1-3-3 Case studies in Systems Biology Goutham Vemuri goutham@chalmers.se Information transmission Fluxome Metabolome flux 1 flux flux 3 Proteome metabolite1 metabolite metabolite3 protein 1 protein protein 3 Interactome Transcriptome mrna A mrna B mrna C mrna D Genome gene A gene B gene C gene D Vemuri and Aristidou (5) Microbiol. Mol Biol Rev. 69():197-16 Tools in metabolic engineering Vemuri and Nielsen (6) 1
Number of publications 1-3-3 1 The post-genomic era 1 8 6 4 1995 1996 1997 1998 1999 1 Year Vemuri and Nielsen (6) 3 4 5 6 Case study 1 Overflow metabolism Overflow Metabolism ATP CO oxaloacetate malate citrate fumarate isocitrate succinate CO a-ketoglutarate ATP CO
1-3-3 Overflow Metabolism ATP CO oxaloacetate malate citrate fumarate isocitrate succinate CO a-ketoglutarate ATP CO Overflow Metabolism ATP CO oxaloacetate malate citrate fumarate isocitrate succinate CO a-ketoglutarate ATP CO Overflow Metabolism ATP CO oxaloacetate malate citrate fumarate isocitrate succinate CO a-ketoglutarate ATP CO 3
ATP CO CO 1-3-3 Overflow Metabolism glycerol (S. cerevisiae) ATP lactate (mammalian cells) ethanol (S. cerevisiae) CO acetate (E. coli) oxaloacetate malate citrate fumarate isocitrate succinate CO a-ketoglutarate ATP CO Hypothesis Limited capacity of TCA cycle enzymes Allosteric inhibition of the TCA cycle enzymes Glucose repression of genes of the TCA cycle oxaloacetate malate citrate fumarate isocitrate succinate CO a-ketoglutarate Overflow metabolism and redox Glucose + ADP + + + Pi NDHI NDHII + ATP + TCA cycle NDHI NDHII Pyruvate + GDP + FAD + 4 + 3CO + GTP + FADH + 4 4
1-3-3 Overflow metabolism and redox Glucose + ADP + + + Pi NDE1 NDE + ATP + TCA cycle NDI Pyruvate + GDP + FAD + 4 + 3CO + GTP + FADH + 4 -dependent product formation Berrios-Rivera et al., Metab. Eng., 4(3):17-9, Hypothesis Glucose + ADP + + + Pi Pyruvate + GDP + FAD + 4 + + ATP + 3CO + GTP + FADH + 4 oxidase nox + O + H O 5
METABOLISM 1-3-3 Methodology Transcription Metabolism Association Metabolic Models Biochemical Experiments New Functions Refinement Modify Interpretations New Hypotheses YES Phenotype explained correctly? NO Methodology Chemostats Physiological quantification (q S, Y x/s, q A, ) Intracellular quantification ([],[], metabolites, ) Global transcriptome profiling Response in E. coli Physiological Response qs (, ), YX/S (, ) 1.6 1.4 1. 1..8.6.4. NOX- NOX+ qo (, ), qco (, ) 4 3 1 NOX - NOX +...1..3.4.5.6 Specific Growth Rate (h-1)...4.6.8 1. 1. 1.4 1.6 Specific consumption rate (g/gh) Vemuri et al. (6) Appl. Environ. Microbiol. 7(5):3653-3661 6
1-3-3 Response in E. coli Overflow Metabolism.4 Specific acetate formation rate (g/gh).3..1....4.6.8 1. 1. 1.4 1.6 Specific uptake rate (g/gh) Vemuri et al. (6) Appl. Environ. Microbiol. 7(5):3653-3661 Response in E. coli Intracellular Response Molar / Ratio (, ).1.8.6.4.....4.6.8 1. 1. 1.4 1.6 Specific consumption rate (g/gh) NOX - 5 15 1 5.1..39.6.85 1.7 1.4 5 NOX + 15 1 5 16.1..47.75.98 1.5 1.34 14 Specific consumption rate (g/lh) 1 G6P (u mol / g DCW) 1 F6P (u mol/ g DCW) 8 PEP (u mol/ g DCW) Pyr (u mol/ g DCW) 6 ACoA (u mol / g DCW) Vemuri et al. (6) Appl. Environ. 4 Microbiol. 7(5):3653-3661 Response in E. coli 1. Transcriptional Response.8 log [ratio].6.4.. -. NOX - NOX + -.4 -.6 Glycolysis TCA cycle -.8 Pentose phosphate Respiration -1...4.6.8 1. 1. 1.4 1.6 qs (g/g DCW h) Vemuri et al. (6) Appl. Environ. Microbiol. 7(5):3653-3661 7
1-3-3 Regulation in E. coli Identifying targets for TFs.5. 1.5 1..5. -.5-1. -1.5. 1.. -1. -. -3. -4. -5. -6. -7. -8. (p = 1.5 E -43) (p = 1 E -17) arca Transport: Aerobic Respiratory Control 65 Cell envelop: Central metabolism: 9 46 Degradation Energy metabolism: of small molecules: 5 48 Unknown Fatty genes: acid biosynthesis: 11 1 Global regulatory functions: 8 Nucleotide Biosynthesis 36 Vemuri et al. (6) Appl. Environ. Microbiol. 7(5):3653-3661 Regulation in E. coli Acetate / ATP ArcA ATP ArcA ArcA TCA cycle genes ArcB ADP ArcB PO 4 Vemuri et al. Submitted Metabolic engineering in E. coli Physiological Response Increased growth rate Faster consumption Faster metabolism (higher q CO ) Reduced acetate formation Transcriptional Response TCA cycle genes not repressed Respiration genes not repressed Induction of amino acid genes Induction of probable global regulators Complete elimination of overflow metabolism in ΔarcA-NOX + US Pat Appl. 74918 8
