Metabolic auxotrophic mutant

Put selective pressure against plasmid-free cells using ctuxotrophic mutants or antibiotic-resistant plasmids (Parker and DiBiasio, 1987). Auxotrophic mutant is the cell which is mutated so that it requires a specific growth substance beyond the minimum required for normal metabolism and reproduction. 

Structural considerations and known enzymic reactions suggest that the reaction of shikimate 5-phosphate with enolpyruvate phosphate is related to prephenate formation, and that the reactions of shikimate 5-phosphate with glutamine are involved in the synthesis of anthranilate and p-amino-benzoate. A common intermediate for these two branches of the main pathway, after shikimate 5-phosphate, appears to be unlikely, since it would require the further transformation of the latter compound before reaction with enolpyruvate phosphate. If shikimate 5-phosphate is the branch-point intermediate, quintuple auxotrophs should be completely blocked in any one reaction before shikimate 5-phosphate. In the absence of secondary metabolic effects, mutants blocked immediately after it should not be quintuple auxotrophs. They should show a requirement for either phenylalanine plus tyrosine, or tryptophan plus p-aminobenzoate (since anthranilate and p-aminobenzoate may have a common intermediate ). p-Hydroxybenzoate might be derived from this intermediate, or, independently, from shikimate 5-phosphate. This possibility is illustrated on page 264, X being the possible common intermediate. 

Mutation. For industrial applications, mutations are induced by x-rays, uv irradiation or chemicals (nitrosoguanidine, EMS, MMS, etc). Mutant selections based on amino acid or nucleotide base analogue resistance or treatment with Nystatin or 2-deoxyglucose to select auxotrophs or temperature-sensitive mutations are easily carried out. Examples of useful mutants are strains of Candida membranefaciens, which produce L-threonine Hansenula anomala, which produces tryptophan or strains of Candida lipofytica that produce citric acid. An auxotrophic mutant of S. cerevisiae that requires leucine for growth has been produced for use in wine fermentations (see also Wine). This yeast produces only minimal quantities of isoamji alcohol, a fusel oil fraction derived from leucine by the Ehrlich reaction (10,11). A mutant strain of bakers yeast with cold-sensitive metabolism shows increased stability and has been marketed in Japan for use in doughs stored in the refrigerator (12). 

The method proposed for scrutinizing these results is analogous to that by which information on biosynthetic pathways has been obtained from auxotrophic mutants. The principle of the latter technique is as follows. Consider a sequence of metabolic reactions wherein a is converted into d through intermediates b and c, by means of enzymes el, e2, and e3. 

Histidine is the Cinderella of plant amino acids having been virtually ignored by plant biochemists a search of Chemical Abstracts over the last 10 years only revealed two papers relevant to histidine metabolism in higher plants. The probable reason for this is the very difficult biochemistry involved in this pathway and the lack of any usable mutants in higher plant systems. Our knowledge of the pathway is therefore solely based on bacterial metabolism and the elucidation of the pathway is a classical example of the use of auxotrophic mutants (see Brenner and Ames, 1971 Hartman ft al., 1971). 

Auxotrophic mutant An organism that has lost the abihty to synthesize one or more metabolically important enzymes through mutation, therefore requires special substances in its growth medium. 

Metabolic block see Mutant technique. Auxotrophic mutants. 

Mutant technique an important area of biochemical methodology, employing Mutants (see), in particular Auxotrophic mutants (see) of microorganisms. An auxotrophic mutant has a metabolic block at a certain point in the biosynthetic pathway of an essential product (e.g. an amino acid or coenzyme). Such a block is caused by the Mutation (see) of a gene, which (via transcription and translation) is responsible for production of an enzyme ... 

Cohen et al. [61] reported that inducible strains of S. aureus were derepressed by 10 minutes of treatment at 42°C, producing a burst of penicillinase synthesis on subsequent growth at 37°C. The increase in rate started immediately but had stopped after 10 minutes. In the presence of puromycin no protein was formed, but, when the puromycin was removed, the same burst of penicillinase synthesis occurred. Cohen et al. conclude that this behavior could be produced by the thermal inactivation of a repressor and that the restoration of the repressed state required protein synthesis. This thermal derepression is sensitive to certain metabolic conditions, for one particular auxotrophic mutant requiring both adenine and guanine was derepressed only if optimal concentrations of the purines were present at lower concentrations derepression was not observed. Constitutive mutants were not stimulated by the heat treatment. 

Whatever the outcome, the availability of a variant like XIIB2 with an altered metabolism is proving useful for investigating questions about the significance of lAA and its metabolites in cells. We hope that further characterization of the broad range of resistant and auxotrophic mutants described in this paper will lead us onto firmer ground, particularly in the areas of hormone biosynthesis and reception. 

For that purpose, leucine and methionine auxotrophic mutants (Leuf and Meh )) devoid of the chromosomal TK-encoding genes were constructed and transformed with a TKLl-harboring plasmid (strain TKl )) or empty vector (strain TKH). As shown in Tables 15.3 and 15.4, leucine and methionine equally met the metabolic requirement of both TK-deficient and TK-expressing bacteria. 

The elucidation of the steps in the common part of the pathway from carbohydrate precursors to the formation of chorismate remains a classic example of the combined use of auxotrophic mutants, isotop-ically labelled precursors and finally enzyme studies to define a metabolic sequence. An auxotrophic mutant of an organism is a nutritional mutant. The genetic change in such mutant strains causes an inability to carry out one of the reactions in a biosynthetic pathway leading to one or more essential metabolites. Analysis of the sequence of steps in the biosynthetic pathway is facilitated by the fact that each mutant may accumulate in its culture fiuid the substrate for the enzymic reaction which is blocked and may utilise later members of the sequence to replace the required metabolites Davis S produced auxotrophic mutants of Escherichia coli, Aerobacter aerogenes. Bacillus subtilis and Salmonella typhitnurium by ultra-violet irradiation of a bacterial culture in a minimal medium of salts plus D-glucose. Selection of the mutant strains was dependent on the use of the observation that penicillin kills bacterial cells only... 

The terminal amino acids are under strict metabolic control. Some act as feedback inhibitors or repressors. Their synthesis is in equilibrium with metabolic requirement. This equilibrium position prevents their accumulation and hence the yield of these compounds is low. By changing the growth requirement (environmental stimulus) or by genetic manipulation, mutants could be found with limited or removed feedback inhibitors and repressors, e.g. auxotrophic and regulatory mutants 49). This needed a better understanding of biosynthesis and regulation of amino acid production. By selection of these mutants it became possible to alter microbial metabolism which led to the accumulation of the desired amino acids. 

Metabolic Implications of Auxotrophs. The levels and ratios of the 2-methoxy-3-alky1 pyrazines synthesized by these various mutants are contained in Table V. For the auxotrophs, the required nutrients... 

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