Download Molecular Biology of the Cell

Supervision 5. L10. Examples of questions
1. Name three activated carriers needed in anabolic reactions and where they
are synthesized.
2. Would a mammalian cell culture be a good system to produce bioethanol? Explain.
3. Possible reasons why some organisms are not able to assimilate specific sugars
(e.g. pentoses; disaccharides such as lactose). Any solution?
4. Net result of complete oxidation of glucose (100% respiratory metabolism).
Could the total ATP per glucose generated by a microorganism (e.g. yeast) be
different compared to animal cells? Explain.
5. Net result and possible phenotypes of complete catabolism of glucose
in absence of oxygen: a) In mammalian cells. b) In yeast.
Supervision 5. L10. Examples of questions
6. Net result of catabolism of glucose by an anaerobe facultative organism
(e.g. Saccharomyces cerevisiae ,‘budding yeast’, able to assimilate carbon by both
respiratory and fermentative pathways, i.e. ‘respirofermentative’ metabolism)
under environmental conditions leading to: 25% of carbon being metabolized by
respiration; 75% by fermentation.
7. Main cause (and consequences) of appearance of lactic fermentation in animal cells.
Among the organisms and conditions mentioned in 4), 5) and 6) :
8. What organisms would be adequate for production of bioethanol/biofuels?
9. Which ones would be preferable as hosts for synthesis of biomass and
coupled-to-growth (type-1) products? (e.g. recombinant proteins).
10. Would an environmental medium containing sugars, fats, nitrogen sources, salts
and vitamins be a good substrate for biotechnological processes? Suggest
possible effects/phenotypes in microorganisms and animal cells.
Supervision 5. L10. Answers to questions. Examples.
1.
a) ATP (main energy carrier). Produced in catabolic reactions such as glycolysis,
alcoholic and/or lactic fermentation, tricarboxilic acid (TCA) cycle + oxidative
phoshorylation and oxidation of fats.
b) NADH. Produced in catabolic reactions such as glycolysis, alcoholic and/or
lactic fermentation, the tricarboxilic acid (TCA) cycle.
c) Acetyl-CoA. Synthesized in reactions such as the tricarboxilic acid (TCA) cycle.
d) NADPH. Synthesized in the pentose-phosphate pathway.
e) FADH2. Synthesized in the TCA cycle.
------------------------------------------------------------------------------------------------------------------------------------------------------
Supervision 5. L10. Answers to questions. Examples.
2. No. In absence of oxygen, mammalian cells can obtain energy converting pyruvate
to lactate (i.e. lactic fermentation) not to ethanol. Some microorganisms (e.g.
Saccharomyces yeasts) have evolved to assimilate sugars (e.g. sucrose, fructose,
glucose, from processed sugar cane, molasses, etc) at a high rate, leading to high
glycolytic fluxes, high fluxes of pyruvate synthesis and high levels of ethanol
production (alcoholic fermentation). These are commonly used in bioethanol
production processes.
------------------------------------------------------------------------------------------------------------------------------------------------------
( Example )
Basso LC, de Amorim HV, de Oliveira AJ, Lopes ML. (2008) Yeast selection for fuel ethanol production in
Brazil. FEMS Yeast Res. 8, 1155-1163.
(….The present paper reports on a yeast selection program performed during the last 12 years aimed at selecting
Saccharomyces cerevisiae strains suitable for fermentation of sugar cane substrates (cane juice and molasses) with
cell recycle, as it is conducted in Brazilian bioethanol plants. As a result, some evidence is presented showing the
positive impact of selected yeast strains in increasing ethanol yield and reducing production costs, due to their
higher fermentation performance (high ethanol yield, reduced glycerol and foam formation, maintenance of high
viability during recycling and very high implantation capability into industrial fermenters).
Supervision 5. L10. Answers to questions. Examples.
3.
With the exception of glucose, not all sugars in nature can be readily assimilated.
Some of them (e.g. polysaccharides such as starch or cellulose, disaccharides such
as lactose, pentoses such as xylose) need to be hydrolyzed, transported into the
cell and/or converted by specific enzymes before entering the glycolytic pathway.
Only a few organisms contain the genes able to express these specific enzymes,
and can grow using these compounds as the main carbon source (e.g.
Saccharomyces yeasts cannot grow on lactose but Kluyveromyces yeasts and
bacteria such as Lactobacillus are able to express the necessary enzymes and
assimilate them). In specific cases metabolic engineering strategies (e.g. genetic
engineering) are needed to allow an organism to grow on a specific carbon and/or
nitrogen source. Example:
---------------------------------------------------------------------------------------------------------------------------------------------------
(Example)
Matsushika A, Watanabe S, Kodaki T, Makino K, Sawayama S. (2008) Bioethanol production from
xylose by recombinant Saccharomyces cerevisiae expressing xylose reductase, NADP(+)-dependent
xylitol dehydrogenase, and xylulokinase. J Biosci. Bioeng. 105, 296-299.
