Engineering of Biological Processes Lecture 1: Metabolic pathways

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Engineering of Biological ProcessesLecture 1
Metabolic pathways

• Mark Riley, Associate Professor
• Department of Ag and Biosystems Engineering
• The University of Arizona, Tucson, AZ
• 2007

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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
Sugars Amino acids Small molecules Oxygen
CO2, NH4, H2S, H2O Energy Protein Large 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 composition
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 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

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Redox reactions of NAD / NADHNicotinamide
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
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Glycolysis
Glucose
Glucose 6-Phosphate
Fructose 6-Phosphate
Dihydroxyacetone phosphate
Fructose 1,6-Bisphosphate
Glyceraldehyde 3-Phosphate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
TCA cycle
Acetyl CoA
Acetate
Citrate
Oxaloacetate
Isocitrate
Malate
a-Ketoglutarate
Fumarate
Succinate
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Glycolysis

• Also called the EMP pathway (Embden-Meyerhoff-Parn
  as).
• 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

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

• pyruvate NAD CoA-SH ?
• acetyl CoA CO2 NADH H
• Occurs in the cytoplasm
• Acetyl CoA is transferred into the mitochondria
  of eukaryotes

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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 consumed
• Aerobic

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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
Succinate
Acetate
Acetyl CoA
Lactate
Malate
Ethanol
Pyruvate
Oxaloacetate
Acetaldehyde
Acetolactate
Acetoacetyl CoA
Formate
CO2
Acetoin
Butanol
H2
Butylene glycol
Butyrate
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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.

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Glycolysis
Glucose
Glucose 6-Phosphate
Fructose 6-Phosphate
Dihydroxyacetone phosphate
Fructose 1,6-Bisphosphate
Glyceraldehyde 3-Phosphate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
Lactate
TCA cycle
Acetyl CoA
Acetate
Ethanol
Citrate
Oxaloacetate
Isocitrate
Malate
Fermentation
a-Ketoglutarate
Fumarate
Succinate
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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 soups
• Saccharomyces species
• Cells are round, oval, or elongated
• Multiply 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
fermentation Alcoholic 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 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