Transcript Metabolic pathways

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
Objectives: Lecture 1
Develop basic metabolic processes
Carbon flow
Energy production
Cell as a black box
Inputs
Outputs
Cell
Sugars
Amino acids
Small molecules
Oxygen
CO2, NH4, H2S, H2O
Energy
Protein
Large 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
6236
962.6
NA
NA
TOTAL
Mosier and Ladisch, 2006
Cell composition
Dry weight vs. wet weight
70% of the composition is water
CHxOyNz
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%
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
fumarate
fumarate → succinate
iron
Fe3+ → Fe2+
Shewanella putrefaciens
nitrate
NO3- → NO2-
Thiobacillus denitrificans
sulfate
SO42+ → HS-
Desulfovibrio desulfuricans
Methanosarcina barkeri
Proteus rettgeri
Energy currency
ATP
NADH
FADH2
Adenosine triphosphate
Nicotinamide adenine dinucleotide
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+ / NADH
Nicotinamide adenine dinucleotide
O
H
O
H
H
CNH2
+ H+
CNH2
+ 2 e-
N+
N
R
R
NAD+
NADH
NAD+ is the electron acceptor in many reactions
Glycolysis
Glucose
Glucose 6-Phosphate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Dihydroxyacetone phosphate
Glyceraldehyde 3-Phosphate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
TCA cycle
NADH
Acetate
Acetyl CoA
Citrate
Oxaloacetate
NADH
Isocitrate
Malate
CO2+NADH
a-Ketoglutarate
Fumarate
GTP
Succinate
FADH2
GDP+Pi
CO2+NADH
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
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 consumed
Aerobic
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
Succinate
Malate
Lactate
Oxaloacetate
Pyruvate
Acetyl CoA
Acetate
Ethanol
Acetaldehyde
Acetoacetyl CoA
Acetolactate
Acetoin
Butanol
Butyrate
Butylene glycol
Formate
CO2
H2
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.
Glycolysis
Glucose
Glucose 6-Phosphate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Dihydroxyacetone phosphate
Glyceraldehyde 3-Phosphate
2-Phosphoglycerate
Phosphoenolpyruvate
Lactate
Pyruvate
TCA cycle
NADH
Acetate
Acetyl CoA
Ethanol
Citrate
Oxaloacetate
NADH
Fermentation
Isocitrate
Malate
CO2+NADH
a-Ketoglutarate
Fumarate
GTP
Succinate
FADH2
GDP+Pi
CO2+NADH
NAD+
NADH
Glycolysis
Glucose
C6H12O6
Lactate
CH3CHOHCOO
Pyruvate
CH3CCOO
O
CO2 + H2O
O2
H+
CO2
Ethanol
CH3CH2OH
+
NAD
Acetaldehyde
CHOCH3
NADH
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
soups
Saccharomyces
species
Cells are round, oval,
or elongated
Multiply by budding
Cell metabolism
If no oxygen is available
Glucose
C6H12O6
→
lactic acid + energy
2 C3H6O3
Anaerobic metabolism
Lactic acid fermentation
Alcoholic fermentation
2 ATP
Cell metabolism
Glucose + oxygen → carbon dioxide + water + energy
C6H12O6
6 O2
6 CO2
If plenty of oxygen is available
Aerobic metabolism
6H2O
36 ATP
Summary of metabolism
Pathway
NADH
FADH2
ATP
Glycolysis
PDH
TCA
2
2
6
0
0
2
2
0
2
Total ATP
(+ ox phos)
6
6
24
Total
10
2
4
36
or,
Fermentation
1-2
0
0-2
1-4