Metabolic rewiring enables ammonium assimilation via a non-canonical fumarate-based pathway

Glutamate serves as the major cellular amino group donor. In Bacillus subtilis, glutamate is synthesized by the combined action of the glutamine synthetase and the glutamate synthase (GOGAT). The glutamate dehydrogenases are devoted to glutamate degradation in vivo. To keep the cellular glutamate concentration high, the genes and the encoded enzymes involved in glutamate biosynthesis and degradation need to be tightly regulated depending on the available carbon and nitrogen sources. Serendipitously, we found that the inactivation of the ansR and citG genes encoding the repressor of the ansAB genes and the fumarase, respectively, enables the GOGAT-deficient B. subtilis mutant to synthesize glutamate via a non-canonical fumarate-based ammonium assimilation pathway. We also show that the de-repression of the ansAB genes is sufficient to restore aspartate prototrophy of an aspB aspartate transaminase mutant. Moreover, in the presence of arginine, B. subtilis mutants lacking fumarase activity show a growth defect that can be relieved by aspB overexpression, by reducing arginine uptake and by decreasing the metabolic flux through the TCA cycle. Glutamate is an abundant cellular metabolite that serves as a major donor of amino groups for the synthesis of nitrogen-containing metabolites. Many bacteria can use either the glutamate dehydrogenase- or the glutamine synthetase/glutamate synthase-dependent route for de novo synthesis of glutamate. Here, we show that the cellular requirement for glutamate in the soil bacterium Bacillus subtilis can be met by a non-canonical fumarate-based ammonium assimilation pathway.image