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