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.

FIG. 1.

Summary of the metabolic pathway from glucose to l-glutamate in C. glutamicum.

FIG. 2.

Map of the odhA gene. Double-headed arrows indicate the fragment used. Arrow A, 4.4-kbp SalI-XhoI fragment containing the odhA gene cloned into pPKSX4.4. Arrow B, 1.5-kbp 5′-terminal fragment used for Southern hybridization. Double-headed dashed arrow C indicates the 1.9-kbp KpnI-KpnI region deleted in the odhA deletion strain.

FIG. 3.

Growth (A) and l-glutamate production (B) of the C. glutamicum wild-type strain ATCC 13869 and its derivative lacking odhA. Batch fermentations were conducted in stirred vessels at 30°C and pH 7.0 until all the glucose was consumed. AJ13133 produced a remarkably high level of l-glutamate. It accumulated l-glutamate during growth, which continued to the end of the fermentation. Solid circles, wild type; open circles, odhA deletion strain.

FIG. 4.

Clones isolated from the original strain AJ13133 lacking odhA. (A) Growth and l-glutamate production of clones isolated from the original odhA deletion strain AJ13133. The isolated clones could be divided into three groups: the first, rapidly growing clones that produced l-glutamate as efficiently as the parent strain (squares); the second, rapidly growing clones that did not overproduce l-glutamate (circles); and the third, clones whose growth was slower than that of the parent and whose l-glutamate production was 60 to 70% of that by the parent (triangles). OD₆₆₀ and l-glutamate levels were measured after glucose was completely consumed, when the cultivation time was 16 to 20 h for the clones in the first and second groups and 40 to 46 h for the clones in the third group. (B and C) By-products of the isolated clones. (B) Amino acids; (C) organic acids. Results for two clones from each group are shown. Solid bars, clones in the first group; open bars, clones in the second group; shaded bars, clones in the third group.

FIG. 5.

Effects of the l-glutamate-producing condition of biotin limitation on l-glutamate production in AJ11024. Wild-type (solid circles) and AJ110214 (open circles) cells were cultured under biotin-limited conditions. Biotin concentrations are shown along the x axis. For biotin-limited conditions, seed cultures were prepared with 10, 30, 60, 100, and 300 μg of biotin liter⁻¹, and 1 ml was inoculated into 20 ml of the main medium lacking biotin so that biotin was depleted during the main cultures. For biotin-rich conditions, a seed culture was prepared in 300 μg of biotin liter⁻¹, and 1 ml was inoculated into 20 ml of the main medium containing 300 μg of biotin liter⁻¹. The biotin concentration was calculated by dividing the total amounts of biotin supplied to the seed and main cultures by the volume of the main culture. The specific l-glutamate production rate (A), the specific glucose consumption rate (B), and the l-glutamate yield (C) are shown. Specific rates were calculated during the periods when they were approximately constant. l-Glutamate yield is defined as millimoles of l-glutamate produced per millimole of glucose at the end of the experiment. Error bars, standard deviations from the means, calculated from three independent experiments.

FIG. 6.

Effects of the l-glutamate-producing condition of Tween 40 addition on l-glutamate production in AJ110214. Wild-type (solid circles) and AJ110214 (open circles) cells were cultured with Tween 40 addition; Tween 40 was added to 0, 0.1, 0.3, or 3 g liter⁻¹. The specific l-glutamate production rate (A), the specific glucose consumption rate (B), and the l-glutamate yield (C) are shown. Specific rates were calculated during the periods when they were approximately constant. l-Glutamate yield is defined as millimoles of l-glutamate produced per millimole of glucose at the end of the experiment. Error bars, standard deviations from the means, calculated from three independent experiments.

FIG. 7.

Effects of the l-glutamate-producing condition of penicillin addition on l-glutamate production in AJ11024. Wild-type (solid symbols) and AJ110214 (open symbols) cells were cultured without penicillin (circles) or with 0.2 U of penicillin ml⁻¹ (triangles). (A) Time courses of growth; (B) glucose consumption; (C) l-glutamate production; (D) l-glutamate yield.

FIG. 8.

Optimization of GDH activity for l-glutamate production in AJ110214. Derivatives of AJ110214 with various GDH activities were cultured in flasks, and production of l-glutamate (A) and 2-oxoglutarate (B) was analyzed at 15 to 16 h, when glucose was completely consumed. GDH activities are shown relative to that of AJ110214 (taken as 1). l-Glutamate production was maximally increased from 108 to 130 mM, at which concentration GDH activity was 7.1- to 13.5-fold higher than that in AJ110214. Two independent experiments were performed. Open diamonds, AJ110214gdh2; open squares, AJ110214gdh3; open triangles, AJ110214gdh7; asterisks, AJ110214gdh7′; solid diamonds, AJ110214/pgdh2; solid squares, AJ110214/pgdh3; small bars, AJ110214/pgdh4; solid triangles, AJ110214/pgdh7; crosses, AJ110214/pgdhwt; open circles, AJ110214; solid circles, AJ110214/pSAC4 (vector).

FIG. 9.

Effects of Tween 40 addition on l-glutamate production in the odhA deletion strains (AJ110214) with wild-type levels of GDH activity. Intracellular and extracellular metabolites were analyzed under conditions of no Tween 40 addition (A and D) or addition of 0.1 (B and E) or 0.3 (C and F) g of Tween 40 liter⁻¹, using the odhA deletion strains (AJ110214) with 1-, 6.0-, 13.5-, and 95.1-fold-higher levels of GDH activity. (A, B, and C) Intercellular concentrations of amino acids (left panels) and organic acids (right panels); (C, D, and E) extracellular production of l-glutamate (left panels), other amino acids (center panels), and organic acids (right panels) per gram of cells (dry weight). Results of two independent experiments are shown. Magenta, AJ110214; pink, AJ11024/pSAC4 (vector); yellow, AJ110214gdh7 (with 6.0-fold-higher GDH levels); light blue, AJ11024/pghdwt (13.5-fold-higher GDH levels); violet, AJ11024/pgdh7 (95.1-fold-higher GDH levels).