1. Engineering of Biological Processes Lecture 1: Metabolic pathways Mark Riley, Associate Professor Department of Ag and Biosystems Engineering The University of Arizona, Tucson, AZ 2007

2. Objectives: Lecture 1 Develop basic metabolic processes Carbon flow Energy production

3. Cell as a black box Cell InputsOutputs Sugars Amino acids Small molecules Oxygen CO 2, NH 4, H 2 S, H 2 O Energy Protein Large molecules

4. Metabolic processes Catabolic = Breakdown: generation of energy and reducing power from complex molecules produces small molecules (CO 2, NH 3 ) for use and as waste products Anabolic = Biosynthesis: construction of large molecules to serve as cellular components such as amino acids for proteins, nucleic acids, fats and cholesterol usually consumes energy

5. Concentration of components in a cell Componentu moles per g dry cell Weight (mg) per g dry cell Approx MW u moles / L Proteins508164350,00012.9 Nucleotides RNA DNA 630 100 216 33 100,000 2,000,000 2.2 0.000016 Lipo-polysaccharides218401,00040 Peptidoglycan16628.410,0002.8 Polyamines412.21,0002.2 TOTAL6236962.6NA Mosier and Ladisch, 2006

6. Cell composition CH x O y N z

7. Inputs (cellular nutrients) Carbon source –sugars glucose, sucrose, fructose, maltose polymers of glucose: cellulose, cellobiose Nitrogen –amino acids and ammonia Energy extraction: –oxidized input → reduced product –reduced input → oxidized product

8. Other inputs to metabolism CompoundsGeneral reactionExample of a species carbonateCO 2 → CH 4 Methanosarcina barkeri fumaratefumarate → succinateProteus rettgeri ironFe 3+ → Fe 2+ Shewanella putrefaciens nitrateNO 3- → NO 2- Thiobacillus denitrificans sulfateSO 4 2+ → HS - Desulfovibrio desulfuricans

9. Energy currency ATPAdenosine triphosphate NADHNicotinamide adenine dinucleotide FADH 2 Flavin adenine dinucleotide The basic reactions for formation of each are: ADP + P i → ATP AMP + P i → ADP NAD + + H + → NADH FADH + H + → FADH 2

10. Redox reactions of NAD + / NADH Nicotinamide adenine dinucleotide N+N+ R H CNH 2 O N R H O H + H + NAD + NADH + 2 e - NAD + is the electron acceptor in many reactions

11. GlucoseGlucose 6-Phosphate Fructose 6-Phosphate Fructose 1,6-Bisphosphate Glyceraldehyde 3-Phosphate Pyruvate Acetate Acetyl CoA Citrate  -Ketoglutarate Succinate Fumarate Oxaloacetate Malate Isocitrate CO 2 +NADH FADH 2 CO 2 +NADH NADH GTP GDP+P i Phosphoenolpyruvate Dihydroxyacetone phosphate 2-Phosphoglycerate Glycolysis TCA cycle

12. Glycolysis Also called the EMP pathway (Embden-Meyerhoff-Parnas). Glucose + 2 P i + 2 NAD + + 2 ADP → 2 Pyruvate + 2 ATP + 2 NADH + 2H + + 2 H 2 O 9 step process with 8 intermediate molecules 2 ATP produced / 1 Glucose consumed Anaerobic

13. Pyruvate dehydrogenase pyruvate + NAD + + CoA-SH → acetyl CoA + CO 2 + NADH + H + Occurs in the cytoplasm Acetyl CoA is transferred into the mitochondria of eukaryotes Co-enzyme A, carries acetyl groups (2 Carbon)

14. Citric Acid Cycle The overall reaction is: Acetyl-CoA + 3 NAD + + FAD + GDP + P i + 2 H 2 O → 3 NADH + 3H + + FADH 2 + CoA-SH + GTP + 2 CO 2 2 ATP (GTP) produced / 1 Glucose consumed Anaerobic

15. Oxidative phosphorylation – (respiration) Electrons from NAD and FADH 2 are used to power the formation of ATP. NADH + ½ O 2 + H + → H 2 O + NAD + ADP + P i + H + → ATP + H 2 O 32 ATP produced / 1 Glucose consumed Aerobic

16. Overall reaction Complete aerobic conversion of glucose Glucose + 36P i + 36 ADP + 36 H + + 6O 2 → 6 CO 2 + 36 ATP + 42 H 2 O

17. Products of anaerobic metabolism of pyruvate Pyruvate Lactate Acetate Acetaldehyde Ethanol Formate Acetolactate Acetoin Butylene glycol Acetoacetyl CoA Butanol Butyrate Oxaloacetate Malate Succinate Acetyl CoA CO 2 H2H2

18. Fermentation No electron transport chain (no ox phos). Anaerobic process Glucose (or other sugars) converted to lactate, pyruvate, ethanol, many others Energy yields are low. Typical energy yields are 1-4 ATP per substrate molecule fermented. In the absence of oxygen, the available NAD + is often limiting. The primary purpose is to regenerate NAD + from NADH allowing glycolysis to continue.

19. GlucoseGlucose 6-Phosphate Fructose 6-Phosphate Fructose 1,6-Bisphosphate Glyceraldehyde 3-Phosphate Pyruvate Acetate Acetyl CoA Citrate  -Ketoglutarate Succinate Fumarate Oxaloacetate Malate Isocitrate CO 2 +NADH FADH 2 CO 2 +NADH NADH GTP GDP+P i Phosphoenolpyruvate Dihydroxyacetone phosphate 2-Phosphoglycerate Glycolysis TCA cycle Lactate Ethanol Fermentation

20. Glucose C 6 H 12 O 6 Glycolysis Pyruvate CH 3 CCOO O Acetaldehyde CHOCH 3 Ethanol CH 3 CH 2 OH NADH NAD + CO 2 + H 2 O Lactate CH 3 CHOHCOO NADH NAD + O2O2 H+H+ CO 2

21. Types of fermentation Lactic acid fermentation (produce lactate) –Performed by: Lactococci, Leuconostoc, Lactobacilli, Streptococci, Bifidobacterium Lack enzymes to perform the TCA cycle. Often use lactose as the input sugar (found in milk) Alcoholic fermentation (produce ethanol)

22. Alcoholic fermentation Operates in yeast and in several microorganisms Pyruvate + H + ↔ acetaldehyde + CO 2 Acetaldehyde + NADH + H + ↔ ethanol + NAD + Reversible reactions Acetaldehyde is an important component in many industrial fermentations, particularly for food and alcohol.

23. Yeasts Only a few species are associated with fermentation of food and alcohol products, leavening bread, and to flavor soups Saccharomyces species Cells are round, oval, or elongated Multiply by budding

24. Cell metabolism If no oxygen is available Glucose → lactic acid + energy C 6 H 12 O 6 2 C 3 H 6 O 3 2 ATP Anaerobic metabolism Lactic acid fermentation Alcoholic fermentation

25. Cell metabolism Glucose + oxygen → carbon dioxide + water + energy C 6 H 12 O 6 6 O 2 6 CO 2 6H 2 O 36 ATP If plenty of oxygen is available Aerobic metabolism

26. Summary of metabolism Pathway NADH FADH 2 ATP Total ATP (+ ox phos) Glycolysis 20 2 6 PDH 20 0 6 TCA 62 224 Total 102 4 36 or, Fermentation 1-20 0-21-4