Lysine auxotroph

After recovery of L-lysine, the residual dl-(49) is epimerized to a mixture of the DL and meso isomers, and the latter is subjected to the same decarboxylation step. This reaction is a part of a microbial process in which glucose is fermented by a lysine auxotroph of E. coli to meso- which accumulates in the medium. Meso-(49) is quantitatively decarboxylated to L-lysine by cell suspensions oi erobacteraerogenes (93). However, L-lysine and some... 

It is possible to assess lysine availability using an Escherichia coli lysine auxotroph,44 but further development is required before such a test can compete with furosine determination. 

X. Li and S. C. Ricke, Influence of soluble lysine maillard reaction products on Escherichia coli amino acid lysine auxotroph growth-based assay, J. Food Sci., 2002, 67, 2126-2128. 

In fungi, although a-aminoadipaie is available fiom the lysine biosyniheTic pathway, it could also be obtained from a lysine catabolic pathway, similar to that fouiKl in the actinomycetes, and be channeled into penicillin biosynthesis. In keeping with this possibility, a lysine auxotroph of Pentdltium cfiryspgenum with a block before a-aminoadi-pate formation was found to produce penicillin in a lysine-supplemented medium, and it showed LAT activity (17). Therefore, in fungi, in addition to the lysine biosynthetic pathway, lysine catabolism may also provide a-aminoadipate for penicillin biosynthesis however, the relative contribution of each pathway is unclear. 

There appear to be two pathways for the biosynthesis of lysine among microorganisms. In E. coli there is strong evidence that the diamino dicarboxylic acid, a,e-diaminopimelic acid, is a precursor of lysine. Diaminopimelic acid has been isolated from bacteria - and found to be decarboxylated by a specific decarboxylase occurring in wild-type E. coli. This enzyme has been found to be constitutive rather than adaptive, and it is absent from certain of the lysine auxotrophs. "... 

Other microorganisms, as evidenced by Neurospora, have a different pathw ay of biosynthesis of lysine. The lysine auxotrophs of this organism do not respond to diaminopimelic acid and do respond to such 6-carbon compounds as DL-a-aminoadipic acid or DL-a-amino-6-hydroxy-caproio acid to satisfy their lysine requirement. The latter compounds are inactive for E, coli. Furthermore, diaminopimelic acid could not be detected in Neurospora. 

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). 

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. 

Lysine is formed from aspartate and pyruvate pyruvate, however, is also consumed for the synthesis of alanine. The discovery of an alanine auxotroph, a mutant which needs external alanine addition for growth because it cannot catalyze a precursor step to alanine, was responsible for a yield increase from 16 to 33 g L 1. 

A fermentation process for producing lysine was made possible by using mutants of Corynebacterium glutamicum or Brevibacterium flavum. Both auxotrophic and regulatory mutants have been obtained for overproduction of lysine. Figure 30.19 shows the biosynthetic... 

The pathway of biosynthesis of L-lysine and L-threonine in Corynebacterium glutamicum is shown in Fig. 1. The first step, the formation of phosphoaspartate from aspartate, is catalyzed by aspertokinase and this enzyme is susceptible to the concerted feedback inhibition by L-lysine and L-threonine. The auxotrophic mutant of homoserine (or threonine plus methionine), lacking homoserine dehydrogenase, was constructed and found to produce L-lysine in the culture medium. Second, the mutants which show the threonine or methionine sensitive phenotype caused by the mutation on homoserine dehydrogenase (low activity) was also found to produce appreciable amounts of L-lysine in the culture medium. Furthermore, a lysine analogue (S-aminoethylcysteine) resistant mutant was obtained as an L-lysine producer and in this strain aspartokinase was insensitive to the feedback inhibition. 

The cells could produce even more lysine if the kinase were to remain active but insensitive to a high lysine concentration. Mutants of the homoserine auxotrophs of C. glutamicum were isolated with this property by growing the organism in the presence of toxic isosteres (close structural analogues) of lysine (e.g. S-(2-aminoethyl)-L-cysteine). One way in which the cells can become resistant to the toxic isostere is to overproduce lysine. This is likely to occur in cells where the mutation alters aspartyl kinase in such a way as to make it insensitive to inhibition by lysine, while allowing it to retain its full catalytic activity. 

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