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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.

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.