Analogue-resistant mutants

Methionine is overproduced by auxotrophic and analogue-resistant mutants. E. coli K-12 mutant resistant to methionine analogues (norleucine, ethionine, and methyl methionine) produced 2mg/ml of methionine. A multianalogue-resistant mutant of Corynebacterium lilium resistant to ethionine, norleucine, and methionine sulfoxide yielded 2.3 g/L methionine in a 5L fermenter.Methionine production from different analogue-resistant yeasts are also reported in the late 1970s and 1980s. ° Mondal et al. have reported methionine production from methionine-analogue-resistant mutants of Brevibacterium heali. 

Mondal S, Chatterjee SP. Enhancement of methionine production by methionine analogue resistant mutants of Brevibacterium heali. Acta Biotechnol 1994 14 199-204. 

Roy A, Murkhopadhyay SK, Chatterjee SP (1997) Production of tyrosine by auxotrophic analogue resistant mutants of Arthrobacter globiformis. J Sci Ind Res 56 727-733... 

In 1958, Kinoshita and Nakayama of Kyowa Hakko Kogyo Co. Ltd. reported that the auxotrophic mutant of Corynebacterium glutamicum, which lacks homoserine dehydrogenase and is defective in L-homoserine (or i-threonine plus i-methionine) biosynthesis, produced L-lysine in the culture medium (Kinoshita et al. 1958). This was the first report on production of an amino acid by an auxotrophic mutant. Subsequently, amino acid production by auxotrophic mutants expanded greatly. Then, the mutants with the L-threonine- or L-methio-nine-sensitive phenotype due to the mutation in homoserine dehydrogenase (low activity) were also found to produce appreciable amounts of L-lysine in the culture medium (Tosaka and Takinami 1986). Furthermore, a lysine analogue (S-aminoethylcysteine)-resistant mutant was obtained as an L-lysine producer. In this strain, aspartokinase was insensitive to feedback inhibition (Tosaka and Takinami 1986). This is the first demonstration of amino acid production by an analogue-resistant mutant. 

L-Threonine is produced by some auxotrophic mutants or threonine-analogue-resistant mutants, and those are created by genetic engineering techniques. The bacteria used are Escherichia coli, Corynebacterium glutamicum, Brevibacterium lactofermentum, B. flavum, Serratia marcescens, and Proteus rettgeri (Nakamori 1986). L-Threonine production by fermentation was started in the 1970s. The auxotrophic mutant and analogue-resistant mutant strains obtained for this purpose were cultured in the presence of amino acids required by the mutant. 

Regulatory mutants, whose aspartate kinase is desensitized to the concerted feedback inhibition, have been obtained by isolation of lysine analogue-resistant mutants, whose growth was not inhibited by a lysine analogue, such as amino ethyl cysteine (AEC). The lysine analogue behaved as a false feedback inhibitor on aspartate kinase and did not allow growth of the parent strain. Only the mutants desensitized to the feedback inhibition grew. 

Other L-amino acids are manufactured much more economically ia thousands of tons per year ia Japan by simplified fermentations direcdy from glucose, ethanol, acetic acid, glycerol, or / -paraffin, by means of selected auxotrophic, regulatory, and analogue-resistant bacterial mutants (94,95). 

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). 

To achieve overproduction of phenylalanine, the micro-organism should be derepressed at the pheA level and free of inhibition at the arcG level. Both genes are located on the chromosomal DNA of the micro-organism and, by means of amino add analogues such as p-fluoro-DL-phenylalanine, it is possible to make (phenylalanine) feedback resistant mutants of E.cdi (pheA and oroF mutants). The following procedure can be used ... 

THYMIDINE KINASE (TK) An enzyme involved in the utilization of the nucleoside thymidine (which ultimately becomes part of the structure of DNA) catalyzes the phosphorylation of thymidine to thymidine monophosphate mutants that lack TK are resistant to the toxic effects of several thymidine analogues, including bromodeoxy-uridine and trifluorothymidine selection of these drug-resistant mutants provides the basis of several... 

For the aromatic pathway (Figure 30.20), the critical control points are the condensation of phosphoenolpyruvate and erythrose-4-phosphate to 3-deoxy-D-arabinoheptulosonate 7-phosphate, DAHP, by DAHP synthase. For tryptophan, the formation of anthranilic acid from chorismic acid by anthranilate synthase is the second critical control point. The transcriptional regulation was overcome through the use of alternative promoters and allosteric regulation was circumvented by the classical technique of selection for feedback-resistant mutants using toxic analogues of the repressing compounds. 

Terbinafin resistance in Necria haematocacca may be caused by a less sensitive squalene epoxidase. The resistant biotypes have at least 10 times more squalene than the normal, and it seems that the epoxidase has less affinity to both squalene and terbinafine in the resistant mutants. Fenpropi-morph causes certain sterols to accumulate (A8,14-sterols) and some to decrease (the A5,7 analogue). Resistance seems to be caused by tolerance to these changes, rather than to less sensitive enzymes. 

The rib operon of Corynebacterium ammoniagenes is structured as the rib operons of B. subtilis or C. glutamicum. Purine analogue-resistant C. ammoniagenes mutants, which were transformed with plasmids encoding the... 

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