The dynamic responses of reserve carbohydrates with respect to shortage of either carbon or nitrogen source was studied to obtain a sound basis for further investigations devoted to the characterization of mechanisms by which the yeast Saccharomyces cerevisiae can cope with nutrient limitation during growth. This study was carried out in well‐controlled bioreactors which allow accurate monitoring of growth and frequent sampling without disturbing the culture. Under glucose limitation, genes involved in glycogen and trehalose biosynthesis (GLG1, GSY1, GSY2, GAC1, GLC3, TPS1 ), in their degradation (GPH1, NTH1 ), and the typical stress‐responsive CTT1 gene were coordinately induced in parallel with glycogen, when the growth has left the pure exponential phase and while glucose was still plentiful in the medium. Trehalose accumulation was delayed until the diauxic shift, although TPS1 was induced much earlier, due to hydrolysis of trehalose by high trehalase activity. In contrast, under nitrogen limitation, both glycogen and trehalose began to accumulate at the precise time when the nitrogen source was exhausted from the medium, coincidentally with the transcriptional activation of genes involved in their metabolism. While this response to nitrogen starvation was likely mediated by the stress‐responsive elements (STREs) in the promoter of these genes, we found that these elements were not responsible for the co‐induction of genes involved in reserve carbohydrate metabolism during glucose limitation, since GLG1, which does not contain any STRE, was coordinately induced with GSY2 and TPS1. Copyright © 1999 John Wiley & Sons, Ltd.

The gene encoding the dextransucrase DsrD from the industrial strain Leuconostoc mesenteroides Lcc4 was isolated by PCR using degenerate primers recognizing conserved regions present in other dextransucrase-encoding genes from Leuconostoc spp. and Southern blot analyses on total genomic DNA. N-terminal sequence analysis of the active protein recovered in the culture showed that the secreted protein of 165 kDa is devoid of a 42 aa prepeptide which is removed post-translationally, most likely by signal peptidase cleavage. Primer extension and Northern blot analysis identified a monocistronic dsrD mRNA with two transcription initiation sites. Expression of the dextransucrase DsrD was investigated in pH-controlled fed-batch cultures via Northern blot analysis and enzyme activity measurement during the experiments. Sucrose levels of 20 g l 21 wereshown to induce the DsrD biosynthesis around 10-fold. The combination of pH-controlled fed-batch fermentation and Northern analysis clearly showed that dsrD expression was related to the growth of the bacteria. dsrD was transferred to and expressed in Lactococcus lactis MG1363. Controlled fed-batch cultures revealed that active dextransucrase was produced and secreted by the recombinant L. lactis strain. The expression was independent of sucrose levels. These results show that dextransucrase can be efficiently expressed and secreted in a non-Leuconostoc, heterologous host and is able to drive dextran synthesis.