Glucose Consumption - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

Glucose is the main source of energy in most organisms, occupying a central position in metabolism. Glycolysis is a sequence of enzymatic reactions that occur in the cytoplasm of cells and allows the cleavage of glucose molecules, resulting in two molecules of pyruvate and a small amount of ATP and reducing equivalents. The fate of the pyruvate molecule depends on the organism and determines the amount of ATP that can be obtained. Pyruvate can follow three different metabolic paths, depending on the organism and environmental conditions.

Under aerobic conditions in in mammals, pyruvate is turned into Acetyl-CoA and directed to the mitochondria.

Acetyl-CoA is is oxidized to CO₂ in the citric acid cycle with the production of reducing power in the form of NADH and FADH₂ and small amounts of GTP.

The potential energy of the electrons in NADH and FADH₂ are used to pump protons out of the mitochondria and ultimately used to reduce O₂ to water.

Under anaerobic conditions pyruvate is instead converted into lactic acid, increasing lactic acid levels in the blood.

Yeasts are unicellular organisms widely used in industrial processes. Saccharomyces cerevisiae is responsible for fermentation during the production of fermented beverages such as wine and beer, as well as in baking.

Under anaerobic conditions, these yeasts can metabolize glucose, diverting pyruvate to the formation of ethanol and carbon dioxide in a process called alcoholic fermentation.

Pyruvate can be directly converted to acetyl-CoA by the pyruvate dehydrogenase (PDH) complex in the mitochondria.

Alternatively, pyruvate can also be converted to acetyl-CoA in the cytosol via the PDH bypass pathway through acetaldehyde and acetate.

In yeasts, this bypass requires the activity of three different enzymes:

1. PDC (pyruvate decarboxylase) which converts pyruvate to acetaldehyde
2. ALD (acetaldehyde dehydrogenase), which converts acetaldehyde to acetate
3. ACS (acetyl-CoA synthetase), which converts acetate to acetyl-CoA

which can then be transported to the mitochondria via the carnitine acetyltransferase system.

At high glucose concentrations, the glycolytic rate increases, forming more pyruvate, saturating the PDH bypass, and redirecting carbon flow through ethanol production, initiating fermentation.

The central carbon metabolism pathways are essentially identical among different yeast species. However, the mechanisms for nutrient absorption, the isoenzymes involved, and the regulation of respiratory and fermentative processes differ substantially.

For S. cerevisiae, high glucose concentrations increase pyruvate decarboxylase activity 3-4 times and decrease acetaldehyde dehydrogenase activity, favoring alcoholic fermentation and thus ethanol formation. Conversely, at low glucose concentrations, pyruvate is primarily converted to acetyl-CoA by the PDH complex.

The acetyl-CoA formed in the PDH complex or the PDH bypass is the link between glycolysis and the tricarboxylic acid (TCA) cycle. The main function of the TCA cycle is to provide reducing equivalents to the respiratory chain (see above). However, the TCA cycle also functions in biosynthetic pathways and, as such, may be partially active even under anaerobic conditions.

Yeasts can be physiologically classified according to the type of energy metabolism involved in sugar metabolism:

• Non-fermentative (aerobic or respiratory)
• Obligatory fermentative
• Facultative fermentative

Non-fermentative yeasts have a respiratory metabolism and cannot perform alcoholic fermentation from glucose (e.g. Rhodotorula glutinis, a famous oleaginous yeast), whereas obligatory fermentative yeasts can only metabolize glucose through alcoholic fermentation (e.g. Piromyces).

Most identified yeasts are facultative fermentative and, depending on growth conditions, sugar type and concentration, and oxygen availability, can exhibit respiratory, fermentative, or mixed metabolism.

There are two main effects associated with energy acquisition through sugar metabolism and/or oxygen availability in yeasts: Pasteur and Crabtree effects.

The Pasteur effect refers to the activation of anaerobic glycolysis to meet cellular ATP requirements due to the low efficiency of ATP production by fermentation compared to aerobic respiration. Louis Pasteur was investigating how yeast converts sugars into alcohol during fermentation. He noticed that when oxygen was present, yeast would produce much less alcohol and consume less sugar compared to when oxygen was absent. This was contrary to the then-prevailing belief that oxygen was necessary for all forms of life to produce energy efficiently.