Supervision 5. L10. Answers to questions. Examples.
4. Reactions involved (100% respiratory metabolism, 0% fermentation):
-- > Glycolysis (glucose to pyruvate) / pyruvate to acetyl-CoA (?) / TCA cycle /
-- > oxidative phosphorylation (variable stoichiometry)
[1]
[2]
[3]
Glycolysis. From glucose (6C) to pyruvate (3C):
Glucose + 2NAD+ + 2ADP + 2Pi -- > 2Pyruvate + 2NADH+ 2ATP
From pyruvate (3C) to acetyl-CoA (2C):
Pyruvate + (? –mitoch. transport-) + CoA + NAD+ -- > acetyl-CoA + NADH+ + CO2
TCA cycle (one turn of the TCA cycle)
acetyl-CoA + 3NAD+ + GDP + FAD -- > 3NADH+ + GTP + FADH2 + 2CO2
Net result:
[1] + 2 [2] + 2 [3] + oxidative phosphorylation (variable stoichiometry).
e.g. (glucose … -- > 2ATP (glyc) + 10NADH+ + 2GTP + 2FADH2 + 6CO2)
(5O2 + 10NADH; O2+ 2FADH2 + variable ATP produced per H+)
Net result:
Glucose (C6H12O6) + 6 O2 (g) → 6 CO2 (g) + 6 H2O + n (?) ATP
( Biology textbooks often state that 38 ATP molecules can be made per oxidized glucose molecule during cellular respiration:
2 from glycolysis, 2 from the Krebs cycle (from GTP), and about 34 from the electron transport system). However, this max.
yield is never quite reached due to losses (leaky membranes), cost of moving pyruvate and ADP into the mitochondrial
matrix AND the variable stoichiometry of the FoF1ATPase. Textbooks estimates range around 29 to 30 ATP per glucose ).
ATP per H+ (2e) generated by the yeast FoF1ATPase ≠ ATP per H+ (2e) generated by mammalian ATPase.
Thus, ATP per glucose generated by yeast ≠ ATP per glucose generated by mammalian cells.
Supervision 5. L10. Answers to questions. Examples.
5. Reactions involved (100% fermentation):
Mammalian cells -- > Glycolysis (glucose to pyruvate) + pyruvate to lactate (Lactic fermentation)
[1]
Glycolysis. From glucose (6C) to pyruvate (3C):
Glucose + 2NAD+ + 2ADP + 2Pi -- > 2Pyruvate + 2NADH+ + 2ATP
[4]
From pyruvate (3C) to lactate (3C)
Pyruvate + 2NADH+ -- > Lactate + 2NAD+
Net result: [1] + 2 [4] -- >
Glucose + 2ADP + 2Pi -- > 2 lactate + 2ATP
Possible phenotypes:
During power exercises in which the rate of demand for energy is high, lactate is produced faster than the ability of the
tissues to remove it, so lactate concentration begins to rise. Contrary to popular belief, this increased concentration of
lactate does not directly cause acidosis, nor is it responsible for delayed onset muscle soreness. This is because lactate
itself is not capable of releasing a proton. The acidosis that is associated with increases in lactate concentration
during heavy exercise arises from a separate reaction. When ATP is hydrolysed, a hydrogen ion is released. ATPderived hydrogen ions are responsible primarily for the decrease in pH. During intense exercise, aerobic metabolism
cannot produce ATP quickly enough to supply the demands of the muscle. As a result, anaerobic metabolism becomes the
dominant energy-producing pathway, as it can form ATP at high rates. Due to the large amounts of ATP being produced
and hydrolysed in a short period of time, the buffering systems of the tissues are overcome, causing pH to fall and creating
a state of acidosis.
Supervision 5. L10. Answers to questions. Examples.
5. Reactions involved (100% fermentation):
Yeast -- > Glycolysis (glucose to pyruvate) + pyruvate to ethanol (Alcoholic fermentation)
[1]
Glycolysis. From glucose (6C) to pyruvate (3C):
Glucose + 2NAD+ + 2ADP + 2Pi -- > 2Pyruvate + 2NADH+ + 2ATP
[5]
From pyruvate (3C) to ethanol (2C)
[5a] Pyruvate -- > CO2 + CH3CHO
(enzyme pyruvate decarboxylase, PDC)
[5b] CH3CHO + NADH+ -- > ethanol + NAD+
Net result: [1] + 2 [5] -- >
(enzyme alcohol dehydrogenase, ADH)
Glucose + 2ADP + 2Pi -- > 2 ethanol + 2ATP + 2CO2
Possible phenotypes: Ethanol excreted into the medium until reaching concentrations that can be toxic to
potential competitors (i.e. S. cerevisiae high tolerance to ethanol). -- > Final high alcoholic concentrations under
anaerobic conditions (e.g. wine fermentations). Under presence of oxygen some specific strains may reconsume
the ethanol produced (diauxic growth).
