Auxotrophs

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... 

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). 

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 ... 

Abbreviations Horn, homoserine AEG, 3 -(2-aminoethyl)-L-cysteine resistant —, auxotroph. ... 

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. 

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. 

The best amino add producers are organisms that are both auxotrophic and regulatory mutants. 

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. 

Addition of antibiotics to the fermentation broth may be used to avoid problems associated with growth revertants (eg auxotrophic back mutation) ensure that genetic material (eg plasmid DNA) is maintained within the process micro-organism. 

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). 

Schneider, J.C., Jenings, A.F., Mun, D.M. et al. (2005) Auxotrophic markers pyrF and proC can replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonas fluorescens fermentation. Biotechnology Progress, 21 (2), 343-348. 

Although iron-sulfur proteins are found in various cellular localizations in eukaryotic cells, mitochondria are the major site of Fe-S cluster biosynthesis (Lill et ah, 1999). Deletions in nuclear genes involved in mitochondrial iron-sulfur cluster formation lead to massive accumulation of iron in mitochondria (Chapter 7). For example, deletion of ATM1, a mitochondrial ATPase, which seems to be responsible for the export of Fe-S clusters, leads to respiratory incompetence, excessive iron accumulation and leucine auxotrophy (Kispal et ah, 1999). In Ayfhl cells there is only partial loss of mitochondrial Fe-S enzymes and the cells are not leucine auxotrophs. 

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... 

Auxin-like herbicides, 13 305 Auxins, 13 35, 38, 284, 304 Auxochromes, 19 425 Auxotrophic mutations, in yeast,... 

Auxotrophic plant pathogens, 13 350-351 Available Chemicals Directory (ACD), 6 19-20... 

---