Modification Of The Permeability

The best example of the modification of the permeability of a micro-organism is provided by the glutamic acid fermentation. Kinoshita et al. (1957a) isolated a biotin-requiring, glutamate-producing organism, subsequently named Corynebacterium glutamicum, the permeability of which could be modified by the level of biotin. Provided that the level of biotin in the production medium was below 5 /ig dm"3 then the organism would excrete glutamate, but at concentrations of biotin optimum for growth the organism produced lactate (Kinoshita and Nakayama, 1978). Thus, the permeability of C. glutamicum may be controlled by the composition of the culture medium and, as such, is not an example of strain improvement in the sense that the term has been used in this chapter. However, the isolation of C. glutamicum was a major advance in the microbial production of amino acids and the demonstration of the role of permeability in the production of glutamate suggested the possibility of the genetic modification of permeability to achieve high levels of productivity. Thus, it is relevant at this stage to consider the physiology of glutamate production by biotin-limited culture.

Kinoshita's isolate was not only deficient in its ability to produce biotin but also in the level of a-keto-glutarate dehydrogenase, which normally converts a-ketoglutarate to succinate in the tricarboxylic acid cycle. Thus, a metabolic block results in the accumulation of a large concentration of a-ketoglutarate which the organism converts to glutamic acid. Oxaloacetate is regenerated in glutamate producers by the activity of the glyoxylate pathway as shown in Fig. 3.11.

When C. glutamicum was grown in a medium containing a high concentration of biotin, the organism synthesized glutamate at a level of 25-36 /igmg"1 dry weight of cells, further production being assumed to be prevented by some form of feedback control by glutamate of its own synthesis (Demain and Birnbaum,

Glucose

Acetate

Acetyl coenzyme A.

Acetyl coenzyme A.

Glutamate Synthesis Glutamicum

Glutamate

Fig. 3.11. Biosynthesis of glutamate by C. glutamicum. Heavy lines indicate the route to glutamate and the light lines indicate the route in the regeneration of oxaloacetate via the glyoxylate cycle.

Glutamate

Fig. 3.11. Biosynthesis of glutamate by C. glutamicum. Heavy lines indicate the route to glutamate and the light lines indicate the route in the regeneration of oxaloacetate via the glyoxylate cycle.

1968). Under conditions of biotin limitation, glutamate was released from the cells and accumulated to a level of up to 50 g dm^3. The increased permeability of the biotin-limited cells to glutamate corresponds with a change in the fatty acid and phospholipid content of the cell envelopes. The crucial factor in the control of permeability appears to be the synthesis of membranes deficient in phospholipids (Nakao et ai, 1973).

The role of biotin in the glutamate fermentation obviously necessitates the inclusion of limiting levels of biotin in the medium. Thus, the use of crude carbon sources such as cane molasses, which are rich in biotin, presented difficulties which have been overcome by the modification of the permeability by means other than biotin limitation. The inclusion of penicillin or fatty acid derivatives such as polyoxyethylene sorbitan mono-oleate (Tween 80) during logarithmic growth in biotin-rich medium caused an aberration of the cell envelope permeability resulting in the release of glutamate (Udagawa et al., 1962).

The production of glutamate from hydrocarbons (as carbon sources) has also presented difficulties in the control of permeability, in that the assimilation of hydrocarbons results in the production of fatty acids which effectively bypasses the site of biotin control (Nakao et al., 1972). The permeability of the producing organism may be controlled by the addition of penicillin (Wang et al., 1979) but a genetic solution to this problem has also been found. Nakao et al. (1970) isolated a glycerol requiring auxotrophic mutant of Coiynebacterium alkanolyticum in which phospholipid synthesis was controlled by the supply of glycerol. The mutant produced about 40 g dm-3 glutamate when grown on n-paraffins in the presence of 0.01% glycerol (Nakao et al., 1972). Thus, an understanding of the mode of action of biotin limitation in the glutamate fermentation has led to the use of genetic modification of permeability as a method to overcome the normal control mechanisms of the producing organism.

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