Auxotrophic mutant

Kao and Puck (1968) have developed a general method for production of auxotrophic mutants. This is based on the observation that DNA containing 5-bromodeoxyuridine (BUdr) is sensitive to visible light produced by fluorescent lamps. This can be used to selectively kill prototrophs growing on restrictive media, i.e. media which restrict the growth of auxotrophic (or other) mutants. The cells that later grow out in supplemented media are auxotrophic mutants. The successive steps involved are given below. 

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Many kinds of amino acids (eg, L-lysine, L-omithine, t-phenylalanine, L-threonine, L-tyrosine, L-valine) are accumulated by auxotrophic mutant strains (which are altered to require some growth factors such as vitamins and amino acids) (Table 6, Primary mutation) (22). In these mutants, the formation of regulatory effector(s) on the amino acid biosynthesis is genetically blocked and the concentration of the effector(s) is kept low enough to release the regulation and iaduce the overproduction of the corresponding amino acid and its accumulation outside the cells (22). 

Mutation. For industrial appHcations, mutations are induced by x-rays, uv irradiation or chemicals (iiitrosoguanidine, 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 Hansenu/a anomala, which produces tryptophan or strains of Candida lipolytica 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 isoamyl alcohol, a fusel oil fraction derived from leucine by the Ehrlich reaction (10,11). A mutant strain of bakers yeast with cold-sensitive metaboHsm shows increased stabiUty and has been marketed in Japan for use in doughs stored in the refrigerator (12). 

Auxotrophic mutants are mutants that miss one or more of the enzymes used in the biosynthetical pathway for one or more amino adds. In practice this means that the mutant needs one or more key metabolites which it cannot synthesise for growth in its growth medium. For example, consider Figure 8.4. 

In the case of a tyrosine auxotrophic mutant, the mutant does not produce at least one of the enzymes to synthesise tyrosine (E6 in Figure 8.4). 

Auxotrophic mutants are used in the production of end products of branched pathways, ie pathways leading to more than one amino add at the same time. This is the case for L-lysine, L-methicmine, L-threonine and L-isoleudne in Brevibacterium flavum and Corynebacterium glutamicum. 

Unlike auxotrophic mutants, regulatory mutants can be grown in inexpensive, complex media and they do not require careful control of growth conditions. 

Auxotrophic mutants of E. cdi are particularly useful for the production of L-phenylalanine by direct fermentation. 

Since auxotrophic mutants and regulatory mutants are widely used in the overproduction of amino adds, this can be a severe problem. In nature, mutation always takes place but this takes some time. However, in fermentation many generations are produced in a relatively short period of time and the chances of back mutation are enhanced. 

Auxotrophic mutant lack one or more enzymes involved in the synthesis of amino acids (such as tyrosine). This prevents accumulation of the amino acid and thus avoids feedback inhibition of enzymatic steps in the L-phenylalanine pathway. 

Protoplast fusion induced by polyethyleneglycol and Ca was carried out between two auxotrophic mutants of Aspergillus sp. CH-Y-1043. The hybrids obtained showed significant differences in endopectinase activity and morphology compared to the prototrophic strain. Strains grown on lemon peel showed production improvement with respect to the parental strain. Since H15 hybrid showed up to 90% higher endopectinase production than the wild type CH-Y-1043, kinetics of enzyme production in Fernbach flasks and Fermentor (14L) by H15 were determined. 

Aspergillus sp. CH-Y-1043 was used as the prototrophic parental strain. The auxotrophic mutants A200 ade- (an adenine-requiring mutant) and A400 pyr (a pyridoxine requiring mutant) were isolated from the parental strain by treatment with N-methyl-N -nitro-nitrosoguanidina (NTG) as described previously (10). 

The introduction of techniques for mutagenesis by UV irradiation or by the use of chemicals considerably extended the applications of microbial studies to nutrition (Davis, 1954-1955). Auxotrophic mutants were produced with nutrient dependencies not shown in the untreated parental strains (Beadle and Tatum, 1940). The fortuitous discovery of penicillin by Fleming and its successful use in the treatment of infections (Florey) promoted exhaustive research into its mode of action. Eventually it was established that penicillin prevented the proliferation of gram-positive bacteria by blocking the synthesis of their cell walls... 

Ames test analy chem A bioassay that uses a set of histidine auxotrophic mutants of Salmomlla typhimurium for detecting mutagenic and possibly carcinogenic compounds. amz, test ... 

Cultivation of Auxotrophic Mutant Microbial processes that use an auxotrophic mutant require control of the concentration of a required nutrient. 

The Ames Test In a nutrient medium that lacks histidine, a thin layer of agar containing 109 Salmonella ty-phimurium histidine auxotrophs (mutant cells that require histidine to survive) produces 13 colonies over a two-day incubation period at 37 °C (see Fig. 25-19). How do these colonies arise in the absence of histidine The experiment is repeated in the presence of 0.4 pg of 2-aminoanthracene. The number of colonies produced over two days exceeds 10,000. What does this indicate about 2-aminoanthracene What can you surmise about its carcinogenicity ... 

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. 

The use of genetic mutants determined the complex pathways that lead to the amino acids. A mutant is an organism that has a different DNA sequence from its parent(s). Mutant bacteria that require a specific compound for growth are called auxotrophs. The first step in pathway determination is to assemble a large collection of auxotrophic mutants that can t make the compound of interest. 

However, recently it has proved possible to positively identify tryptophan radicals in cytochromec peroxidase[147] and tyrosine radicals in ribonucleotide reductase, prostaglandin H synthase and photosystem II of chloroplasts [148], This has been achieved by a combination of the techniques discussed already, but with the powerful, additional non-invasive tool of isotopic substitution. As deuterons (5=1) give different splitting than protons (S = 1/2), substituting different labelled amino-acid residues into the enzyme should reveal the nature of the radical-containing residue. This is easily achieved in an auxotrophic mutant that requires this amino acid to be supplied in the medium. The specific residue can then be identified by site-directed mutagenesis of the evolutionary conserved amino-acid residues [108,149-151]. 

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