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Switching the mode of sucrose utilization by Saccharomyces cerevisiae.

Badotti F, Dário MG, Alves SL, Cordioli ML, Miletti LC, de Araujo PS, Stambuk BU - Microb. Cell Fact. (2008)

Bottom Line: Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes.It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields.Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil. bstambuk@mbox1.ufsc.br.

ABSTRACT

Background: Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures.

Results: We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source.

Conclusion: Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae.

No MeSH data available.


Related in: MedlinePlus

Biomass yields by yeast strains. The relative biomass yields, normalized to the values obtained by the wild-type 1403-7A strain in medium containing 20 g/L glucose, were determined during growth of strain 1403-7A (black symbols) or strain LCM001 (red symbols) in synthetic yeast nitrogen medium containing 5 (triangles), 10 (squares) or 15 (inverted triangles) g/L ammonium sulfate (open symbols) or peptone (close symbols) as nitrogen source, and 20 g/L of the indicated carbon sources. Results obtained with rich YP medium (circles) in the absence (open symbols) or presence (close symbols) of antimycin A are also shown. For the wild-type 1403-7A strain the Yx/s values varied between 0.20 ± 0.02 and 0.48 ± 0.03 g biomass (g glucose)-1 when ammonium sulfate or peptone were used as nitrogen source, respectively, or 0.14 g biomass (g sucrose)-1 when antimycin A was added to the medium.
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Figure 6: Biomass yields by yeast strains. The relative biomass yields, normalized to the values obtained by the wild-type 1403-7A strain in medium containing 20 g/L glucose, were determined during growth of strain 1403-7A (black symbols) or strain LCM001 (red symbols) in synthetic yeast nitrogen medium containing 5 (triangles), 10 (squares) or 15 (inverted triangles) g/L ammonium sulfate (open symbols) or peptone (close symbols) as nitrogen source, and 20 g/L of the indicated carbon sources. Results obtained with rich YP medium (circles) in the absence (open symbols) or presence (close symbols) of antimycin A are also shown. For the wild-type 1403-7A strain the Yx/s values varied between 0.20 ± 0.02 and 0.48 ± 0.03 g biomass (g glucose)-1 when ammonium sulfate or peptone were used as nitrogen source, respectively, or 0.14 g biomass (g sucrose)-1 when antimycin A was added to the medium.

Mentions: We thus analyzed the biomass yield of these two strains during growth using three different carbon sources (glucose, maltose and sucrose) and different amounts (and quality) of nitrogen sources (Figure 6). It is evident that strain LCM001 produces from 1.5- to 2-fold more biomass during batch growth on sucrose (compared to the parental strain), while the biomass yields on glucose or maltose were unaffected. Conversely, while the ethanol yields on glucose by both the wild-type and the agt1Δ strain were similar and varied from Ye/s values between 0.16 ± 0.02 and 0.31 ± 0.03 g ethanol (g glucose)-1 when ammonium sulfate or peptone were used as nitrogen source, respectively, the agt1Δ strain LCM001 produced only 10 to 40% of the ethanol produced by the wild-type strain 1403-7A when sucrose was used as carbon source. In order to confirm that this increase in biomass production by the LCM001 strain is due to respiration of the sugar, we added amtimycin A to the medium. Under this condition (respiration blocked), the biomass yield on sucrose of strain LCM001 was exactly the same as for the parental strain 1403-7A (Fig. 6), and both strains fermented sucrose efficiently (Ye/s = 0.50 ± 0.01 g ethanol [g sucrose]-1).


Switching the mode of sucrose utilization by Saccharomyces cerevisiae.

Badotti F, Dário MG, Alves SL, Cordioli ML, Miletti LC, de Araujo PS, Stambuk BU - Microb. Cell Fact. (2008)

Biomass yields by yeast strains. The relative biomass yields, normalized to the values obtained by the wild-type 1403-7A strain in medium containing 20 g/L glucose, were determined during growth of strain 1403-7A (black symbols) or strain LCM001 (red symbols) in synthetic yeast nitrogen medium containing 5 (triangles), 10 (squares) or 15 (inverted triangles) g/L ammonium sulfate (open symbols) or peptone (close symbols) as nitrogen source, and 20 g/L of the indicated carbon sources. Results obtained with rich YP medium (circles) in the absence (open symbols) or presence (close symbols) of antimycin A are also shown. For the wild-type 1403-7A strain the Yx/s values varied between 0.20 ± 0.02 and 0.48 ± 0.03 g biomass (g glucose)-1 when ammonium sulfate or peptone were used as nitrogen source, respectively, or 0.14 g biomass (g sucrose)-1 when antimycin A was added to the medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268662&req=5

Figure 6: Biomass yields by yeast strains. The relative biomass yields, normalized to the values obtained by the wild-type 1403-7A strain in medium containing 20 g/L glucose, were determined during growth of strain 1403-7A (black symbols) or strain LCM001 (red symbols) in synthetic yeast nitrogen medium containing 5 (triangles), 10 (squares) or 15 (inverted triangles) g/L ammonium sulfate (open symbols) or peptone (close symbols) as nitrogen source, and 20 g/L of the indicated carbon sources. Results obtained with rich YP medium (circles) in the absence (open symbols) or presence (close symbols) of antimycin A are also shown. For the wild-type 1403-7A strain the Yx/s values varied between 0.20 ± 0.02 and 0.48 ± 0.03 g biomass (g glucose)-1 when ammonium sulfate or peptone were used as nitrogen source, respectively, or 0.14 g biomass (g sucrose)-1 when antimycin A was added to the medium.
Mentions: We thus analyzed the biomass yield of these two strains during growth using three different carbon sources (glucose, maltose and sucrose) and different amounts (and quality) of nitrogen sources (Figure 6). It is evident that strain LCM001 produces from 1.5- to 2-fold more biomass during batch growth on sucrose (compared to the parental strain), while the biomass yields on glucose or maltose were unaffected. Conversely, while the ethanol yields on glucose by both the wild-type and the agt1Δ strain were similar and varied from Ye/s values between 0.16 ± 0.02 and 0.31 ± 0.03 g ethanol (g glucose)-1 when ammonium sulfate or peptone were used as nitrogen source, respectively, the agt1Δ strain LCM001 produced only 10 to 40% of the ethanol produced by the wild-type strain 1403-7A when sucrose was used as carbon source. In order to confirm that this increase in biomass production by the LCM001 strain is due to respiration of the sugar, we added amtimycin A to the medium. Under this condition (respiration blocked), the biomass yield on sucrose of strain LCM001 was exactly the same as for the parental strain 1403-7A (Fig. 6), and both strains fermented sucrose efficiently (Ye/s = 0.50 ± 0.01 g ethanol [g sucrose]-1).

Bottom Line: Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes.It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields.Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil. bstambuk@mbox1.ufsc.br.

ABSTRACT

Background: Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures.

Results: We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source.

Conclusion: Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae.

No MeSH data available.


Related in: MedlinePlus