D-Psicose production of 8 g/l and 52 g/l were obtained from the recombinant strains harboring pS208-DPE and pS208cT-DPE, respectively, for 24 hours of bioconversion reaction at 30℃. The plasmid pS208cT-dpe was selected for further use in optimization process of D-psicose production.
The bioconversion reaction conditions including working volume, cell concentration, temperature, substrate and media were optimized to increase the D-psicose production. In particular, the production titer and rate of D-psicose were significantly increased with the increase of the reaction temperature. D-Psicose was produced up to 120 g/l for 6 hours at 50℃, or 3hours at 60℃. The production rates were dependant on the reaction temperature with 2.5 g/l/h at 30℃, 20 g/l/h at 50℃, and 40 g/l/h at 60℃. There was a 16-fold increase of production rate in the reaction of 60℃ compared to that of 30℃.
The high temperature reaction was suspected to be possible due to the protection of DPE enzyme from heat by cellular barriers, e.g. cell envelopes and components. In order to confirm it, in vitro enzyme reaction was performed by using crude cell lysate. The enzyme activity was found to being decreased as the increase of the reaction temperature, especially above 50℃. Additionally, the same temperature experiment was carried out using a recombinant E. coli overexpressing the DPE to see the applicability of the high temperature conversion reaction to other microorganisms other than C. glutamicum known as a robust microorganism with rigid cell wall. The D-psicose production using the E. coli was improved with the increase of the temperature in a similar manner with that of C. glutamicum. Thereafter, it was studied the recycled use of the resting cell biomass in the high temperature reaction using the recombinant C. glutamicum and E. coli. A severe decrease of the resting cell biocatalyst activity was observed in E. coli during there cycle whereas not in C. glutamicum. The results suggest that the high temperature conversion reaction is highly efficient for D-psicose production, but only suitable for the resting cell conversion reaction, using the robust Gram-positive C. glutamicum.
Taken together, it is concluded that the resting cell bioconversion process for D-psicose production is significantly superior compared to invitro enzymatic bioconversion. The highly efficient process of D-psicose production based on the high temperature reaction can be established, with a combined use of the robust C. glutamicum as a host and some novel thermostable DPE as an enzyme.
The enzyme containing a Mn²⁺ metal ion catalyzes the interconversion of D-psicose and D-fructose by epimerizing the C-3 position. The catalytic residues, Glu149 and Glu243, are responsible for deprotonation and protonation at the C-3 epimerization center. The metal ion in DPEase plays an important role in an epimerization reaction at the C-3 position by anchoring the bound fructose and the catalytic residues. The metal-binding site is formed by Glu 149, Asp 182, His 208, and Glu 243. Since biological production of D-psicose is lower than D-fructose, we applied computational protein design strategies in order to improve catalytic activity of Clostridium hylemonae DPEase. The 3D structure of C. hylemonae DPEase was predicted using homology modeling method and refined by molecular dynamics (MD) simulation. A series of DPEase mutants obtained from mutation energy calculation were used for MD simulations. The structural comparison between wild type and mutants indicated that D-fructose in mutants showed persistent interactions with Glu243 and Glu149 during the simulation time. Furthermore, binding free energies between the protein and D-fructose were calculated. We hope that mutant candidates suggested from this study can be helpful to mass production of D-psicose through biosynthesis.
Recombinant Echerichia coli haboring heterologous DPEase gene has been used to produce the D-psicose by whole cell biotransformation. However, food grade microbial host for industrial D-psicose production is needed to avoid potential safety problems as a food ingredient. Corynebacterium glutamicum is widely used for the industrial production of useful metabolite, and it has been identified as a ‘Generally Recognized As Safe’ (GRAS) strain in the food industry. In addition, C. glutamicum has a unique mechanism of fructose metabolism. We are interested in reconstruction of the fructose metabolic pathway in C. glutamicum via metabolic engineering for psicose production. In C. glutamicum, fructose is mainly transported and phosphorylated by fructose-specific phosphotranferase system (PTS) However, it has been reported that fructose could be transported as an unphosphorylated form via a PTS-indepedent uptake mechanism, myo-inositol transporter (IolT). When expressing heterologous DPEase gene from Agrobacterium tumerfaciences in C. glutamicum, the recombinant strain could directly convert the intracellular fructose to psicose. D-psicose production is limited by the uptake of D-fructose in its unphosphorylated form. Therefore, to further increase the intracellular unphosphorylated D-fructose concentration we deleted the fructose enzyme II gene (ptsF), and overexpressed myo-inositol transporter gene (iolT) as a non-PTS fructose uptake system. We have co-expressed DPEase and IolT gene in ΔptsF mutant. Using whole cell biotransformation 110 gL^-1 psicose was produced from initial cell concentration (OD600nm, 40) and fructose concentration of 40% (w/v) in 24 hours of culture. These genetic modification of fructose metabolism via reconstruction of fructose metabolic pathways could be a good strategy for producing rare sugars like D-psicose in C. glutamicum.
The study was performed to develop the analytical method of sugar and its phosphate using liquid chromatography/tandem mass spectrometry (LC-MS/MS). The chromatographic separation of sugar including fructose, glucose, psicose, and sucrose was carried out on Luna amine column (150 × 2.0 mm, 3 μm, 100 Å) at a flow rate of 0.2 mL/min, using mixture of acetonitrile and water as the mobile phase. Carbohydrate mainly produced deprotonated [M-H]- in negative ion ization mode. Sugar phosphate such as fructose monophosphate, fructose bisphosphate and sucrose phosphate was separated on Reprosil-Pur BasicC18column (150 × 2.0 mm, 2.5 μm) at a flow rate of 0.2 mL/min, using mixture of acetonitrile and water as the mobile phase. The ion transitions monitored in selected reaction monitoring mode were m/ z 179>89 for psicose, fructose, and glucose, m/ z 341>179 for sucrose, m/ z 259>97 for fructose monophosphate, m/ z 339>97 for fructose bisphosphate, and m/ z 421>241 for sucrose phosphate. This analytical method is a very simple, sensitive and accurate to determine carbohydrates in biological samples.