engineering of biological processes lecture 1: metabolic pathways mark riley, associate professor...
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Engineering of Biological Processes
Lecture 1: Metabolic pathways Mark Riley, Associate Professor
Department of Ag and Biosystems Engineering
The University of Arizona, Tucson, AZ2007
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Objectives: Lecture 1
Develop basic metabolic processes
Carbon flow
Energy production
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Cell as a black box
Cell
Inputs Outputs
SugarsAmino acidsSmall moleculesOxygen
CO2, NH4, H2S, H2OEnergyProteinLarge molecules
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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
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Concentration of components in a cell
Component u moles per g dry cell
Weight (mg) per g dry cell
Approx MW
u moles / L
Proteins 5081 643 50,000 12.9
Nucleotides
RNA
DNA
630
100
216
33
100,000
2,000,000
2.2
0.000016
Lipo-polysaccharides 218 40 1,000 40
Peptidoglycan 166 28.4 10,000 2.8
Polyamines 41 2.2 1,000 2.2
TOTAL 6236 962.6 NA NA
Mosier and Ladisch, 2006
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Cell compositionDry weight vs. wet weight
70% of the composition is water
Dry weight consists of:
Element E. coli Yeast
C O N H P S K
Na Others
50% 20% 14% 8% 3% 1% 1% 1%
<1%
50% 34% 8% 6% 1%
<1% <1% <1% <1%
CHxOyNz
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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
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Other inputs to metabolism
Compounds General reaction Example of a species
carbonate CO2 → CH4 Methanosarcina barkeri
fumarate fumarate → succinate Proteus rettgeri
iron Fe3+ → Fe2+ Shewanella putrefaciens
nitrate NO3- → NO2- Thiobacillus denitrificans
sulfate SO42+ → HS- Desulfovibrio desulfuricans
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Energy currency
ATP Adenosine triphosphateNADH 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
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Redox reactions of NAD+ / NADHNicotinamide adenine dinucleotide
N+
R
H
CNH2
O
N
R
H
CNH2
OH
+ H+
NAD+ NADH
+ 2 e-
NAD+ is the electron acceptor in many reactions
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Glucose Glucose 6-Phosphate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Glyceraldehyde 3-Phosphate
Pyruvate
Acetate Acetyl CoA
Citrate
-Ketoglutarate
Succinate
Fumarate
Oxaloacetate
MalateIsocitrate
CO2+NADHFADH2
CO2+NADH
NADH
NADH
GTP
GDP+Pi
Phosphoenolpyruvate
Dihydroxyacetone phosphate
2-Phosphoglycerate
Glycolysis
TCA cycle
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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 molecules2 ATP produced / 1 Glucose consumedAnaerobic
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Pyruvate dehydrogenase
pyruvate + NAD+ + CoA-SH →
acetyl CoA + CO2 + NADH + H+
Occurs in the cytoplasm
Acetyl CoA is transferred into the mitochondria of eukaryotes
Co-enzyme A, carries acetyl groups(2 Carbon)
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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
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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 consumedAerobic
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Overall reaction
Complete aerobic conversion of glucose
Glucose + 36Pi + 36 ADP + 36 H+ + 6O2→
6 CO2 + 36 ATP + 42 H2O
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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
CO2
H2
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Fermentation
No electron transport chain (no ox phos).Anaerobic processGlucose (or other sugars) converted to
lactate, pyruvate, ethanol, many othersEnergy 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.
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Glucose Glucose 6-Phosphate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Glyceraldehyde 3-Phosphate
Pyruvate
Acetate Acetyl CoA
Citrate
-Ketoglutarate
Succinate
Fumarate
Oxaloacetate
MalateIsocitrate
CO2+NADHFADH2
CO2+NADH
NADH
NADH
GTP
GDP+Pi
Phosphoenolpyruvate
Dihydroxyacetone phosphate
2-Phosphoglycerate
Glycolysis
TCA cycle
Lactate
Ethanol
Fermentation
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GlucoseC6H12O6
Glycolysis PyruvateCH3CCOO
O
AcetaldehydeCHOCH3
EthanolCH3CH2OH
NADHNAD+
CO2 + H2O
LactateCH3CHOHCOO
NADH
NAD+
O2
H+
CO2
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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)
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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.
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Yeasts Only a few species are
associated with fermentation of food and alcohol products, leavening bread, and to flavor soupsSaccharomyces
speciesCells are round, oval,
or elongatedMultiply by budding
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Cell metabolism
If no oxygen is available
Glucose → lactic acid + energy
C6H12O6 2 C3H6O3 2 ATP
Anaerobic metabolism
Lactic acid fermentationAlcoholic fermentation
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Cell metabolism
Glucose + oxygen → carbon dioxide + water + energy
C6H12O6 6 O2 6 CO2 6H2O 36 ATP
If plenty of oxygen is available
Aerobic metabolism
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Summary of metabolismPathway NADH FADH2 ATP Total ATP
(+ ox phos)Glycolysis 2 0 2 6PDH 2 0 0 6TCA 6 2 2 24
Total 10 2 4 36
or,Fermentation 1-2 0 0-2 1-4