Tsuyoshi Osumi

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.

Medium composition suitable for L-lysine production by Methylophilus methylotrophus in fed-batch cultivation

L-Lysine production was investigated in fed-batch fermentation using L-lysine producer of Methylophilus methylotrophus. By the addition of nutrient composition, containing L-methionine, K(2)HPO(4), NaH(2)PO(4), CuSO(4).5aq, MnSO(4).5aq, ZnSO(4).7aq, FeCl(3), MgSO(4).7aq and CaCl(2).2aq, in the feed medium, cell growth could be maintained through the cultivation, and L-lysine production reached to 7.86 g. In addition, the effect of counter ion for NH(4)(+) (Cl(-), SO(4)(2-), glutamate, succinate and citrate) was examined. The result showed that the cell growth in the medium using Cl(-) and glutamate were improved compared with that using SO(4)(2-), succinate and citrate, and L-lysine production in the medium using Cl(-) and glutamate reached to more than 9.0 g. In this experiment, there was a clear correlation between ionic strength and growth rate in the cultivation. In order to examine the influence of ionic strength on growth rate, the activity of enzymes in central metabolic pathway from methanol to pyruvate were assayed using samples at the log-phase and the stationary phase in fed-batch cultivation using (NH(4))(2)SO(4) and (NH(4))Cl as ammonium source. It was found that the higher ionic strength inhibited methanol oxidation activity, which linked to cell growth. In this report, it was revealed that maintaining a relatively low ionic strength had a positive effect on L-lysine production using L-lysine producer of M. methylotrophus.

Mechanical damage to Escherichia coli cells in a model of amino-acid crystal fermentation

We investigated the mechanical damage to the Escherichia coli cell caused by polyvinyl chloride particles as a model of amino-acid crystal fermentation. Our results indicated that the glucose-consumption rate and the intracellular ATP concentration temporarily increased by the mechanical damage, and decreased after considerable damage had occurred on cell membrane.