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1 Metabolic Engineering Department of ioinformatic Engineering, Graduate School of nformation Science and Technology, Osaka University Dr. Hiroshi Shimizu Professor No. Experimental Determination Method of Flux Distribution with sotope Labeling April th July 7th, 5

2 Experimental Determination of Metabolic Flux with sotope Labeling Simple Network Assumption No accumulation of and d d alance Eqs of and d d ( ( ) ( ) ( ) ) Measurement:(onsumption) (i) (ii)

3 Use of : : ombination of two Atoms incorporated A () Ratio Met Pattern of () () () - - A() A() A() A (): ratio of - and - in Metabolite A () A() ( ) ( ) ( ) A i i i i i i i D ( i)

4 Material alance of and in Atomic Level d d (Assumption ) d () A () () () d () A () () () d () A () () () d () A () () () d () () d () () () () d () () () () d () () ( ) ( ) ( ) ( ) ( ) () ( ) ( ) ( ) 5 6 ( ) () ( ) 7 8 9

5 5 ( ) ( ) ( ) ( ) () () () () () () () () () () () () A A A A Subsequently A()~A():Measure (GMS or NMR) ()~() ()~() ( (i)): Determined Unknown ars:

6 フラックス決定までのフローチャートフラックス (,,) の仮定 ()~(), ()~(),, の最適化上記 8 個の値を用いて測定データと比較できる値に変換 N 変換値と測定データの比較 Y 測定データフラックス決定 6

7 代謝解析技術 ( メタボロミクス ) NMR の原理外から磁場のかからない場合核スピンは勝手な方向を向いている核核スピン強い外部磁場外から強い外部磁場をかけた場合核スピンの向きは磁場方向とその反対方向にきれいに分かれる β スピンエネルギー順位でみると低い状態が多い α スピンエネルギー差に相当するエネルギーを与えてやると下から上へ励起され ( 核磁気共鳴 ) その後緩和する 7

8 代謝解析技術 ( メタボロミクス ) GMS の原理ガスクロマトグラフ (G) 移動相 : ガス (He) と固定相 ( キャピラリカラム ) への分配を繰り返し物質の移動速度の差により分離質量分析 (MS) 気化された試料分子の電子をイオン化電圧を与えることによりたたき出して ( 電子衝撃法 ) 電圧を変化させる四重極を安定に通り抜けるイオンを検出イオンビーム四重極直流と高周波交流が重ね合わせて印加 8

9 代謝解析技術 ( メタボロミクス ) 代謝物質分子内の同位体濃縮の決定法 G-MS NMR 分析法 G-MSデータ NMRデータ () d s () () () 炭素番号は図も二進表記も左から位位とする ( M i) ntensity of ( M i) ntensity of ( M j) j [ M ] () [ M ] () () [ M ] () s d Area( S) () Area( S) Area( d) () () Area( d) () Area( S) Area( d) () () 9

10 A A Question Make molecule balance of and. Make atom balance of and. D D A-> (r); -> (r); -> (r); ->D (r)

11 ( ) ( ) () () () () () () / () / () () () () () () () () () () () () () () () () () () () () () () () d d d d d d A A A A A-> (r); -> (r); -> (r); ->D (r) : : A A D D

12 Determination of Flux Distribution of coryneform bacteria by -NMR

13 ack Ground of This Study orynebacterium glutamicum: Glutamate Producing Strain (urrently used in ndustry) Fermentation Temperature: o orynebacterium efficiens: Glutamate Producing Strain Ability to grow at higher temperature (7 o ) than. glutamicum ( o ) Saving Energy in Fermentation Process

14 ackground Flux analysis at α-ketoglutarate branch point PEP v Oxaloacetate Pyruvate Acetyl-oA v v TAcycle v v α-ketoglutarate Effect of anaplerotic pathway distribution on Glutamate production omparison between.efficiens and. glutamicum v glutamate Material balance Oxaloacetate: v v v α-ketoglutarate: v v v v v

15 Glycolytic Pathway Glucose G6P PEP Pyruvate Acetyl-oA Pentose -P Pathway Objectives Determination of Flux distributions at PEP/pyruvate G6P branch points y -NMR Oxaloacetate TAcycle α-ketoglutarate glutamate 5

