Engineering of Biological Processes Lecture 1: Metabolic pathways

1. Engineering of Biological ProcessesLecture 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 Inputs Outputs Sugars Amino acids Small molecules Oxygen CO2, NH4, H2S, H2O Energy Protein Large molecules

4. Metabolic processes • Catabolic = Breakdown: • generation of energy and reducing power from complex molecules • produces small molecules (CO2, NH3) 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 Mosier and Ladisch, 2006

6. Cell composition CHxOyNz

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 Compounds General reaction Example of a species carbonate CO2 → CH4Methanosarcina barkeri fumarate fumarate → succinate Proteus rettgeri iron Fe3+ → Fe2+Shewanella putrefaciens nitrate NO3- → NO2-Thiobacillus denitrificans sulfate SO42+ → HS-Desulfovibrio desulfuricans

9. Energy currency • ATP Adenosine triphosphate • NADH Nicotinamide adenine dinucleotide • FADH2 Flavin adenine dinucleotide • The basic reactions for formation of each are: • ADP + Pi → ATP • AMP + Pi → ADP • NAD+ + H+ → NADH • FADH + H+ → FADH2

10. Redox reactions of NAD+ / NADHNicotinamide adenine dinucleotide O O H H H CNH2 CNH2 + H+ + 2 e- N+ N R R NAD+ NADH NAD+ is the electron acceptor in many reactions

11. NADH NADH CO2+NADH GTP CO2+NADH GDP+Pi FADH2 Glycolysis Glucose Glucose 6-Phosphate Fructose 6-Phosphate Dihydroxyacetone phosphate Fructose 1,6-Bisphosphate Glyceraldehyde 3-Phosphate 2-Phosphoglycerate Phosphoenolpyruvate Pyruvate TCA cycle Acetyl CoA Acetate Citrate Oxaloacetate Isocitrate Malate a-Ketoglutarate Fumarate Succinate

12. Glycolysis • Also called the EMP pathway (Embden-Meyerhoff-Parnas). • Glucose + 2 Pi + 2 NAD+ + 2 ADP → • 2 Pyruvate + 2 ATP + 2 NADH + 2H+ + 2 H2O • 9 step process with 8 intermediate molecules • 2 ATP produced / 1 Glucose consumed • Anaerobic

13. Pyruvate dehydrogenase Co-enzyme A, carries acetyl groups (2 Carbon) • pyruvate + NAD+ + CoA-SH → • acetyl CoA + CO2 + NADH + H+ • Occurs in the cytoplasm • Acetyl CoA is transferred into the mitochondria of eukaryotes

14. Citric Acid Cycle • The overall reaction is: • Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O → • 3 NADH + 3H+ + FADH2 + CoA-SH + GTP + 2 CO2 • 2 ATP (GTP) produced / 1 Glucose consumed • Anaerobic

15. Oxidative phosphorylation – (respiration) • Electrons from NAD and FADH2 are used to power the formation of ATP. • NADH + ½ O2 + H+ → H2O + NAD+ • ADP + Pi + H+ → ATP + H2O • 32 ATP produced / 1 Glucose consumed • Aerobic

16. Overall reaction • Complete aerobic conversion of glucose • Glucose + 36Pi + 36 ADP + 36 H+ + 6O2→ • 6 CO2 + 36 ATP + 42 H2O

17. Products of anaerobic metabolism of pyruvate Succinate Acetyl CoA Acetate Lactate Malate Ethanol Pyruvate Oxaloacetate Acetaldehyde Acetolactate Acetoacetyl CoA Formate CO2 Acetoin Butanol H2 Butylene glycol Butyrate

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. NADH NADH CO2+NADH GTP CO2+NADH GDP+Pi FADH2 Glycolysis Glucose Glucose 6-Phosphate Fructose 6-Phosphate Dihydroxyacetone phosphate Fructose 1,6-Bisphosphate Glyceraldehyde 3-Phosphate 2-Phosphoglycerate Phosphoenolpyruvate Pyruvate Lactate TCA cycle Acetyl CoA Acetate Ethanol Citrate Oxaloacetate Isocitrate Malate Fermentation a-Ketoglutarate Fumarate Succinate

20. Lactate CH3CHOHCOO NAD+ NADH Glycolysis Glucose C6H12O6 Pyruvate CH3CCOO CO2 + H2O O O2 H+ Ethanol CH3CH2OH CO2 NAD+ Acetaldehyde CHOCH3 NADH

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 + CO2 • 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 C6H12O6 2 C3H6O3 2 ATP Anaerobic metabolism Lactic acid fermentation Alcoholic fermentation

25. Cell metabolism Glucose + oxygen → carbon dioxide + water + energy C6H12O6 6 O2 6 CO2 6H2O 36 ATP If plenty of oxygen is available Aerobic metabolism

26. Summary of metabolism • Pathway NADH FADH2 ATP Total ATP (+ ox phos) • Glycolysis 2 0 2 6 • PDH 2 0 0 6 • TCA 6 2 2 24 • Total 10 2 4 36 • or, • Fermentation 1-2 0 0-2 1-4