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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
Objectives: Lecture 1
Develop basic metabolic processes
Carbon flow
Energy production
Cell as a black box
Cell
Inputs Outputs
SugarsAmino acidsSmall moleculesOxygen
CO2, NH4, H2S, H2OEnergyProteinLarge molecules
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
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
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
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
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
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
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
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
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
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)
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
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
Overall reaction
Complete aerobic conversion of glucose
Glucose + 36Pi + 36 ADP + 36 H+ + 6O2→
6 CO2 + 36 ATP + 42 H2O
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
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.
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
GlucoseC6H12O6
Glycolysis PyruvateCH3CCOO
O
AcetaldehydeCHOCH3
EthanolCH3CH2OH
NADHNAD+
CO2 + H2O
LactateCH3CHOHCOO
NADH
NAD+
O2
H+
CO2
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)
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.
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
Cell metabolism
If no oxygen is available
Glucose → lactic acid + energy
C6H12O6 2 C3H6O3 2 ATP
Anaerobic metabolism
Lactic acid fermentationAlcoholic fermentation
Cell metabolism
Glucose + oxygen → carbon dioxide + water + energy
C6H12O6 6 O2 6 CO2 6H2O 36 ATP
If plenty of oxygen is available
Aerobic metabolism
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