The Crabtree effect is defined as the occurrence of alcoholic fermentation even under aerobic conditions (where it is not expected). In Crabtree-negative yeasts, there is no significant ethanol production under aerobic conditions. Saccharomyces cerevisiae is considered Crabtree-positive.

Torulaspora delbrueckii is yeast species that has gained attention due to its unique properties and applications in various industrial processes, especially in food and beverage production. It is closely related to Saccharomyces cerevisiae but differs significantly in its metabolic and physiological characteristics. Torulaspora delbrueckii can ferment a variety of sugars, including glucose, fructose, and sucrose. Torulaspora delbrueckii generally produces less ethanol and more glycerol compared to S. cerevisiae, resulting in less alcoholic beverages, making it suitable for low-alcohol wines and beers.

In this practical class, we will assess glucose consumption by Saccharomyces cerevisiae or Torulaspora delbrueckii yeast cells during short time in the presence or absence of oxygen.

In this class, two different yeast species will be used: Saccharomyces cerevisiae and Torulaspora delbrueckii. Before the class, the yeast suspension was prepared by dissolving the respective cells in a buffered saline solution (PBS) at pH 7.4.

Yeast cells will be incubated for a short time (up to 15 min). This means that we do not have to worry about sterility of the experiment. It is important to work rapidly.

Group	Respiratory Condition	Yeast	Container	PBS (µL)	10% Glucose (µL)	Cell suspension (µL)
1	Anaerobic	S. cerevisiae	Closed Eppendorf tube (1.5 mL)	400	80	200
2	"	T. delbrueckii	"	"	"	"
3	Aerobic	S. cerevisiae	Open culture plate	"	"	"
4	"	T. delbrueckii	"	"	"	"

1. Prepare eight Eppendorf tubes (1.5 mL) per group. The experiment should be performed in duplicate.
2. Mark them with 0, 5 10 and 15 on the lid. These are the incubation times in minutes.
3. Add 400 µL PBS to each tube.
4. Add 80 µL Glucose solution to each tube.
5. Mix the tube with cell suspension by inversion so that content is uniformly mixed.
6. Add 200 µL cell suspension to each Eppendorf tube with PBS+glucose.
7. Remove the t=0 tube and put this tube on ice immediately.
8. Incubate the remaining six tubes in the 37°C water bath.
9. At each time point, remove a tube from the bath and put it on ice.
10. When the last incubation is over, centrifuge all tubes at >10,000 rpm for 5 minutes.
11. Transfer the supernatant from each tube to a clean, labeled Eppendorf tube and freeze at -20°C.

1. Add 400 µL PBS to each of eight wells in two open culture plates. The experiment should be performed in duplicate.
2. Add 80 µL Glucose solution to each of the wells.
3. Mix the tube with cell suspension by inversion so that content is uniformly mixed.
4. Add 200 µL cell suspension to each of four wells
5. Remove the contents of the t=0 well and add it to an Eppendorf tube, put this tube on ice immediately
6. Incubate the cells in an incubator at 37°C and 120 rpm for 5, 10, and 15 minutes, respectively.
7. At each time point, remove the contents of a well and add it to an Eppendorf tube, put this tube on ice immediately.
8. When the last incubation is over, centrifuge all tubes at >10,000 rpm for 5 minutes.
9. Transfer the supernatant from each tube to a clean, labeled Eppendorf tube and freeze at -20°C.

• 30 mL S. cerevisiae cell suspension (15 g/100 mL) in PBS
• 30 mL T. delbrueckii cell suspension (15 g/100 mL) in PBS

Freeze 5 mL in six different tubes.

• 4 FALCON tube with 10 mL of 10% glucose solution
• 4 FALCON tubes with 50 mL PBS
• P1000 pipettes
• P200 pipettes
• Blue tips (not sterile)
• yellow tips (not sterile)
• Freezer box for storing Eppendorf tubes with supernatants
• 4 Iceboxes
• 4 culture plates with 6 wells

• 24 Eppendorf tubes (2 mL)

• 100 Eppendorf tubes
• Incubator at 37°C
• Water batch at 37°C
• Eppendorf centrifuge