Microsoft PowerPoint - ME12.ppt

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1 Osaka Univ Metabolic Engineering Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University Dr. Hiroshi Shimizu Professor No.2 Metabolic Engineering with Bioinformatic Data April 3 July 27, 2005

2 バイオプロセスネットワークのシステムと要素目的生産性収率プロセスネットワーク発酵プロセスデータ ph, DO, CO 2, O 2 Cell conc. Sub.conc 細胞間ネットワーク基質細胞ゲノム細胞内ネットワーク遺伝子トランスクリプトーム遺伝子発現プロセス運転最適化制御生産計画共培養工学代謝代謝物質微生物活性の応答計測増殖基質消費代謝物質生産代謝工学プロテオームメタボローム代謝物質 Bioinformatics 網羅的解析タンパク質代謝制御 PHA 品質最適制御乳酸菌ー酵母の相互作用大腸菌ー大腸菌相互作用アミノ酸代謝工学代謝フラックス解析 MCA 酵母細胞モデリング / 育種と生産 2

3 微生物細胞内の多階層ネットワーク表現型の変化表現型に直結したレイヤー環境の変化代謝ネットワーク Metabolic Flux Analysis ストレス高い浸透圧高い CO 2 Conc 高い EtOH Conc etc タンパク質ネットワーク Proteome Analysis 遺伝子ネットワーク Transcriptome Analysis Clustering Gene Expression Data バイオインフォマティクスデータの統合新規機能を持つ細胞の体系的な創製工学目的にかなう細胞の創製 3

4 Osaka Univ 細胞トランスポータ transpoter receptor レセプタ DNA シグナルトランスダクション mrna タンパク質代謝反応 receptor 4

Expression Data Transcriptome Data, Proteome data Metabolic Flux Analysis Data

5 ストレスに対して異なる強さの細胞を比較する実験室酵母 Normal Conditions Stress Conditions 醸造酵母 Difference of Physiological Characteristics Growth, Substrate Uptake,.. Etc. Expression Data Transcriptome Data, Proteome data Metabolic Flux Analysis Data Systematic design of stress tolerant strain based on the integration of the difference of the two strains 5

6 DNA マイクロアレイによる遺伝子発現解析 Microorganisms Lab Strain Saccharomyces cerevisiae FY834 (MATα hisδ200 ura3-52 leu2δ lys2δ202 trpδ63) Brewing StrainSaccharomyces cerevisiae IFO2347 (Brewing Strain for Japanese Rice Wine) Cultivation Conditions Sakaguchi Flask Culture on YPD medium ( % Bacto yeast extract, 2 % Bacto peptone, 2 % glucose) at 30 o C, 20 spm DNA Microarray Analysis Osmotic pressure was added at mid-log phase by addition of NaCl and Sorbitol (OD 660 =). Total RNA was extracted by hot-phenol method. Cy3 or Cy5 labeled cdna was prepared by reverse transcriptation reaction. Yeast gene chip ver.2 (DNA Chip Research Inc.) Intensities of spots were measured by GenePix 4000A (Axon Instruments). 6 Normalization by LOWESS (locally weighted linear regression ) method.

7 増殖の比較 Lab and Brewing Strains (Under High Osmotic Pressure: NaCl Added) Lab Strain Brewing Strain 4 FY M NaCl 00 FY IFO OD : : M : +0.5 M : M : +.0 M ln (X t /X 0 ) ln(x t /X 0 ) IFO M NaCl

