Switching the mode of sucrose utilization by Saccharomyces cerevisiae

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.

Figure 1

Kinetics of active H⁺-sucrose symport activity in yeast strains. The initial rates of active H⁺ co-transport with sucrose were determined in yeast cells from strain 1403-7A (solid circles), or its agt1Δ counterpart strain LCM001 (hollow squares), pre-grown in rich YP media containing 20 g/L sucrose.

Figure 2

Detection of active sucrose transporter genes in yeast strains. Separation of chromosomes of yeast strains by PFGE and staining with ethidium bromide (A), and detection of sucrose transporter genes after chromosomes were blotted onto a nylon membrane and hybridized with a probe for AGT1 (B) or MALx1 (C). Lanes 1, strain 1403-7A; lanes 2, the agt1Δ strain LCM001. The values on the right in panel A are the sizes (in kilobase pairs) of selected chromosomes, the Roman numerals between panels A and B are chromosome numbers, and the positions of chromosomes carrying AGT1, MAL21, MAL31 and MAL41 are indicated on the right of panel C. Both chromosome sizes and numbers, and locations of transporter genes, were based on results obtained with the reference strains S288C and MC966A (see Table 1, data not shown).

Figure 3

Batch fermentations of 20 g/L sucrose by yeast strains. The consumption of sucrose, and ethanol and biomass production during sucrose batch fermentations with 10 g/L yeast cells (dry weight) of strain 1403-7A (black symbols) or strain LCM001 (open symbols), were determined using synthetic YNB (circles) or rich YP (squares) medium. Data shown are means (± range) from two independent experiments.

Figure 4

Batch fermentations of 250 g/L sucrose by yeast strains. The consumption of sucrose (circles), and ethanol (triangles) and biomass (squares) production during sucrose batch fermentations with 10 g/L yeast cells (dry weight) of strain 1403-7A (black symbols) or strain LCM001 (open symbols) were determined with rich YP medium. Data shown are means (± range) from two independent experiments.

Figure 5

Batch growth of yeast strains on 20 g/L sucrose. The consumption of sucrose and the ethanol and biomass production by strain 1403-7A (solid circles) or strain LCM001 (hollow circles) were determined during growth in rich YP medium. For each strain a representative growth curve is shown.

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 Y_x/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.