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: 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 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, [1-13C]- or [2-13C]acetate provides relatively inexpensive, readily available precursors for the production of selectively labeled, highly enriched L-glutamate. The preparation of L-[15N]glutamate from [15N]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.