Fermentation Method

Excretion of L-amino acids by Escherichia coli when an excess of ammonium salt was added was reported in 1950 (6). In 1957, a fermentation process was successfully commercialized (3) that used a strain of bacteria isolated from soil, later found to be Corynebacteriumglutamicum, which is able to excrete considerable amounts of L-glutamic acid.

Numerous microorganisms have been isolated and found to be able to overproduce L-glutamic acid, including Brevibacterium flavum, B. lactofermentum, and Microbac-terium ammoniaphlum. Because of minor differences in the character of these bacteria, which are all Gram positive, non-spore forming, nonmotile, and require biotin for growth, the name of genus Corynebacterium was suggested for these coryneform bacteria (7).

For production of L-glutamic acid, the key factors in controlling the fermentation are as follows:

1. The presence of biotin in the range of 5 to 10 ig/L, which is optimal for the excretion of L-glutamic acid through cell walls

2. A sufficient supply of oxygen to reduce the accumulation of lactic acid and succinic acid as by-products

When a biotin-rich medium, such as molasses, is used as the carbon source, the addition of penicillin or cepha-losporin C favors the overproduction of L-glutamic acid, supposingly due to the repression of peptide glycan synthesis on the cell membrane. The supplementation of a fatty acid or surfactant also results in an increased permeability of the cell wall, thus enhancing L-glutamic acid excretion. Kramer (8) reports the existence of specific carrier mechanisms that are responsible for active L-glutamic acid secretion in C. glutamicum.

Generally, the intracellular accumulation of L-glutamic acid does not reach levels sufficient for feedback control in glutamate overproducers due to rapid excretion of glutamate. However, the regulatory mechanisms of L-glutamic acid biosynthesis have been studied intensively to obtain mutants with increased productivity, as shown in Figure 1.

Shiio (9) discussed two enzymes that have played key roles in the biosynthesis of L-glutamic acid. The first, phos-phoenolpyruvate carboxylase, catalyzes carboxylation of phosphoenolpyruvate to yield oxaloacetate; it is inhibited by L-aspartic acid and repressed by both L-aspartic acid and L-glutamic acid. Second, a-ketoglutaric acid dehydrogenase converts a-ketoglutaric acid to succinyl-CoA; in L-glutamic acid-overproducing strains, a-ketoglutaric acid dehydrogenase limits further oxidation of a-ketoglutaric acid to carbon dioxide and succinic acid, thus favoring the formation of L-glutamic acid.

In L-glutamic acid-overproducing strains, the Km value of a-ketoglutaric acid dehydrogenase was nearly two magnitudes lower than that of L-glutamic acid dehydrogenase, which catalyzes the last step to L-glutamic acid. Consequently, vmax of L-glutamic acid dehydrogenase proved to be about 150 times higher than that of a-ketoglutaric acid dehydrogenase. Borman (10) isolated and characterized the C. glutamicum glutamic acid dehydrogenase.

A strain of Microbacterium ammoniaphlum cultured under biotin-deficient conditions produced 58% of L-glutamic acid formed from glucose via phosphoenolpyr-vate, citrate, and a-ketoglutaric acid; the other 42% was formed via the TCA cycle or glyoxylate cycle (11).

The mutants are reported as either having sensitivity in cell permeability (12), having the capability for increased carbon dioxide fixation (13), or having a too-low activity level of pyruvate dehydrogenase (14).

Another approach focused on the development of ther-mophilic mutants. A strain of Corynebacterium thermo-aminogenes is reported to accumulate L-glutamic acid at temperatures above 43 °C (15).

Glucose

L-Aspartic acid

Phosphoenol pyruvate (2)

- Pyruvate

Acetyl-CoA

Oxalactate cis-aconitate

cis-aconitate t (1>Glyoxylate<"^ Malate - x \ '

Isocitrate

Fumarate

Succinate

L-Glutamate

Feedback inhibition Repression

Figure 1. Regulation of l-glutamic acid biosynthesis in Corynebacterium glutamicum (5). Regulatory enzymes: 1, phosphoenolpyruvate carboxylase; 2, pyruvate kinase; 3, pyruvate carboxylase; 4, pyruvate dehydroge-nase; 5, citrate synthetase; 6, aconitase; 7, isocitrate de-hydrogenase; 8, l-glutamate dehydrogenase; 9, a-ketoglutarate dehydrogenase; 10, isocitrate lyase; 11, malate synthetase.

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