Pathway analysis and metabolic engineering in Corynebacterium glutamicum

The Gram-positive bacterium Corynebacterium glutamicum is used for the indu strial production of amino acids, e.g. of L-glutamate and L-lysine, During the last 15 years, genetic engineering and amplification of genes have beco me fascinating methods for studying metabolic pathways in greater detail an d for the construction of strains with the desired genotypes. In order to o btain a better understanding of the central metabolism and to quantify the in vivo fluxes in C. glutamicum, the [C-13]-labelling technique was combine d with metabolite balancing to achieve a unifying comprehensive pathway ana lysis. These methods can determine the flux distribution at the branch poin t between glycolysis and the pentose phosphate pathway. The in vivo fluxes in the oxidative part of the pentose phosphate pathway calculated on the ba sis of intracellular metabolite concentrations and the kinetic constants of the purified glucose-6-phosphate and g-phosphogluconate dehydrogenases det ermined in vitro were in full accordance with the fluxes measured by the [C -13]-labelling technique. These data indicate that the oxidative pentose ph osphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH/ NADP concentrations and the specific activity of glucose-6-phosphate dehydr ogenase. The carbon flux via the oxidative pentose phosphate pathway correl ated with the NADPH demand for L-lysine synthesis. Although it has generally been accepted that phosphoenolpyruvate carboxylas e fulfills a main anaplerotic function in C. glutamicum, we recently detect ed that a biotin-dependent pyruvate carboxylase exists as a further anapler otic enzyme in this bacterium. In addition to the activities of these two c arboxylases three enzymes catalysing the decarboxylation of the C-4 metabol ites oxaloacetate or malate are also present in this bacterium. The individ ual flux rates at this complex anaplerotic node were investigated by using [C-13]-labelled substrates. The results indicate that both carboxylation an d decarboxylation occur simultaneously in C. glutamicum so that a high cycl ic flux of oxaloacetate via phosphoenolpyruvate to pyruvate was found. Furthermore, we detected that in C. glutamicum two biosynthetic pathways ex ist for the synthesis of DL-diaminopimetate and L-lysine, As shown by NMR s pectroscopy the relative use of both pathways in vivo is dependent on the a mmonium concentration in the culture medium. Mutants defective in one pathw ay are still able to synthesise enough L-lysine for growth, but the L-lysin e yields with overproducers were reduced. The luxury of having these two pa thways gives C. glutamicum an increased flexibility in response to changing environmental conditions and is also related to the essential need for DL- diaminopimelate as a building block for the synthesis of the murein sacculu s.