Eiichiro Kimura

Altered Metabolic Flux due to Deletion of odhA causes l-Glutamate Overproduction in Corynebacterium glutamicum

ABSTRACT l-Glutamate overproduction in Corynebacterium glutamicum, a biotin auxotroph, is induced by biotin limitation or by treatment with certain fatty acid ester surfactants or with penicillin. We have analyzed the relationship between the inductions, 2-oxoglutarate dehydrogenase complex (ODHC) activity, and l-glutamate production. Here we show that a strain deleted for odhA and completely lacking ODHC activity produces l-glutamate as efficiently as the induced wild type (27.8 mmol/g [dry weight] of cells for the ohdA deletion strain compared with only 1.0 mmol/g [dry weight] of cells for the uninduced wild type). This level of production is achieved without any induction or alteration in the fatty acid composition of the cells, showing that l-glutamate overproduction can be caused by the change in metabolic flux alone. Interestingly, the l-glutamate productivity of the odhA-deleted strain is increased about 10% by each of the l-glutamate-producing inductions, showing that the change in metabolic flux resulting from the odhA deletion and the inductions have additive effects on l-glutamate overproduction. Tween 40 was indicated to induce drastic metabolic change leading to l-glutamate overproduction in the odhA-deleted strain. Furthermore, optimizing the metabolic flux from 2-oxoglutarate to l-glutamate by tuning glutamate dehydrogenase activity increased the l-glutamate production of the odhA-deleted strain.

Metabolic engineering of glutamate production

Since the discovery of Corynebacterium glutamicum as an efficient glutamate-overproducing microorganism in 1957, the production of L-amino acids by the fermentative method has become one of the most important research-target of industrial microbiology. Several research groups have developed metabolic engineering principles for L-amino acid-producing C. glutamicum strains over the last four decades. The mechanism of L-glutamate-overproduction by the microorganism is very unique and interesting. L-Glutamate overproduction by this bacterium, a biotin auxotroph, is induced by a biotin limitation and suppressed by an excess of biotin. Addition of a surfactant or penicillin is known to induce L-glutamate overproduction under excess biotin. After the development of the general molecular biology tools such as cloning vectors and DNA transfer technique, genes encoding biosynthetic enzymes were isolated. With those genes and tools, recombinant DNA technology can be applied in analysis of biosynthetic pathways and strain construction of C. glutamicum. In this review, key points of the L-glutamate biosynthetic pathways are summarized and the recent studies about triggering mechanism of L-glutamate overproduction by C. glutamicum are introduced. Then the metabolic flux analysis of L-glutamate overproduction is explored.

Corynebacterium efficiens sp. nov., a glutamic-acid-producing species from soil and vegetables.

Three glutamic-acid-producing coryneform strains were isolated from soil and vegetable samples. Chemotaxonomic investigations indicated that these strains belonged to the genus Corynebacterium. Phylogenetic studies, based on 16S rDNA analysis, demonstrated that the three strains formed a distinct cluster within the genus Corynebacterium and that their nearest relatives were Corynebacterium glutamicum and Corynebacterium callunae, also known as glutamic-acid-producing species. The data from 16S rDNA sequence and DNA-DNA relatedness studies clearly indicated that the three isolates represented a new species within the genus Corynebacterium. All of the isolates could grow at 45 degrees C and produced acid from dextrin; these were the most significant characteristics differentiating the three isolates from their neighbours. On the basis of the data presented here, it is proposed that the three glutamic-acid-producing isolates together be classified as Corynebacterium efficiens sp. nov., the type strain of which is YS-314T (= AJ 12310T = JCM 11189T = DSM 44549T).

Triggering mechanism of L-glutamate overproduction by DtsR1 in coryneform bacteria.

The mechanism of L-glutamate-overproduction by Corynebacterium glutamicum, a biotin auxotroph, is very unique and interesting. L-Glutamate overproduction by this bacterium is induced by biotin-limitation and suppressed by an excess of biotin. Addition of a surfactant or penicillin is also induces L-glutamate overproduction even under excess biotin. After the development of general molecular biological tools such as cloning vectors and DNA transfer techniques, genes encoding biosynthetic enzymes were isolated. With those genes and tools, recombinant DNA technology can be applied to the analysis of biosynthetic pathways and the construction of C. glutamicum strains. In this review, recent studies on the triggering mechanism of L-glutamate overproduction by C. glutamicum are discussed. Disruption of the dtsR1 gene, which encodes a putative component of a biotin-containing enzyme complex that is involved in fatty acid synthesis, causes constitutive overproduction of L-glutamate. As in the case of biotin-limitation, i.e., addition of a surfactant or penicillin, dtsR1-disruption also reduces the activity of the 2-oxoglutarate dehydrogense complex (ODHC). These results indicate that the DtsR1 level affects the activity of ODHC. In our recent studies, a novel regulatory factor that suppresses the expression of DtsR1 was determined. Based on these findings, the triggering mechanism of L-glutamate overproduction is expected to be clarified in more detail.

