Transcriptional responses to glucose in Saccharomyces cerevisiae strains lacking a functional protein kinase A

Background The pattern of gene transcripts in the yeast Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA), triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. In S. cerevisiae three genes, TPK1, TPK2, and TPK3, encode catalytic subunits of PKA. The lack of viability of tpk1 tpk2 tpk3 triple mutants may be suppressed by mutations such as yak1 or msn2/msn4. To investigate the requirement for PKA in glucose control of gene expression, we have compared the effects of glucose on global transcription in a wild-type strain and in two strains devoid of PKA activity, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4. Results We have identified different classes of genes that can be induced -or repressed- by glucose in the absence of PKA. Representative examples are genes required for glucose utilization and genes involved in the metabolism of other carbon sources, respectively. Among the genes responding to glucose in strains devoid of PKA some are also controlled by a redundant signalling pathway involving PKA activation, while others are not affected when PKA is activated through an increase in cAMP concentration. On the other hand, among genes that do not respond to glucose in the absence of PKA, some give a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways. We show also that, for a number of genes controlled by glucose through a PKA-dependent pathway, the changes in mRNA levels are transient. We found that, in cells grown in gluconeogenic conditions, expression of a small number of genes, mainly connected with the response to stress, is reduced in the strains lacking PKA. Conclusions In S. cerevisiae, the transcriptional responses to glucose are triggered by a variety of pathways, alone or in combination, in which PKA is often involved. Redundant signalling pathways confer a greater robustness to the response to glucose, while cooperative pathways provide a greater flexibility.BT/BiotechnologyApplied Science

Switching the mode of sucrose utilization by <it>Saccharomyces cerevisiae</it>

<p>Abstract</p> <p>Background</p> <p>Overflow metabolism is an undesirable characteristic of aerobic cultures of <it>Saccharomyces cerevisiae </it>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 <it>S. cerevisiae </it>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.</p> <p>Results</p> <p>We have deleted from the genome of a <it>S. cerevisiae </it>strain lacking invertase the high-affinity sucrose-H⁺symporter encoded by the <it>AGT1 </it>gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H⁺symport activity mediated by the <it>MALx1 </it>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.</p> <p>Conclusion</p> <p>Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a <it>S. cerevisiae </it>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 <it>S. cerevisiae</it>.</p