Biosynthetic preparation of L-[13C]- and [15N]glutamate by Brevibacterium flavum

Abstract

The biosynthesis of isotopically labeled L-glutamic acid by the microorganism Brevibacterium flavum was studied with a variety of carbon-13-enriched precursors. The purpose of this study was twofold: (i) to develop techniques for the efficient preparation of labeled L-glutamate with a variety of useful labeling patterns which can be used for other metabolic studies, and (ii) to better understand the metabolic events leading to label scrambling in these strains. B. flavum, which is used commercially for the production of monosodium glutamate, has the capability of utilizing glucose or acetate as a sole carbon source, an important criterion from the standpoint of developing labeling strategies. Unfortunately, singly labeled glucose precursors lead to excessive isotopic dilution which reduces their usefulness. Studies with [3-13C]pyruvate indicate that this problem can in principle be overcome by using labeled three-carbon precursors; however, conditions could not be found which would lead to an acceptable yield of isotopically labeled L-glutamate. In contrast, [1-_3C]or [2-13C]acetate provides relatively inexpensive, readily available precursors for the production of selectively labeled, highly enriched L-glutamate. The preparation of L-[1s5Nglutamate from ['5N]ammonium sulfate was carried out and is a very effective labeling strategy. Analysis of the isotopic distribution in labeled glutamate provides details about the metabolic pathways in these interesting organisms.

Metabolite and isotopomer balancing in the analysis of metabolic cycles: II. Applications

“…Therefore, enrichment of glutamate should be (1 − z)/2 at C-1, 1 at C-5, and 0 at C-2, C-3, and C-4. This is exactly what is observed in proline synthesis by E. coli (Crawford et al, 1987) and glutamate synthesis by B. flavum (Walker and London, 1987). Since the system of (Crawford et al, 1987) was similar to that of (Walsh and Koshland, 1984), one can assume the same value for z ‫ס‬ 0.2 that yields an estimate for glutamate C-1 enrichment of 0.4.…”

“…They contain no information for the value of the flux ratio z, however, they are very consistent with the above patterns. Similar labeling patterns were observed in glutamate by Brevibacterium flavum (Walker and London, 1987) and proline, which also reflects exact labeling pattern of ␣-ketoglutarate by E. coli (Crawford et al, 1987).…”

Metabolite and isotopomer balancing in the analysis of metabolic cycles: I. Theory

“…Walsh and Koshland (1984) were able to determine the flux split ratio at the branch point of the tricarboxylic acid cycle and the glyoxylate shunt, based on the 13 C NMR spectrum of intracellular glutamate in Escherichia coli. den Hollander et al (1986) studied aerobic and anaerobic glycolysis in yeast Saccharomyces cerevisiae, whereas Walker and London (1987) studied the metabolic pathways leading to the biosynthesis of glutamate by Brevibacterium flavum. Using isotopically labeled acetate, Crawford et al (1987) monitored acetate metabolism in a proline-overproducing strain of E. coli and Narbad et al (1989) studied acetate and methanol metabolism of methylotrophic Pseudomonas strains.…”

Microbial Upgrading of Acetate into Value-Added Products—Examining Microbial Diversity, Bioenergetic Constraints and Metabolic Engineering Approaches

“…Table 6) [36,[105][106][107][109][110][111]. The generation of isotope-labeled L-glutamate or electricity via microbial fuel cells seem to be special cases and constitute less common ways to upgrade acetate into value-added products [108,112].…”