Yoshio Kawahara

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.

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.

Effect of glycine betaine, an osmoprotective compound on the growth of Brevibacterium lactofermentum

SummaryOsmoregulation of Brevibacterium lactofermentum was examined. Exogenous glycine betaine was found to stimulate the growth rate of the bacterium in media of inhibitory osmotic strength. The stimulation was independent of any specific solute, electrolyte, or non-electrolyte. The bacterium did not utilize glycine betaine as a sole carbon source or nitrogen source, or degrade it even in complete medium. The changes in intracellular proline and glycine betaine concentrations were measured in media of different osmolarity. Brevibacterium lactofermentum grown in media without glycine betaine did not accumulate it, but synthesized several hyndred millimoles of proline inside the cells. On the other hand, when glycine betaine was added to the growth media, it accumulated in the cell instead of proline. These data indicate that glycine betaine is an osmoprotective compound for B. lactofermentum.

Stimulatory effect of glycine betaine on l-lysine fermentation

SummaryThe growth rate, sugar consumption rate, and production rate of an l-lysine producing Brevibacterium lactofermentum mutant were stimulated by addition of exogenous glycine betaine. Glycine betaine stimulated the growth rate especially in media of inhibitory osmotic stress, and the stimulation was independent of any specific solute. Therefore growth stimulation by glycine betaine was considered to be an osmoprotective effect. A strong enhancement of the sugar consumption rate and the l-lysine production rate was observed even with resting cells under osmotic stress as well as in a fermentation with growing cells. These data indicated that the osmoprotective effects of glycine betaine on l-lysine production can be independent of protein synthesis.

In vivo NMR system for evaluating oxygen-dependent metabolic status in microbial culture.

To optimize an appropriate microbial culture in a fermentor, precise control of the mediums dissolved oxygen tension (DOT) is crucial. In particular, to study the effect of DOT on cellular metabolic status by using in vivo nuclear magnetic resonance (NMR) measurements, the set-up of the experiment must be optimized to maintain DOT in the culture. In the conventional method, DOT is monitored by a sensor inside a fermentor and is controlled by changing the agitation rate. Here, we report a novel and accurate system that minimizes time lag by an automated aeration flow control device, allowing an NMR spectrometer to monitor representative metabolites in real-time. To fulfill these two objects, the fermentor was composed of a fermentation vessel and two outer tubes, through which the medium was circulated by rotary pumps. One tube monitored DOT in via a sensor, and at the same time the other tube monitored metabolites via an NMR spectrometer. In this study, we used this system to analyze the responses of Escherichia coli cells under various oxygen conditions. The results validated the use of this system in the study of microbial metabolism.

Effect of glycine betaine on the sucrose catabolism of an l-lysine producing mutant of Brevibacterium lactofermentum

SummaryEffect of exogenous betaine on the growth of an l-lysine-producing mutant of Brevibacterium lactofermentum was examined in a medium containing different carbon sources such as glucose, fructose, or sucrose. The growth rate decreased significantly with a rise in temperature when sucrose was the carbon source. Both the specific sucrose consumption rate and the invertase activity of the mutant decreased with the culture period when the cultivation temperature was 35°C. The addition of betaine restored both growth and invertase activity on medium containing sucrose as the carbon source at 35°C. Betaine protected the invertase activity against the inactivating effects of high temperature in vitro. Furthermore, the addition of exogenous invertase into production medium at 35°C restored the growth rate to that at 32°C. These results indicated that growth decreased on medium containing sucrose at 35°C due to a decrease in invertase activity, and that addition of betaine was an effective way to enhance growth on this medium at a higher temperature.

A convenient NMR method for in situ observation of aerobically cultured cells

We observed P31-NMR signals of intracellular phosphorylated metabolites, i.e. ATP, NAD(H) and UDPG, in the aerobically cultured cells of AJ13375 (a derivative of E. coli K-12) without a cell culture circulating system, which needs much more cells. Each NMR spectrum of 40 ml cell-culture was obtained with a 25 mm Phi sample tube mini-fermenter equipped with a pH electrode and three supply routes for O2 gas, aqueous ammonia and glucose. Spectra were measured at 15-min intervals. When glucose in the culture medium was consumed, P31-NMR signals of ATP disappeared first and then those of ADP decreased to below the limit of detection. The intracellular concentration of ATP was estimated to be approximately 7 mM.

>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.