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 CO2, NH4, H2S, H2O
Amino acids Energy
Small molecules Protein
Oxygen 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 Weight (mg) Approx u moles / L

g dry cell per g dry cell MW
Proteins 5081 643 50,000 12.9

Nucleotides
RNA 630 216 100,000 2.2
DNA 100 33 2,000,000 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 composition
Dry weight vs. wet weight
70% of the composition is water

CHxOyNz
Dry weight consists of:
Element E. coli Yeast

C 50% 50%
O 20% 34%
N 14% 8%
H 8% 6%
P 3% 1%
S 1% <1%
K 1% <1%
Na 1% <1%
Others <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 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 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
Redox reactions of NAD+ / NADH
Nicotinamide 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

 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
GDP+Pi CO2+NADH
FADH2
 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
Co-enzyme A,
carries acetyl groups
pyruvate + NAD+ + CoA-SH → (2 Carbon)

acetyl CoA + CO2 + NADH + H+

Occurs in the cytoplasm

Acetyl CoA is transferred into the
mitochondria of eukaryotes
 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

Lactate Acetyl CoA Acetate

Malate

Oxaloacetate Pyruvate Ethanol

Acetaldehyde

Acetoacetyl CoA Acetolactate

Acetoin Formate CO2

Butanol
H2
Butyrate Butylene glycol
 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

Pyruvate
Lactate

TCA cycle
NADH

Acetate Acetyl CoA

Ethanol
Citrate
Oxaloacetate
NADH

Isocitrate
Malate
Fermentation CO2+NADH

a-Ketoglutarate
Fumarate
GTP
Succinate
GDP+Pi CO2+NADH
FADH2
 NAD+ Lactate
CH3CHOHCOO
NADH
Glycolysis
Glucose Pyruvate CO2 + H2O
C6H12O6 CH3CCOO O2
H+
O
CO2 Ethanol
+
CH3CH2OH
NAD
Acetaldehyde NADH

CHOCH3
 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 → lactic acid + energy

C6H12O6 2 C3H6O3 2 ATP

Anaerobic metabolism

Lactic acid fermentation

Alcoholic 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 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