Metabolic Engineering
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1 saka Univ Metabolic Engineering Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, saka University Dr. Hiroshi Shimizu Professor No. 2 Metabolic Engineering for Bioproduction April 13 July27,
2 saka Univ What is Metabolic Pathway? Sequence of biochemical reactions in the cell 2
3 saka Univ What is Role of Metabolic Pathway? Uptake nutrients from outside of the cell Energy generation (Catabolism) Synthesis of building blocks in polymerization reactions Polymerization of DNA, RNA, proteins, lipids, carbohydrates etc (Anabolism) Replication 3
4 saka Univ Components in Cells Dry Cell 30% Ion Small Molecules: 4% Phospholipids: 2% DNA: 1% RNA: 6% Proteins: 15% Polysaccharides: 2% Water 70% Bacterial Cells 4
5 saka Univ Building Blocks and Macromolecules Building Blocks of the Cell Sugars Fatty Acids Amino Acids Nucleotides Larger Units of the Cell Polysaccharides Lipids/ Membranes Proteins Nucleic Acids (DNA RNA) 5
6 saka Univ Catabolic Pathway Degradation Uptake Proteins Amino Acids NH3 2 xidative Phosphorelation Energy Formation ATP Glycolysis Polysaccharides Sugars Gluc Pyr ATP AcCoA TCA cycle Fats Fatty Acids Glycerol Cell C2 6
7 saka Univ Anabolic Pathway Gluc DNA RNA Nucleotides G6P Polysaccharides Amino Acids F6P Cell Proteins Lipids DHAP 3PG Amino Acids PEP Amino Acids Cholesterol/Fatty Acids Amino Acids Cit ΑKG Pyr xa TCA cycle SucCoA 7
8 saka UnivCurrency Metabolites and Metabolic Coupling ATP and + ATP: energy currency metabolite in the cell : Electron transport PH: Electron transport 8
9 (Currency Metabolites ATP) ATP High energy state Adenine - P P P CH2 ATP production Ribose Use in Synthetic Pathway Adenine - P - P P CH2 Ribose Inorganic Phosphate ADP Low energy state 9
10 P- (Currency Metabolites: ) + (oxidation form) H C NH 2 Nicotineamide Ring Ribose + N H - P- Electron transport (reduction form) H H C NH 2 N Ribose Adenine ( H + +2e-) Adenine P- Ribose P- Ribose Nicotine Amide Adenine Dinucleotide 10
11 (Currency Metabolites PH) Electron Transfer P + (xidation form) H H H Nicotineamide Ring C PH(Reduction C NH Form) 2 + N N P- P- Ribose Ribose H - NH 2 P- Ribose P ( H + +2e-) Adenine Adenine P- Ribose P Nicotine Amide Adenine Dinucleotide Phosphate 11
12 Elements in the Cell Transcription, Translation Networks in the Cell Enzyme Reactions Genes Proteins Metabolites Replication Number of Elements ~1,000 ~1, 000 ~1,000 Roles Blueprint encoding sequence of proteins Number of observable variables in experiments DNA Array:~1,000 Catalysis of metabolic reaction Players in bioreactions 2D-electrophoresis:~100 Uptake of nutrients from Env. Catabolism:Production of Energy(ATP PH) Synthesis of Building Blocks (amino acids, nucleic acids, lipids, carbohydrates) Bioreactions MFA ~10 Physiological Data~
13 Deleted based on copyright concern. 13
14 Courtesy of ffice of Energy Efficiency and Renewable Energy (EERE) 2004 Aug 14
15 Courtesy of ffice of Energy Efficiency and Renewable Energy (EERE) 15
16 Courtesy of ffice of Energy Efficiency and Renewable Energy (EERE) 16
17 Courtesy of ffice of Energy Efficiency and Renewable Energy (EERE) 17
18 saka Univ Metabolic Flux Distribution Analysis 7 Ribu5P Gluc 42 Gluc6P 12 Fruc6P 14 GAP 14 G3P 11 PEP EtH 8 Pyr AcCoA AC 129 Analysis of distribution of reaction rates in metabolic pathway Use of information of stoichiometry of the cell and measurements xa 94 AcCoA Mal 81 Fum Glyx Suc IcCyt 91 6 AKG 66 SucCoA Many applications Understanding cell physiology Process operation Molecular breeding 18
