Strains, auxotrophic

Exper- iment No. Bacterial strain Auxotrophic for tryptophan Other run parameters ... 

Investigations with different carbon sources (Table IV) showed that although the amounts of the two pyrazines varied according to the carbon source used, the ratio of the isopropyl to secbutyl pyra-zine remained relatively constant. This suggested that either availability of precursor followed a set ratio, or else enzyme specificity for the different side chains played a role in determining the relative amounts of each compound synthesized. Accordingly, strains auxotrophic for leucine (leu-), isoleucine (ilu-), or leucine, valine, isoleucine, and pantothenic acid (leu"> val-, ilu-, pant-) were isolated from cultures treated with... 

The translational activity of various unsaturated analogs of L-isoleucine was evaluated using an Escherichia coli strain auxotrophic for isoleucine. It was observed that the alkene [2-amino-3-methyl-4-pentenoic acid (2)] and alkyne [2-amino-3-methyl-4-pentynoic acid (3)] derivatives of L-isoleucine can support protein synthesis at levels approximately 50% of that observed in cultures supplemented with isoleucine. However, no incorporation of the ocC or /JC methylated derivatives could be detected. In order to examine the stereoselectivity of incorporation, the (2S, 3S) and (2S, 3R) diastereomers of 2 and 3 were prepared. The extents of isoleucine substitution in vivo were 80% and 70% for (2S, 3S)-2 and (2S, 3S)-3, respectively, under the conditions examined in this study. 

Refs. 21, 22, 155. Abbreviations AHV, a-amino-(3-hydroxyvaleric acid Horn, L-homoserine AEG, (3 -(2-aminoethyl)-L-cysteine ppc, phosphoenolpymvate carboxylase the strain improvement largely depends on the transduction technology sensitive resistant —, auxotroph or deficient , leaky auxotroph +, prototrophic revertant. Table 7. Amino Acid Production from Hydrocarbons ... 

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). 

Two types of mutants have been used for amino add overproduction auxotrophic and regulatory mutants. In some cases, mutant strains have been further improved through DNA-recombination. 

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). 

Compared to conventional NMR isotopes (13C, 15N, 2H), 19F-labels cannot be readily placed into proteins in a versatile manner by any biosynthetic expression strategy. Certain auxotrophic bacterial strains can be used to incorporate iso-steric 19F-labelled amino acids (e.g. Fluoro-Phe, Fluoro-Trp, Fluoro-Leu, Fluoro-Ile see Fig. 3), but yields tend to be low. Many other fluoro-organics are toxic if they get converted into fluoroacetic acid, which blocks the enzyme aconitase in the citric... 

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... 

In addition to using auxotrophic strains of naturally occurring amino acids, the laboratory of Peter Schultz has developed very elegant methods to incorporate labeled amino acids in specific positions of a protein by extending the genetic code of prokaryotic as well as eukaryotic cells [26,27]. The... 

B834 B strain methionine auxotroph used for and selenomethionine labelling... 

If a methionine auxotroph is not used, the methionine within the culture can be replaced by SeMet using a methionine biosynthesis inhibition or poisoning method such as that outlined in Protocol 2.7. This method has the advantage of using any E. coli strain available and thus the initial growth of the culture is not reduced, however the method is more laborious than using the methionine auxotroph. 

Prokaryotes are sensitive towards aminoglycosides. In E. coli sublethal levels suppress auxotrophic mutations and increase the frequency of misincorporation obtained in vitro (Gorini and Kataja 1964 Davies et al. 1965). Yeast is normally quite resistant toward aminoglycosides, but mutant strains expressing translational suppressors display increased sensitivity in vivo and increased mistranslation frequencies in vitro (Palmer et al. 1979 Singh et al. 

This assay employs a number of different strains of Salmonella typhimurium mutants that are histidine auxotrophs. The first tester strains developed and tested on extracts of diesel and ambient POM in the mid to late 1970s included TA1535, which responds to base-pair substitution chemical mutagens (e.g., /f -propiolac-tone), and TA1537 and TA1538, which respond to frameshift mutagens, e.g., BaP and other PAHs. 

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