The amino acid glutamate plays important roles in cellular processes in the Gram-positive and ‑negative model bacteria Bacillus subtilis and Escherichia coli, respectively. It is the major amino group donor in anabolic reactions and a key molecule connecting carbon with nitrogen metabolism (1,2). B. subtilis synthesises glutamate via the glutamate synthase (GOGAT)/glutamine synthetase (GS) pathway (1), while E. coli can utilize the GOGAT/GS pathway, as well as the glutamate dehydrogenase (GDH) for glutamate biosynthesis (1,2). The GDHs of B. subtilis are strictly catabolically active. Recently, a non-canonical, fumarate-based pathway – involving the ansB-encoded L-aspartase and the aspB- encoded aspartate transaminase – was found that restored glutamate-prototrophy of a glutamate-auxotrophic B. subtilis strain lacking the GOGAT-encoding genes gltAB (3). The aim of this project is to better understand the mechanism and regulations of the different glutamate synthesizing pathways. To further analyse the fumarate-based pathway for glutamate biosynthesis, we constructed plasmids allowing for ansB and aspB expression in B. subtilis and E. coli glutamate-auxotrophic strains and further we characterized the novel pathway by growth experiments under defined growth conditions with different carbon and nitrogen sources. Results show that the novel pathway is able to restore glutamate-prototrophy not only in B. subtilis but also in E. coli and highlight different needs of the novel-pathway to the canonical glutamate biosynthetic pathway.

(1) Gunka, K., & Commichau, F.M. (2012) Mol Microbiol. 85 (2): 213–224. doi: 10.1111/j.1365-2958.2012.08105.x.

(2) Schulz-Mirbach, H., et al. (2022) 11: e77492. doi: 10.7554/eLife.77492.

(3) Mardoukhi, M.S.Y., et al. (2024) Microb Biotechnol. 17 (3): e14429. doi: 10.1111/1751-7915.14429.