Fundamentals of Biochemical Engineering, Part 1 - Bailey, J., Ollis, D. 1989

Molecular Genetics and Regulatory Systems
Industrial Applications of Research Results on Microbial Genetics and Mutant Populations
Applications of Auxotrophic Mutants

Auxotrophic mutants (Methods for their Selection and identification are given in Table 6.3) are characterized by a lack of the enzymatic activity required to carry out one or more steps in a biosynthetic pathway. As a result, auxotrophs are unable to synthesize the end product of that metabolic pathway. Such a mutant can survive only if the non-synthesized metabolite is present in the growth medium. For example, a Tryptophan auxotroph will grow only in the presence of tryptophan. If a strain synthesizes tryptophan on its own, it is referred to as tryptophan-prototrophic.

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FIG. 6.18. Auxotrophic mutants can be used to increase the yield of intermediate products in a metabolic pathway. In the hypothetical example shown here, the mutant lacks enzyme c and, consequently, does not synthesize repressor E. (Reproduced from: Demain A. L., Overproduction of Microbial Metabolites and Enzymes due to Alteration of Regulation, in Advances in Biochemical Engineering 1, Ghose T. K., Fiechter A. (eds.), p. 120, Springer-Verlag, New York, 1971.)

Since the missing metabolites are supplied in the growth medium rather than synthesized by the auxotroph, their concentrations are regulated, essentially, not by the microorganism, but by the biochemical engineer. From a practical standpoint, this situation can be very convenient if the non-synthesized metabolic product is a repressor. As shown in Fig. 6.18, auxotrophic mutants unable to synthesize a repressor can be forced to overproduce* a particular metabolic intermediate. By maintaining a low concentration of repressor E in the medium, Feedback inhibition and enzyme repression (affecting enzymes involved in this pathway) can be minimized. Under these conditions, intermediate compound C (usually the substrate for enzyme c, which is absent in this mutant) is produced at a much higher concentration than in the native Organism.

* "Overproduction" here refers to an increased rate of substance Biosynthesis compared to the optimal rate required for Cell growth. However, the optimal rate of biosynthesis for The Cell does not yield enough product for industrial manufacturing. This is precisely why mutant strains with disrupted regulatory systems are used industrially.

FIG. 6.19. A homoserine dehydrogenase-deficient mutant of C. glutamicum produces elevated amounts of L-Lysine. (Reproduced from: Demain A. L., Overproduction of Microbial Metabolites and Enzymes due to Alteration of Regulation, in Advances in Biochemical Engineering 1, Ghose T. K., Fiechter A. (eds.), p. 122, Springer-Verlag, New York, 1971.)

As we noted at the end of the previous section, such a situation may require additional operations to ensure the diffusion of intermediate compound C across The cell membrane into the medium.

Fig. 6.19 illustrates the mechanism underlying the industrial production of L-lysine using an auxotrophic mutant of C. glutamicum. The high-yielding mutant lacks the enzyme homoserine dehydrogenase; therefore, the inhibition of lysine synthesis by Threonine (via aspartokinase) is not observed in this mutant. Because the auxotrophic mutant synthesizes neither threonine nor Methionine, both of these Amino Acids must be added to the growth medium.

A comparison of The regulation of aspartate-family amino acid biosynthetic pathways in C. glutamicum (Fig. 6.19) and E. coli (Fig. 5.12) leads to an important Conclusion: organisms with similar biosynthetic pathways do not necessarily have identical regulatory systems. Note, in particular, that in the scheme of Fig. 5.12, lysine auto-inhibits its own biosynthesis via a reaction leading to dihydrodipicolinic acid. In the wild-type (non-mutant) strain of C. glutamicum, the corresponding inhibition step is absent. The aspartokinase system found in Corynebacterium appears to differ from that of E. coli in that the former is inhibited only in the simultaneous presence of lysine and threonine (concerted or multivalent feedback inhibition), whereas the aspartokinase Isoenzymes in E. coli are inhibited by lysine alone. For these very reasons, it is more advantageous from an industrial standpoint to use Corynebacterium.

A similar approach has been successfully applied to develop highly efficient microbiological processes for producing flavoring agents—the purine NUCLEOTIDES guanosine monophosphate (GMP), inosine monophosphate (IMP), and xanthine monophosphate (XMP). As shown in Fig. 6.20, minimizing AMP and GMP concentrations using an auxotrophic mutant of Brevibacterium ammoniagenes makes it possible to enhance The biosynthesis of inosine and hypoxanthine. The concentration of the latter in the medium increases upon The addition of small amounts (about 10 μುತ್ತೇವೆ/L) of manganese ions (Mn²⁺). Available data indicate that manganese alters cell membrane permeability.