19 What can we do by MFA? 1. Understanding physiology of the cell by interpreting flux distribution. 2. Development of operational strategy and control policy according to determined flux distribution. 3. Systematic molecular breeding by analysis of impact of improvement of metabolic pathway by comparison of flux distribution of recombinant microorganism with parent strain. 19
20 1. Metabolic Pathway Modeling Metabolic flux distribution is analyzed with intracellular and extracellular metabolites, based on the information of metabolic reactions. Metabolic Pathway: Sequence of Biochemical Reactions r1 A (Substrate) B D r3 r2 r4 C (Target Product) E (By-Product) Cell r5 F (By-Product) r 1 : A->B r 2 : B+PH->C r 3 :B->D r 4 : D->E+PH r 5 :D->F 20
21 2. Material Balances in Biochemical Reactions Pseudo-Steady State Assumption Intracellular metabolites concentrations: constant (Linear Equations) B: r 1 =r 2 +r 3 D: r 3 =r 4 +r 5 PH: -r 2 +r 4 =0 r1 A (Substrate) Cell B D r3 r2 r5 r4 C (Target Product) E (By-Product) F (By-Product) (Vector Form) r 0 r 1 r 0 r r =
22 ATP ADP ATP ADP Glucose Glucose6P Fructose6P Fructose1,6bisP Question 1 Figure shows a metabolic pathway of glycolysis. Make one stoichiometric equation, summarizing up from glucose to pyruvate. ADP ATP Glyceraldehyde3P 1,3Diphosphoglycerate 3Phosphoglycerate 1. Gluc+2ADP+2=2PYR+2ATP+2 2. Gluc+ADP+=2PYR+ATP+ 3. Gluc+=2PYR+ ADP ATP Phosphoenolpyruvate pyruvate Fig. A metabolic pathway of glycolysis. 22
23 Answer of Q1 1. Gluc+2ADP+2=2PYR+2ATP+2 Gluc+ATP=G6P+ADP Gluc6P=F6P F6P+ATP=F1,6BP+ADP F1,6BP=2(G3P) 2(G3P)+2=2(1,3PG)+2 2(1,3PG)+2ADP=2(3PG)+2ATP 2(3PG)=2PEP 2PEP+2ADP=2PYR+2ATP Gluc+(4-2)ADP+2=2PYR+2ATP+(4-2) 23
24 rgluc r1:gluc+2adp+2=2pyr+2atp+2 ADP ATP rc2 C2 Acetyl-P Acetate rac r5 r1 r3 Pyruvate Acetyl-CoA r2 Formate Lactate Acetaldehyde Ethanol reth rlac rf Fig. Metabolism from glucose in homo- and hetero-fermentativelactococci. r4 r6 Question 2 Figure Shows a fermentative metabolism from glucose in homo- and hetero-fermentative Lactococci. 2-1 Make balance eq. of pyruvate with reaction rates ri. 2-2 Make balance eq. of AcCoA 2-3 Make balance eq. of / 2-4 Determine all the ri fluxes when rgluc=100, rlac=80, and rc2=20 are measured. 2-5 How much ATP is totally formed? 2-6 Confirm that in homofermentative metabolism from 1mol gluc 2mol Lac and 2mol ATP are produced to make / balance 24
25 rgluc r1:gluc+2adp+2=2pyr+2atp+2 ADP C2 rc2 Acetyl-P r5 r1 Pyruvate Lactate r2 rlac r3 r4 Formate rf Acetyl-CoA r6 Question 2 Fig. Shows a fermentative metabolism from glucose in homo- and hetero-fermentative Lactococci. 2-1 Make balance eq. of pyruvate 2-2 Make balance eq. of AcCoA 2-3 Make balance eq. of / ATP Acetate rac reth Fig. Metabolism from glucose in homo- and hetero-fermentativelactococci. 25
26 rgluc r1 Pyruvate r2 Lactate rlac Stoichiometric Equations C2 rc2 r3 r4 Acetyl-CoA Formate rf r1:gluc+2adp+2=2pyr+2a TP+2 r2:pyr+=lac ADP ATP Acetyl-P r5 Acetate rac Acetaldehyde r6 Ethanol reth r3: PYR=AceCoA++C2 r4: PYR=FR+AcCoA r5:accoa=ac+atp r6: AcCoA+2=EtH Fig. Metabolism from glucose in homo- and hetero-fermentativelactococci. 26
27 rgluc r1:gluc+2adp+2=2pyr+2atp+2 C2 rc2 r1 Pyruvate r2 r3 r4 Formate Acetyl-CoA Answer Balance eq. of pyruvate 2r1-r2-r3-r4=0 2-2 Balance eq. of AcCoA r3+r4-r5-r6=0 2-3 Balance eq. of / 2r1-r2+r3-2r6=0 ADP Acetyl-P r5 r6 ATP Acetate rac reth Fig. Metabolism from glucose in homo- and hetero-fermentativelactococci. 27
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