Functional Characterization of Key Enzymes involved in l -Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

Abstract

bBacillus methanolicus wild-type strain MGA3 secretes 59 g/liter ؊1 of L-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in L-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low K m value for ammonium (10 mM) and a high K m value for L-glutamate (250 mM), and the V max value is 7-fold higher for L-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar K m values (1 to 1.4 mM) and V max values (4 U/mg) for both L-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating L-glutamate production in this organism.T he production of L-glutamate is a fast-growing industry, and approximately 2 million tons are produced annually, almost exclusively by Corynebacterium glutamicum fermentation using sugar-based raw materials (1). Genetic and biochemical insight into L-glutamate synthesis and degradation is of fundamental importance to understand nitrogen metabolism in the cells, as well as for metabolic engineering of L-glutamate overproduction. In C. glutamicum, L-glutamate is synthesized by both glutamate dehydrogenase (GDH) and glutamate synthase (GOGAT) (2), and L-glutamate overproduction can be achieved by downregulating the 2-oxoglutarate dehydrogenase (OGDH) activity (3, 4).In Bacillus subtilis, the gltAB operon encodes the large (GltA) and the small (GltB) subunit of GOGAT. GOGAT catalyzes a reaction where the NH 2 group from the amide group in L-glutamine is transferred to 2-oxoglutarate, forming two molecules of L-glutamate. Glutamine synthetase (GS) is encoded by glnA and catalyzes the synthesis of L-glutamine from L-glutamate and ammonia. Together, these two enzymes form the GOGAT/GS cycle ( Fig. 1) that is responsible for ammonium assimilation and L-glutamate degradation and synthesis, and B. subtilis mutants deficient in GOGAT are L-glutamate auxotrophs (5, 6). To our knowledge, the kinetic properties of GOGAT in B. subtilis have not been reported in the scientific literature.B. subtilis harbors two genes, rocG and gudB, that both encode GDHs. In the most-studied B. subtilis strain, strain 168, GudB is found to be...

Proteomic analysis of the thermophilic methylotroph Bacillus methanolicusMGA3

“…It is unknown which of these pathways is employed by B. methanolicus for l ‐glutamate production. However, recent findings have demonstrated that overexpression of glutamate synthase and glutamate dehydrogenase in B. methanolicus MGA3 has no or a marginally positive effect on glutamate production , while overproduction of the native Odh protein led to a fivefold reduction in glutamate. Interestingly, Odh overexpression in an l ‐lysine‐overproducing B. methanolicus host resulted in a twofold increase in l ‐lysine production yield, suggesting that Odh activity plays an important role at the branching point between glutamate synthesis and the TCA cycle, eventually regenerating the l ‐lysine precursor molecule oxaloacetate .…”

“…However, recent findings have demonstrated that overexpression of glutamate synthase and glutamate dehydrogenase in B. methanolicus MGA3 has no or a marginally positive effect on glutamate production , while overproduction of the native Odh protein led to a fivefold reduction in glutamate. Interestingly, Odh overexpression in an l ‐lysine‐overproducing B. methanolicus host resulted in a twofold increase in l ‐lysine production yield, suggesting that Odh activity plays an important role at the branching point between glutamate synthesis and the TCA cycle, eventually regenerating the l ‐lysine precursor molecule oxaloacetate . Combining these findings with the proteome data generated for the presence of Odh in this study suggests that the potential for amino acid production could differ on both carbon sources, with methanol being the more suitable substrate for the production of glutamate and mannitol being the preferred substrate for the accumulation of lysine.…”

Proteomic profiling of Bacillus licheniformis reveals a stress response mechanism in the synthesis of extracellular polymeric flocculants

“…Similarly, under tryptophan starvation, B. subtilis increased the expression of the trp operon (Tam et al, ). In addition, GdhA , GlnA , and GltA which have crucial roles in ammonium assimilation and l ‐glutamate metabolism, were increased in γ‐PGA medium‐cultured cells (Krog et al, ; Zhu et al, ).…”

Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3

“…The cell debris was removed by centrifugation at 4 °C for 10 min at 8,000g. Measurement of GDH and OGDH activities was performed as described by Krog et al [11] with slight modifications. Briefly, the GDH assay mixture generally contained 100 mM glycine-KOH (pH 7.5), 200 mM L-glutamate, 10 mM MgSO 4 , and 0.2 mM NAD + ; the OGDH assay mixture generally contained 50 mM TrisHCl (pH 7.…”

“…a Chromosome structures of deletion-carrying strains were analyzed by agarose gel electrophoresis of PCR products generated with primers RocROUT-F/RocROUT-R(lanes 1-3); with primers RocGOUT-F/RocGOUT-R (lanes 4-6); with primers OdhAOUT-F/OdhAOUT-R (7-9); with primers GudBOUT-F/GudBOUT-R (lanes[10][11][12]. M DNA marker, and the size of each band is reported on the left.…”