Metabolic flux distribution for the optimized production of L-glutamate Serpil Takac ¸,* Gu ¨zide C ¸ alık,* Ferda Mavituna, ² and George Dervakos ² *Industrial Biotechnology Department, Ankara University Biotechnology Research Center, Ankara, Turkey; ² Department of Chemical Engineering, UMIST, Manchester, U.K. A comprehensive metabolic network was proposed for glutamic acid bacteria and used in a stoichiometrically based flux balance model for L-glutamate production. Theoretical metabolic pathways leading to optimized glutamate overproduction were determined for several specific cell growth rates; variation in the fluxes was obtained. The off-line extracellular analyses throughout the batch fermentation with Brevibacterium flavum showed the accumulation of arginine, aspartate, lysine, alanine, proline, lactate, -ketoglutarate, succinate, pyruvate, and gluconate in the medium in addition to glutamate. Metabolic flux distributions in the cells throughout the fermentation were determined using the model in combination with the extracellular analyses of the metabolites. The flux distribution maps showed that the cells utilized the TCA cycle in part whereas the glyoxylate bypass was active throughout the fermentation. The results also indicated that the phosphate pentose shunt played an important role in the glutamate fermentation. These diversions in the pathways and certain metabolic reactions depending on the fermentation periods and conditions are also presented in this paper. © 1998 Elsevier Science Inc. Keywords: Metabolic pathways; flux distribution; optimization; l-glutamate; glutamic acid bacteria Introduction l-Glutamate (Glu) which is in widespread use throughout the world as a seasoning as well as a starting material for the synthesis of various chemicals is produced industrially by fermentation using Brevibacterium and Corynebacterium strains. 1 The wild strains of glutamic acid bacteria seem to be able to produce only small amounts of the amino acids extracellularly; therefore, several methods have been em- ployed to alter the cellular metabolism and regulatory controls of the bacteria including cell mutation, cell fusion, and genetic manipulation techniques. 2 However, since the analysis of metabolic flux distribution in an organism can provide information about the bottlenecks in the overpro- duction of the desired metabolite, studies should now focus on metabolic pathway engineering. In this field, stoichio- metrically based flux balance models have attracted a great deal of interest. Escherichia coli is well studied in compar- ison with other bacteria and a lot of information regarding its metabolic system leading to amino acid production is readily available in the literature. This availability of infor- mation has become a good reason to apply such models primarily to E. coli. The central metabolic pathways of E. coli during the growth on several carbon sources were investigated by Holms 3 using the stoichiometric flux analysis that depends on the knowledge of biochemical stoichiometry, biosynthe- sis requirements, and on the measurements of the flows in and out of the cells. The metabolic capabilities of E. coli to produce some precursors and key cofactors were reported in detail by Varma and Palsson. 4 The fueling reactions and electron transfer system occurring in E. coli were presented by these authors. Linear optimization was used to determine the bounds of the flux distribution that the bacteria could achieve. In their following article, Varma and Palsson 5 investigated the effect of metabolic stoichiometry on the cellular function and behavior of E. coli. The optimal growth patterns on glucose (Glc) and acetate (Ac) as well as the effects of pathways utilized, metabolic demands, P/O ratio, and maintenance requirement on the solution of the Address reprint requests to Dr. Serpil Takac ¸, Ankara University, Depart- ment of Chemical Engineering, Tandog ˘an, Ankara, 06100 Turkey Received 30 July 1997; revised 24 March 1998; accepted 31 March 1998 Enzyme and Microbial Technology 23:286 –300, 1998 © 1998 Elsevier Science Inc. All rights reserved. 0141-0229/98/$19.00 655 Avenue of the Americas, New York, NY 10010 PII S0141-0229(98)00047-7