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An improved glutamate-producing fed-batch process, using a temperature-sensitive strain of Corynebacterium glutamicum, has been characterized. By a tight control of the culture temperature, it was possible to get industrially interesting performance as regards glutamate concentration, yield and productivity. A 24 h fermentation period enabled the production of 85 g/l of glutamate in the production phase induced after a temperature shift from 33°C to 39°C. The maximum specific production rate of glutamate was 0.63 g/g/h with a yield of 0.46 g of glutamate/gram of glucose. The two main co-products of the fermentation were lactate (11 g) and trehalose (12 g). Only trace amounts of other organic acids accumulated in the culture medium. This process offers an interesting alternative to currently employed fermentation strategies in which biotin limitation and/or surfactant addition is used to induce glutamate production. Simple control of fermentor cooling can be used to control the onset of the production phase, offering significant advantages from both an economic and a process robustness viewpoint.

The dynamic behavior of the metabolism of Corynebacterium glutamicum during L-glutamic acid fermentation, was evaluated by quantitative analysis of the evolution of intracellular metabolites and key enzyme concentrations. Glutamate production was induced by an increase of the temperature and a final concentration of 80 g/l was attained. During the production phase, various other compounds, notably lactate, trehalose, and DHA were secreted to the medium. Intracellular metabolites analysis showed important variations of glycolytic intermediates and NADH, NAD coenzymes levels throughout the production phase. Two phenomena occur during the production phase which potentially provoke a decrease in the glutamate yield: Both the intracellular concentrations of glycolytic intermediates and the NADH/NAD ratio increase significantly during the period in which the overall metabolic rates decline. This correlates with the decrease in glutamate yield due in part to the production of lactate and also to the period of the fermentation in which growth no longer occurred.

We have determined the nucleotide sequence of a 3.3 kb fragment containing the gene (ADE2) encoding phosphoribosylaminoimidazole carboxylase (AIRC) from the yeast Schwanniomyces occidentalis. Translation of a 1671 bp open reading frame predicts a protein of 557 amino acids which has significant homology to AIRC from Saccharomyces cerevisiae and Schizosaccharomyces pombe. The 5' untranslated region of the S. occidentalis gene contains a sequence corresponding to the consensus binding site of the S. cerevisiae transcription regulatory proteins GCN4, BAS1 and BAS2.

Genome-wide analysis of transcriptional regulation is generally carried out on well-characterized reference laboratory strains; hence, the characteristics of industrial isolates are therefore overlooked. In a previous study on the major cheese yeast Kluyveromyces lactis , we have shown that the reference strain and an industrial strain used in cheese making display a differential gene expression when grown on a single carbon source. Here, we have used more controlled conditions, i.e., growth in a fermentor with pH and oxygen maintained constant, to study how these two isolates grown in glucose reacted to an addition of lactose. The observed differences between sugar consumption and the production of various metabolites, ethanol, acetate, and glycerol, correlated with the response were monitored by the analysis of the expression of 482 genes. Extensive differences in gene expression between the strains were revealed in sugar transport, glucose repression, ethanol metabolism, and amino acid import. These differences were partly due to repression by glucose and another, yet-unknown regulation mechanism. Our results bring to light a new type of K. lactis strain with respect to hexose transport gene content and repression by glucose. We found that a combination of point mutations and variation in gene regulation generates a biodiversity within the K. lactis species that was not anticipated. In contrast to S. cerevisiae , in which there is a massive increase in the number of sugar transporter and fermentation genes, in K. lactis , interstrain diversity in adaptation to a changing environment is based on small changes at the level of key genes and cell growth control.

Samples of the cheese yeast Rhodotorula glutinis were analysed during storage at +4 degrees C for cultivability, viability, vitality (metabolic activity), membrane potential state, intracellular pH, and carbohydrate content. The results have allowed to describe cellular events occurring during storage. The loss of vitality came with the decrease of carbohydrate content. The fall of trehalose content under a threshold value induced the deterioration of the membrane potential. Later, when all the cells were depolarised, the intracellular pH decreased and the cultivability dropped, whereas viable cells still decreased slowly. Then, it led to an intermediate physiological state similar to the viable but non-cultivable state. Finally, the fall of viability dropped. In this work, we have defined rapid methods relevant to describe the sequence of intracellular events in the cheese yeast R. glutinis during storage, and we applied them to understand the weak vitality without fall of viability of yeast samples. These methods might allow to rapidly test yeast sample quality before use and to predict, at the moment of the harvesting, the conservation of the yeast.

International audience; Glucose uptake by Corynebacterium glutamicum is predominantly assured by a mannose phosphotransferase system (PTS) with a high affinity for glucose (Km = 0.35 mM). Mutants selected for their resistance to 2-deoxyglucose (2DG) and lacking detectable PEP-dependent glucose-transporting activity, retained the capacity to grow on media in which glucose was the only carbon and energy source, albeit at significantly diminished rates, due to the presence of a low affinity (Ks = 11 mM) non-PTS uptake system. During growth in media of different osmolarity, specific rates of glucose consumption and of growth of wild type cells were diminished. Cell samples from these cultures were shown to possess similar PTS activities when measured under standard conditions. However, when cells were resuspended in buffer solutions of different osmolarity measurable PTS activity was shown to be dependent upon osmolarity. This inhibition effect was sufficient to account for the decreased rates of both sugar uptake and growth observed in fermentation media of high osmolarity. The secondary glucose transporter was, however, not influenced by medium osmolarity. During industrial fermentation conditions with accumulation of glutamic acid and the corresponding increase in medium osmolarity, similar inhibition of the sugar transport capacity was observed. This phenomenon provokes a major process constraint since the decrease in specific rates leads to an increasing proportion of sugar catabolised for maintenance requirements with an associated decrease in product yields. (C) 2003 Elsevier B.V. All rights reserved.

We have analyzed ARS elements linked to homologous and heterologous ADE2 loci functioning in Schwanniomyces occidentalis. We have identified a region of the ADE2 locus of S. occidentalis which promotes autonomous replication of plasmids in S. occidentalis cells. This region is within 385 bp preceding the ATG codon of the S. occidentalis ADE2 gene. It contains sequences similar to ARS core consensus sequences, ARS boxes, and a potential transcription activator binding site characterized in Saccharomyces cerevisiae. The ADE2 gene of S. cerevisiae was found to complement the ade2 mutation in S. occidentalis cells and the 5' UTR region of this gene is capable of supporting autonomous replication of plasmids in S. occidentalis. Furthermore, we confirmed that the origin of replication of the 2 microm plasmid and the ARS1 sequence of S. cerevisiae are also functional in S. occidentalis cells. Plasmids carrying either ARS, the SwARSA element of S. occidentalis, the ARS linked to the ADE2 gene of S. cerevisiae, and the ARS1 sequence or the 2 microm ori, were found to be maintained in S. occidentalis cells as episomal monomers or oligomers. However, their stability was low as already reported for the ARS in S. occidentalis.