16 Materials and Methods Microorganisms: orynebacterium glutamicum AT869 orynebacterium efficiens FERM96 Reagent:[- ]Glucose [6- ]Glucose(sotec nc., MA) Fermentation: olume onditions Flask ultures ml 5ml Sakaguchi Measurements Flasks Temp.5 (. glutamicum) Temp 7 (. efficiens) Agitation strokes/min ph Adjustment with ao [- ]Glucose [6- ]Glucose Addition initially Principles Glucose Enzymatic Method Glutamate Enzymatic Method -NMR MHz-NMR 6

17 Results and Discussion. Fermentations: OD66 glucose(g/l) glutamate(g/l) [- ]Glucose [6- ]Glucose [- ]Glucose [6- ]Glucose Time(h) Time(h) OD66 glucose(g/l) glutamate(g/l) 5 Time(h) Time(h). glutamicum (.5 ). efficiens(7 ). Efficiens grows in production phase as well. Glutamate production by. efficiens is less than that by. glutamicum. 7

18 . alculation of Enrichment Definition of Enrichment {( mol Abundant mol) / ( mol mol)} How much amount of from [- ] and [6- ] glucose? -NMR Analysis in Glu from - Gluc alculation Enrichment by from area peaks TSP (nternal Std) Abundant in Glu:.% ppm 8

19 incorporated in Glucose (%) [- ]Glucose [6- ]Glucose. glutamicum. efficiens.8 ±.. ±.. ±.6. ±.9 enrichment of Glu in. glutamicum culture(%) Glu- Glu- Glu- [- ] Glucose [6- ]Glucose enrichment of Glu in. efficiens culture(%) Glu- Glu- Glu- [- ]Glucose [6- ]Glucose

21 PEP/Pyruvate AcoA itrate αkg O Glu Fum O O OxA/ Mal O 5 5 5

22 PEP/Pyruvate AcoA itrate αkg O Glu Fum O O OxA/ Mal O 5 5 5

23 . Determination of Flux Distribution Flux Distribution at G6P ranch Point Pyruvate Oxaloacetate Glutamate [- ]Glucose [6- ]Glucose v [- ]PEP/Pyr Enrichment of Glu- should be equal to Pyruvate- v [- ]O Glucose v Glc v G6P v glutamicum v Glc Glucose v G6P v efficiens

24 O Flux Distribution at PEP/pyruvate ranch Point v Oxaloacetate フラックスモデル PEP/ Pyruvate v TAcycle α-ketoglutarate v 5 v v v Glutamate Material alance v v v v v v alance At Oxaloacetate Pyr() Pyr() v Pyr() O /Glu() /Glu() /Glu() /Glu(5) /Glu(5) /Glu() /Glu() /Glu() At α-ketoglutarate Oxa() Glu() Oxa() Glu() Oxa() Glu() Pyr() Glu() Pyr() Glu(5) -v Oxa() Oxa() Oxa() Oxa() v - (v v). glutamicum. efficiens Anaplerotic Path (v) TAcycle(v).% 57.% 5.7% 6.%

25 Summary of Flux Distributions in corynebacteria Glucose Glucose 75. G6P.6 9. G6P 6.. Oxaloacetate PEP /pyruvate Oxaloacetate PEP /pyruvate 6. TAcycle TAcycle Glu Glu. glutamicum. efficiens 5

26 Flux Distribution of PEP/Pyr and Production Yield of Glutamate Assumption: Gluc Uptake Pyr Production Yield : (Glu Production / Gluc onsumption(mm)) (f (-f) 5/6) Where f: ratio of PPP/Glycolitic Path Glutamate Production Yiled and Flux Distribution at PEP/Pyr. glutamicum. efficiens Yield Ratio of Anaplerotic Path Only 6 % (/5.7) Gluc is converted to Pyr in. efficiens 6

第６２巻　第１号　平成２４年４月／石こうを用いた木材ペレット

第６２巻　第１号　平成２４年４月／石こうを用いた木材ペレット Bulletin of Japan Association for Fire Science and Engineering Vol. 62. No. 1 (2012) Development of Two-Dimensional Simple Simulation Model and Evaluation of Discharge Ability for Water Discharge of Firefighting