8 M 0.5 M 0.75 M M 5 min 30 min 45 min 60 min 20 min 高浸透圧下の実験室酵母の遺伝子発現スキャッタープロット

9 FY834 (Laboratory strain) STL YHR087W HSP2 HSP42 PHM7 SSA4 YGR243W SPS00 HXT5 SOL4 YDL023C GPD GLK BTN2 YOL053C ALD3 YBR6C SPI TKL2 HSP26 IFO2347 (Brewing strain) STL YGR243W YHR033W YJL07C BTN2 ENA2 ENA5 PHM7 ENA CUP- YJL44W CUP-2 ARO0 SSA4 GRE2 PRM0 ARO9 PUT4 HSP2 SPI 遺伝子発現変化のトップランキング ( 浸透圧ショック ) Gene name Function of the gene product Fold induction Sugar transporter-like protein Heat shock protein, induced by heat shock, entry into stationary phase, depletion of glucose, and addition of lipids Heat shock protein, similar to HSP26; expression is regulated by stress conditions Unknown, transcription is regulated by PHO system Heat shock protein, member of 70 kda heat shock protein family response to stress Unknown, involved in spore development Hexose transporter, member of superfamily of monosaccharide transporters Unknown, similar to SOL3 Glycerol-3-phosphate dehydrogenase Glucokinase Unknown, gene/protein whose expression is elevated in a btn - /Btnp lacking yeast strain, ph regulated Unknown, DNA damage responsive (DDR2) Aldehyde dehydrogenase, expression induced in response to heat shock, oxidative and osmotic stress, NAD + is preferred coenzyme Strongly expressed during stationary phase, and trancription is dependent on MSN2/MSN4 Transketolase, similar to TKL Heat shock protein 26 Sugar transporter-like protein Unknown, gene/protein whose expression is elevated in a btn - /Btnp lacking yeast strain, ph regulated P-type ATPase Na + pump, plasma membrane ATPase Na + ATPase Unknwon, transcription is regulated by PHO system P-type ATPase Na + pump, plasma membrane ATPase Cupper binding metallothionein Cupper binding metallothionein Pyruvate decarboxylase Heat shock protein, member of 70 kda heat shock protein family response to stress NADPH-dependent methylglyoxal reductase (D-lactaldehyde dehydrogenase); stress induced (osmotic, ionic, oxidative, heat shock and heavy metals); regulated by the HOG pathway Pheromone-regulated membrane protein Aromatic amino acid aminotransferase II Proline specific permease Heat shock protein, induced by heat shock, entry into stationary phase, depletion of glucose, and addition of lipids Strongly expressed during stationary phase, and trancription is dependent on MSN2/MSN ( M NaCl addition; 60 min)

10 グリセロール合成経路の強化株の評価 FPS gene encodes the transporter of glycerol, which involves in the export of glycerol from the cells, especially after high osmotic shock. OD FPS::kanMX4 /paurδcenars Lag + M NaCl OD FPS::kanMX4 /pgpd-4i Lag FY834 + M NaCl Lag + M NaCl FPS + /pgpd-4i Lag FY834 FPS::kanMX4 + M NaCl Lag M NaCl 0

11 ENA- 過剰発現株の評価 ENA T ADH Amp r P ADH pena--0i ColEori ENA gene encodes P-type Na + ion efflux pump protein. Expression level of ENA gene was higher in the brewing strain than in the laboratory strain. AUR-C 00 0 paurδcenars (vector) + M NaCl pena--0i (ENA) + M NaCl ln(x t /X 0 ) paurδcenars M NaCl Lag pena--0i

Extraction of Essential Information for Breeding of Osmotic Stress Tolerant Strains DNA microarray data

12 情報抽出の体系的な手法の確立に向けて Transcriptome Analysis Transcriptome Data of several stress conditions (high osmotic pressure, high EtOH conc, high CO 2 concentration Development of Systematic Methods to Upgrade Information Extraction of Essential Information for Breeding of Osmotic Stress Tolerant Strains DNA microarray data 6,000 遺伝子の時系列データ! Expression データをまとめるデータをまとめるクラスタリングクラスタリング Time 巨大なデータ量巨大なデータ量浸透圧に対して重要な遺伝子の探索浸透圧に対して重要な遺伝子の探索 2

13 レポート課題次のような分野で代謝情報工学はどのような発展をもたらすか () 微生物を用いた物質生産 (2) 代謝に関わる疾患の診断治療 (3) 生物による環境修復 What progress is expected by metabolic engineering in the field shown below? () bioproduction by microorganisms (2) medical diagnosis and treatment for diseases related to metabolism (3) bioremediation by living organisms Summarize one or two A4 pages. 3

Microsoft PowerPoint - ME11.ppt

Microsoft PowerPoint - ME11.ppt Metabolic Engineering Department of ioinformatic Engineering, Graduate School of nformation Science and Technology, Osaka University Dr. Hiroshi Shimizu Professor No. Experimental Determination Method