Supervision 5. L10. Answers to questions. Examples.
5. Reactions involved (100% fermentation):
Yeast -- > Glycolysis (glucose to pyruvate) + pyruvate to ethanol (Alcoholic fermentation)
[1]
Glycolysis. From glucose (6C) to pyruvate (3C):
Glucose + 2NAD+ + 2ADP + 2Pi -- > 2Pyruvate + 2NADH+ + 2ATP
[5]
From pyruvate (3C) to ethanol (2C)
[5a] Pyruvate -- > CO2 + CH3CHO
(enzyme pyruvate decarboxylase, PDC)
[5b] CH3CHO + NADH+ -- > ethanol + NAD+
Net result: [1] + 2 [5] -- >
(enzyme alcohol dehydrogenase, ADH)
Glucose + 2ADP + 2Pi -- > 2 ethanol + 2ATP + 2CO2
Possible phenotypes: Ethanol excreted into the medium until reaching concentrations that can be toxic to
potential competitors (i.e. S. cerevisiae high tolerance to ethanol). -- > Final high alcoholic concentrations under
anaerobic conditions (e.g. wine fermentations). Under presence of oxygen some specific strains may reconsume
the ethanol produced (diauxic growth).
Supervision 5. L10. Answers to questions. Examples.
6. Reactions involved:
(25% respiration): From 4):
0.25 (Glucose + 6 O2 → 6 CO2 + 6 H2O + n ATP) = 0.25 glucose + 1.5 O2 → 1.5 CO2 + 1.5 H2O + 0.25n ATP
(75% fermentation) -- > (In yeast, alcoholic fermentation). From 5:
0.75 (Glucose +2ADP+ 2Pi - > 2 ethanol + 2ATP + 2CO2 ) =
0.75 glucose+1.5ADP+1.5Pi -> 1.5 ethanol+1.5ATP + 1.5CO2
Net result: Glucose + 1.5 O2 + 1.5ADP + 1.5Pi -- > 1.5 ethanol + 3CO2 + 1.5 H2O + 0.25n ATP + 1.5ATP
Glucose + 1.5 O2 + 1.5ADP + 1.5Pi -- > 1.5 ethanol + 3CO2 + 1.5 H2O + 0.25 (n +6) ATP
7.
Shortage of oxygen (for example, under intense exercise) -- > high glycolytic fluxes with low availability of
oxygen, -- > concentration of pyruvate in the cytosol can increase and a % be diverted towards lactic
fermentation in order to obtain more ATP. If complete depleted of oxygen (e.g in the muscle), lactate fermentation
can become the only pathway able to generate ATP from glucose. Consequences/phenotypes explained in answer
to question 5.
Supervision 5. L10. Answers to questions. Examples.
8.
Organisms assimilating sugars with high glycolytic rates towards alcoholic fermentation (e.g. the
yeast S. cerevisiae, in the presence of very low levels of oxygen).
9.
The productivity of ‘coupled-to-growth’ compounds, is by definition, proportional to the amount
of biomass (cell mass) obtained (i.e. biomass yield). This is higher in organisms assimilating
sugars under respiratory metabolism (e.g. ‘respiratory’ yeasts not able to produce ethanol, and/or
yeast or mammalian cells in the presence of excess of oxygen which results in a 100%
respiratory metabolism. This fully respiratory metabolism will also minimize the synthesis of
byproducts that may be toxic or delay growth (e.g. acetate, ethanol, organic acids)
10.
In a biotechnological process (e.g. with microorganisms), a rich medium resulting in
simultaneously consumption of sugars and fats may result in an internal metabolic imbalance
(the catabolism of sugars and fats converge at the level of acetyl-CoA). If this is not transitory
but sustained, may affect cell growth and final yields.
Rich diets in mammals (e.g. with high amounts of sugars and proteins) result in storage of
excess of nutrients as fats (in fat cells), to be reused in the future under conditions of food
shortage. A diet reach in sugars and fats (intrinsic metabolic imbalance) may result in maximum
storage of fats which, if sustained, -not counteracted-, may lead to increased imbalances and
health problems (e.g. diabetes).