Glutamate Overproduction in Corynebacterium glutamicum Triggered by a Decrease in the Level of a Complex Comprising DtsR and a Biotin-containing Subunit

Glutamate overproduction in Corynebacterium glutamicum is induced by Tween 40, biotin-limitation, or sublethal amounts of penicillin. Disruption of the dtsR gene, which encodes a putative component of a biotin-containing enzyme complex involved in fatty acid synthesis, causes constitutive overproduction of glutamate. We report here that overexpression of dtsR inhibits the induction of glutamate overproduction. In contrast, the level of DtsR in the wild type strain was found to decrease in the presence of Tween 40 or limited amounts of biotin. Tween 40, biotin-limitation, or dtsR disruption also reduced the activity of 2-oxoglutarate dehydrogenase complex (ODHC), which is involved in the synthesis of succinate from 2-oxoglutarate. These results indicate that decrease in the level of DtsR or a complex containing DtsR triggers the increased synthesis of glutamate from 2-oxoglutarate by lowering the ODHC activity.

Supplementing chicken broth with monosodium glutamate reduces energy intake from high fat and sweet snacks in middle-aged healthy women ☆

Monosodium L-glutamate (MSG) and inosine monophosphate-5 (IMP) are flavor enhancers for umami taste. However, their effects on appetite and food intake are not well-researched. The objective of the current study was to test their additions in a broth preload on subsequent appetite ratings, energy intake and food choice. Eighty-six healthy middle-aged women with normal body weight received three preload conditions on 3 test days 1 week apart - a low-energy chicken flavor broth (200 ml) as the control preload, and broths with added MSG alone (0.5 g/100 ml, MSG broth) or in combination with IMP (0.05 g/100 ml) (MSG+ broth) served as the experimental conditions. Fifteen minutes after preload administration subjects were provided an ad libitum testing meal which consisted of 16 snacks varying in taste and fat content. MSG and MSG+ enhanced savory taste and broth properties of liking and pleasantness. In comparison with control, the MSG preload resulted in less consumption of total energy, as well as energy from sweet and high-fat snacks. Furthermore, MSG broth preload reduced added sugar intake. These findings were not observed after MSG+ preload. Appetite ratings were not different across the three preloads. Results suggest a potential role of MSG addition to a low-energy broth preload in subsequent energy intake and food choice. This trial was registered at clinicaltrials.gov as NCT01761045.

Monosodium l -glutamate in soup reduces subsequent energy intake from high-fat savoury food in overweight and obese women

The umami seasoning, monosodium L-glutamate (MSG), has been shown to increase satiety in normal body weight adults, although the results have not been consistent. The satiety effect of MSG in overweight and obese adults has not been examined yet. The objective of the present study was to investigate the effect of MSG in a vegetable soup on subsequent energy intakes as well as food selection in overweight and obese adult women without eating disorders. A total of sixty-eight overweight and obese women (BMI range: 25·0-39·9 kg/m²), otherwise healthy, were recruited to our study. A fixed portion (200 ml) of control vegetable soup or the same soup with added MSG (0·5 g/100 ml) was provided 10 min before an ad libitum lunch and an ad libitum snack in the mid-afternoon. The control soup had equivalent amount of Na to the soup with added MSG. Energy intakes at the ad libitum lunch and ad libitum snack time after the soup preload were assessed using a randomised, double-blind, two-way cross-over design. The soup with MSG in comparison with the control soup resulted in significantly lower consumption of energy at lunch. The addition of MSG in the soup also reduced energy intake from high-fat savoury foods. The soup with MSG showed lower but no significant difference in energy intake at mid-afternoon. The addition of umami seasoning MSG in a vegetable soup may decrease subsequent energy intake in overweight and obese women who do not have eating disorders.

>Studies of L-Glutamate-Production in Coryneform Bacteria by Using Metabolic-Flux Analysis

Abstract An odhA-disrupted mutant extremely produce glutamate into the medium in the presence of excess amounts of biotin. This result means the mechanism of glutamate production in coryneform bacteria is irreverent to the permeability of a cell membrane. Furthermore, a noble gene, dtsR, was cloned as a multi-copy suppresser gene of a detergent sensitive mutant derived from a wildtype strain of coryneform bacteria. DtsR showed significant homologies to some biotin enzymes. A dtsR-disrupted mutant produce glutamte. The mechanism of glutamate production in coryneform bacteria was discussed.

Pilot intervention study of a low‐salt diet with monomagnesium di‐L‐glutamate as an umami seasoning in psychiatric inpatients

Schizophrenia patients have an elevated prevalence of stroke and cardiovascular risk factors, such as elevated body mass index, hypertension, and hyperlipidaemia. This pilot study investigated the influence of a low‐sodium diet using umami seasoning on food intake and clinical parameters in schizophrenia patients.