Engineering of Biological Processes - Metabolic pathways

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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, AZ 2007

Objectives: Lecture 1
Develop basic metabolic processes Carbon flow Energy production

Cell as a black box
Inputs Cell
Sugars Amino acids Small molecules Oxygen CO2, NH4, H2S, H2O Energy Protein Large molecules

Outputs

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 Proteins Nucleotides RNA DNA
Lipo-polysaccharides Peptidoglycan Polyamines TOTAL

u moles per g dry cell 5081

Weight (mg) per g dry cell 643

Approx MW 50,000

u moles / L 12.9

630 100 218 166 41 6236

216 33 40 28.4 2.2 962.6

100,000 2,000,000 1,000 10,000 1,000 NA

2.2 0.000016 40 2.8 2.2 NA

Mosier and Ladisch, 2006

Cell composition
Dry weight vs. wet weight 70% of the composition is water Dry weight consists of: Element C O N H P S K Na Others E. coli 50% 20% 14% 8% 3% 1% 1% 1% <1% Yeast 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 carbonate CO2 → CH4 Example of a species Methanosarcina barkeri

fumarate iron
nitrate

fumarate → succinate Fe3+ → Fe2+
NO3- → NO2-

Proteus rettgeri

Shewanella putrefaciens
Thiobacillus denitrificans

sulfate

SO42+ → HS-

Desulfovibrio desulfuricans

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
H O H

O H
CNH2

CNH2

+ H+
N+
R

+ 2 eN
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
NADH

Acetate

Acetyl CoA Citrate

TCA cycle

Oxaloacetate
NADH

Malate

Isocitrate
CO2+NADH

Fumarate Succinate
FADH2

a-Ketoglutarate
GTP CO2+NADH

GDP+Pi

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+
Co-enzyme A, carries acetyl groups (2 Carbon)

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

Malate
Oxaloacetate

Lactate Pyruvate

Acetyl CoA

Acetate Ethanol

Acetaldehyde

Acetoacetyl CoA
Butanol

Acetolactate Acetoin Formate
H2

CO2

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
NADH

Lactate

Acetate
Ethanol

Acetyl CoA Citrate

TCA cycle

Oxaloacetate
NADH

Fermentation

Malate

Isocitrate
CO2+NADH

Fumarate Succinate
FADH2

a-Ketoglutarate
GTP CO2+NADH

GDP+Pi

NAD+

Lactate CH3CHOHCOO

Glycolysis
Glucose C6H12O6

NADH

Pyruvate CH3CCOO O

CO2 + H2O
O2 H+ CO2 NAD

Ethanol CH3CH2OH +

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 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
Glycolysis PDH TCA

NADH
2 2 6

FADH2
0 0 2

ATP
2 0 2

Total ATP (+ ox phos) 6 6 24 36

Total or, Fermentation

10

2

4

1-2

0

0-2

1-4


				
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