Flexibility of the metabolism of Corynebacterium glutamicum 2262, a glutamic acid-producing bacterium, in response to temperature upshocks S Delaunay 1 , P Lapujade 2 , JM Engasser 1 and JL Goergen 2 1 Laboratoire Bioproce ´de ´ s Agroalimentaires, Institut National Polytechnique de Lorraine - 2, Avenue de la Fore ˆt de Haye, BP 172, F - 54505 Vandoeuvre - le ` s - Nancy, France; 2 Laboratoire des Sciences du Ge ´ nie Chimique — CNRS, Institut National Polytechnique de Lorraine - 2, Avenue de la Fore ˆt de Haye, BP 172, F-54505 Vandoeuvre-le ` s - Nancy, France In order to test the temperature sensitivity of glutamate production metabolism, several temperature shifts, from 33 to 37, 38, 39, 40 or 418C, were applied to the temperature - sensitive strain, Corynebacterium glutamicum 2262, cultivated in a 24 - h fed - batch process. Whereas glucose was entirely dedicated to biomass synthesis when cells were grown at 338C, applying temperature upshocks, whatever their range, triggered a redistribution of the carbon utilisation between glutamate, biomass and lactate production. Although increasing the culture temperature from 33 to 37, 38, 39 or 408C resulted in final glutamate titers superior to 80 g / l, temperatures resulting in the best chanelling of the carbon flow towards glutamic acid synthesis were 39 and 408C. Moreover, this study showed that the higher the temperature, the slower the growth rate and the higher the lactate accumulation. Journal of Industrial Microbiology & Biotechnology (2002) 28, 333 – 337 DOI: 10.1038/sj/jim/7000251 Keywords: glutamate; temperature; fed - batch; Corynebacterium glutamicum; carbon flow partitioning Introduction The production of glutamate by Corynebacterium glutamicum is an important part of the industrial exploitation of microbiology [ 12 ]. The current annual production of glutamate reaches approximately 900,000 tons. In view of this fermentation’s economic impact, extensive studies were dedicated to selecting new C. glutamicum strains able to produce ever - increasing amounts of glutamate. Bacteria sensitive to various means of triggering glutamate excretion have been discovered. So far, the major processes developed industrially are biotin limitation [ 1,3,11,19], surfactant addition [ 14,17 ] and penicillin addition [ 16,17 ]. Glutamate production is strongly dependent on the treatment that induces glutamate excretion. Slight modifications often result in a decreased glutamate accumulation. In biotin - limited con- ditions, glutamate excretion triggering is closely related to biotin concentration in the culture medium. Whereas 20 g / l of biotin are sufficient to allow C. glutamicum growth, glutamate excretion occurs only when the biotin external concentration falls to 3 g/l [ 19 ]. In a process using surfactant addition, the amount of added surfactant, or more precisely the ratio of surfactant concentration to biomass concentration, is a key parameter. It has to be sufficient to increase membrane permeability; however, too great an elevated surfactant concentration should lead to cellular death and thus to the decrease of glutamate production [ 4 ]. To date, few data are available on the effect of temperature on glutamate production and on C. glutamicum metabolism. In 1978, Momose and Takagi [ 15 ] isolated several temperature -sensitive mutants of Brevibacterium lactofermentum. Performing either of two temperature shifts ( from 30 to 378C or from 30 to 408C ), they showed that, in their culture conditions, the less temperature sensitive the mutant was, the more severe condition had to be adopted for glutamic acid production. A temperature increase from 34 to 388C was used as well during a process in which glutamate excretion was induced by Tween -40 addition [ 9]. This increase occurred 2 h after surfactant addition, in order to limit biomass accumulation and thus to favor the redirection of the carbon flow towards glutamic acid synthesis. Recently, we presented a fed - batch process enabling the production of 85 g / l of glutamate using C. glutamicum in a biotin - rich medium by increasing the culture temperature from 33 to 398C [ 6]. However, the effect of different temperature shifts was not reported. In the present paper, we investigated the sensitivity of the metabolism of C. glutamicum 2262 to various temperature shifts. Materials and methods Bacterial strain and medium composition The strain used throughout this study was C. glutamicum 2262 [ 6 ]. The composition of the glutamate production medium used was based on MCGC medium [ 20 ] although citrate ( used as chelating agent ) was replaced by deferoxamine. Glucose was used as the sole carbon substrate. This medium consisted of: 60 g / l glucose, 3 g/l Na 2 HPO 4 , 6 g/l KH 2 PO 4 , 2 g/l NaCl, 8 g/l (NH 4 ) 2 SO 4 , 0.4 g/l MgSO 4 7H 2 O, 40 mg/l FeSO 4 7H 2 O, 3.9 mg/l FeCl 3 , 0.9 mg/l ZnSO 4 7H 2 O, 0.3 mg/l CuCl 2 2H 2 O, 3.9 mg/l MnSO 4 H 2 O, 0.1 mg/l (NH 4 ) 6 Mo 7 O 24 4H 2 O, 0.3 mg/l Na 2 B 4 O 7 10H 2 O, 84 mg / l CaCl 2 , 4 mg / l biotin, 20 mg / l thiamine, 3 mg / l deferoxamine, 2 g / l glycine betaine. In fed - batch cultures, this medium was supplemented with 1.3 g / l polypropylene glycol Correspondence: Dr S Delaunay, Laboratoire Bioproce ´de ´s Agroalimentaires, Institut National Polytechnique de Lorraine - 2, Avenue de la Fore ˆt de Haye, BP 172, F - 54505 Vandoeuvre - le `s - Nancy Cedex, France Received 26 September 2001; accepted 23 February 2002 Journal of Industrial Microbiology & Biotechnology (2002) 28, 333 – 337 D 2002 Nature Publishing Group All rights reserved 1367-5435/02 $25.00 www.nature.com / jim