Download Role of TCA cycle and glyoxylate shunt for succinic acid production

Role of TCA cycle and glyoxylate shunt for succinic acid
production in yeast during dough fermentation
1. Introduction
Saccharomyces cerevisiae or ,,baker’s yeast’’ is an eukaryotic microorganism with an
extremely important role in the food and beverage industry. Because its ability to produce
ethanol, via alcoholic fermentation of different sugars as carbon sources, it’s widely used for
the industrial production of alcoholic beverages like beer, wine or sake.
During the fermentation process CO2 is formed as a by-product. This gas is important in the
fermentation of dough and is responsible for the volume increase of dough and therefore gives
bread its characteristic shape.
The TCA cycle plays an important role for oxidative growth of Saccharomyces cerevisiae. In
the presence of high glucose concentration though, the corresponding enzymes are down
regulated. In this scenario yeast prefers alcoholic fermentation to aerobic respiration for the
metabolism of glucose even under aerobic conditions. This behavior is especially interesting
as alcoholic fermentation gains the organism merely 2 mol of ATP out of 1 mol of Glucose,
whereas the gain using TCA cycle is 32 ATP.
Even dough the TCA cycle is down regulated, a residual activity remains. By using the TCA
cycle many organic acids like succinate, malate and citrate are produced and excreted by
yeast. Especially succinate is known to give beverages like sake its characteristic bitterness.
For the case of bread, the fermentation in dough is considered to be generally anaerobic. Yet
there are traces of succinate found in bread and may even influence its quality. Therefore this
is the first time that the production of succinate and the contribution of the oxidative and
reductive TCA cycle and the glyoxylate shunt during dough fermentation are being
investigated.
2. Impact of mutations on growth and production
In order to investigate the 3 possible pathways for production of succinate, mutants were
constructed by knocking out of important enzymes in the TCA-cycle and glyoxylate shunt.
These mutants were than analyzed for their production of succinate and their fermentation
capacity as well as their growth profile.
Knockout of Aconitase and Isocitrate Lyase leads to blocking of oxidative TCA cycle and
glyoxylate shunt. It was shown that double deletion of both enzymes resulted in a significant
decrease of succinate and the mutants lost their ability to enter post-diauxic respiratory growth
phase after depletion of glucose.
Similar results have been obtained for the deletion of NAD+ and NADP+ dependent Isocitrate
Dehydrogenase whereas knockout of both enzymes was necessary for significant decrease of
succinate production. Deletion of α-Ketoglutarate Dehydrogenase led only to a slight
decrease. Knockout of both subunits of Succinate Dehydrogenase was necessary to
significantly increase succinate levels. These results suggest that both oxidative pathways
(oxidative TCA and glyoxylate shunt) interact in the formation of succinate.
In order to investigate the importance of reductive TCA cycle Fumarate Reductase has been
deleted. The results showed no effect on succinate production, suggesting that reductive TCA
cycle isn’t involved in succinate formation.
3. Impact of succinic acid on dough fermentation
Knockout strains with increased (ΔAconitase and Isocitrate Lyase) and decreased succinate
production (ΔIsocitrate Dehydrogenase) were investigated using a rheofermentometer in order
to investigate the impact of succinate on dough fermentation characteristics.
It was shown that the level of succinate doesn’t significantly affect maximum dough height,
fermentation rate, total dough volume or pH.
4. Conclusion
Even though the conditions in dough were believed to be anaerobic, the results shown in this
experiment proof that succinate is being produced via oxidative TCA cycle and glyoxylat
shunt instead of reductive TCA cycle. The presence of oxygen necessary for these reactions to
happen goes against the general belief of anaerobic conditions in dough. The authors of the
paper suggest that O2 is physically adsorbed by dough matrix which is composed of hydrated
starch granules and fibers. This small amount of O2 seems to be sufficient for functionality of
oxidative TCA cycle and glyoxylate shunt enzymes for production of succinate. On the other
it was shown that the reductive TCA cycle isn’t involved in succinate production. Besides
changes in the production of succinate, deletion of key enzymes of the oxidative TCA cycle
and glyoxylate shunt also affect fermentation rate and growth profile of Saccharomyces
cerevisiae.
Comparison of dough fermentation using yeast mutants with increased and decreased
succinate production showed that the concentration of succinate doesn’t affect the properties
of the resulting dough. The concentration differences of succinate in these mutants might be
too little to observe a significant change. The fact that pH in both resulted dough didn’t vary
suggests a high buffering capacity of dough. Changes of dough properties might only be
achieved with higher concentration differences outside the pH buffering range. To further
investigate this, it would be interesting to knock out a high producing commercial yeast strain
with four time’s higher succinate concentration yields.
Sources:
•
Rezaei M.N et al: Contribution of the tricarboxylic acid (TCA) cycle and the
glyoxylate shunt in Saccharomyces cerevisiae to succinic acid production during
dough fermentation. Int J Food Microbiol. 2015 Jul 2;204:24-32. doi: 10.1016
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https://de.wikipedia.org/wiki/Alkoholische_G%C3%A4rung
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https://en.wikipedia.org/wiki/Diauxie
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http://faculty.bsc.edu/phanson/yen/culture.htm