1-3-3 Metabolic engineering in E. coli Recombinant protein production 4 1.4 3 1. -gal (U/g DCW) 1..8.6 Acetate (g/l) 1.4.. 4 6 8 1 1 14 Time (h) Vemuri et al. (6) Biotechnol. Bioeng. 94(3):538-54 Physiological Response 7 Ethanol 1.4 Glycerol 6 1. 5 CON NOX 1. CON NOX Ethanol (g/l) 4 3 Glycerol (g/l).8.6.4 1. 1 3 4 5 Time (h). 1 3 4 5 Time (h) Vemuri et al. PNAS, 7 Glycerol G3P DHAP TCA cycle Overkamp et al.,, J. Bacteriol. 18:83-83 9
1-3-3 Glycerol generation 5 4 Glycerol (g/l) 3 1 1 3 4 5 Time (h) NDE1 NDE GUT NDE1 NDE GUT - NOX Vemuri et al. PNAS, 7 Vemuri et al. PNAS, 7 Altering mitochondrial level 7 Ethanol 1.4 Glycerol 6 1. Ethanol (g/l) 5 4 3 CON NOX AOX Glycerol (g/l) 1..8.6 CON NOX AOX.4 1. 1 3 4 5 Time (h). 1 3 4 5 Time (h) Vemuri et al. PNAS, 7 1
Specific Activity (U/mg prot) / Ratio 1-3-3 Critical dilution rate DECISION SIGNAL Feed Reservoir Ethanol Sensor AOX =.33 h -1 CON =.9 h -1 NOX =.7 h -1 Vemuri et al. PNAS, 7 Steady-state response Strain Dilution Rate (h-1) q S Yield b q O q CO q eth q gly.1 C-limited 1.1.49.75.7 ND ND CON.7 C-limited 3.7.46 7.46 8.3 ND ND.1 N-Limited 4.83.11 4.4 1.11 6.16.4.1 C-limited 1.5.45 3.1 3.14 ND ND NOX.6 C-limited 4.4.39 1.77 11.7 ND ND.1 N-Limited 5.73.9 5.8 17.7 5.6..1 C-limited 1.18.47.99 3. ND ND AOX.3 C-limited 3.87.43 9.8 1.9 ND ND.1 N-Limited 5.7.11 5.54 17. 4.1.5 ND: Not Detected Vemuri et al. PNAS, 7 Intracellular response 1.8.5 1.5 1..9.6.3.4.3..1. C-lim (.1 h -1 ) N-lim (.1 h -1 ) C-lim (D crit) C-lim (.1 h -1 ) N-lim (.1 h -1 ) C-lim (D crit) CON NOX AOX Vemuri et al. PNAS, 7 11
1-3-3 Transcriptional response Metabolic pathways PGI F6P FBP PFK Fructose 6P NH3 Glucose 6P GABAxt NH3xt GABAxt Metabolic graph Reporter metabolites Transcription data TCA Cycle PGI Enzyme interaction FBP PFK graph Subnetwork structures Patil and Nielsen (5) PNAS 1(8):685-689 Transcriptional response Reporter metabolites in response to oxidase sn-glycerol 3-phosphate 3-Phosphonooxy - mitochondrial Acetaldehyde - mitochondrial 3-Phospho-D-glyceroyl phosphate Acetaldehyde Myristic acid Palmitate Stearate CYS xt - mitochondrial (3S)-3-Hydroxyacyl-CoA Glycerone phosphate p-value a 5.38E-9.18E-8 3.16E-3 3.98E-3 5.17E-3 6.6E-3 9.18E-3 9.5E-3 1.E- 1.E- 1.E- 1.E- 1.48E- 1.49E- 1.78E- Reporter metabolites in response to alternate oxidase p-value a - mitochondrial 7.48E-8 - mitochondrial 6.E-5 Orotate 1.37E-3 CoA - mitochondrial 1.53E-3 Oxaloacetate - mitochondrial.3e-3 Acetaldehyde - mitochondrial.5e-3 Oxygen - mitochondrial.85e-3 -Nonaprenyl-3-methyl-6- methoxy-1,4-benzoquinone.85e-3 mitochondrial -Phospho-D-glycerate 4.4E-3 Glyoxylate 4.6E-3 Ubiquinone-9 mitochondrial 4.74E-3 H+ - mitochondrial 6.58E-3 Malate 7.8E-3 Orotidine 5'-phosphate 8.8E-3 Isocitrate 9.8E-3 Vemuri et al. PNAS, 7 Subnetwork analysis NOX vs CON Glycerol pathway Ethanol generation Fatty acid oxidation oxidation Glyoxylate shunt Glycolysis Gluconeogenesis Vemuri et al. PNAS, 7 1
1-3-3 Subnetwork analysis AOX vs CON Glycolysis Gluconeogenesis Ethanol synthesis TCA cycle oxidation ATP translocation Fatty acid synthesis Glyoxylate shunt Nucleotide biosynthesis Vemuri et al. PNAS, 7 Enzymatic analysis.9 G3P DH.6 Glucose.3 6 ADH CON NOX AOX 4 Glycerol CON NOX AOX Pyruvate Ethanol.1 h -1 D crit Oxaloacetate Isocitrate 1 8 4 IDH -Ketoglutarate CON NOX AOX Vemuri et al. PNAS, 7 Conclusion Integrating data is context-dependent Transcriptional regulation is responsible for acetate formation in E. coli Allosteric inhibition of the TCA cycle enzymes in S. cerevisiae Compartmentalization causes overflow of different metabolites Absence of PDC in E. coli is likely why the acetate is overflow metabolite 13