Auxotrophs production

For better control of fermentation and to reduce production costs, complex media components are avoided and mostly refined carbon sources are used for the industrial production of L-lysine. Sucrose can be obtained from cane or beet molasses, and glucose is provided in hydrolysates of corn, cassava, or wheat starch [30, 83]. Ammonia, as nitrogen source, can be added pure or as salts [32]. Further media components are vitamins, in particular biotin, as well as salts and trace elements. Amino acids for auxotrophic production strains can be provided by peptones, corn steep liquors, or soybean meal hydrolysates [30]. Preferably, media are sterilized continuously, whereas carbon sources and nitrogen sources are typically sterilized separately to avoid Maillard-type reactions [32]. Sterility is important for processes with the mesophilic and neutrophilic C. glutamicum with bacilli as main contamination risk [84], while phage infection is hardly a problem. 

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 ... 

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. 

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

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. 

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. 

Nucleotide Biosynthesis in Amino Acid Auxotrophic Bacteria Normal E. coli cells can synthesize all 20 common amino acids, but some mutants, called amino acid aux-otrophs, are unable to synthesize a specific amino acid and require its addition to the culture medium for optimal growth. Besides their role in protein synthesis, some amino acids are also precursors for other nitrogenous cell products. Consider the three amino acid auxotrophs that are unable to synthesize glycine, glutamine, and aspartate, respectively. For each mutant, what nitrogenous products other than proteins would the cell fail to synthesize ... 

AUXOTROPH An organism or cell that is unable to carry out some particular synthesis and hence requires the product of that synthesis to be present in the growth media. (See also PR0TOTROPH)... 

By use of synthetic medium the formation of 3,4-trans-CH D was maintained for a 36-h cultivation period, resulting in accumulation of up to 790 mg L 1 3,4-trans-CHD 1 [11]. With this strain, however, it is necessary to separate the growth and production phases, because the substances for which E. coli strain BN117 is known to be auxotrophic, i.e. tryptophan, tyrosine, phenylalanine, proline, arginine, histidine, and p-aminobenzoate, also partially inhibit entry to the chorismate biosynthesis pathway (feedback inhibition of DAHP-synthase). 

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. 

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. 

The terminal amino acids are under strict metabolic control. Some act as feedback inhibitors or repressors. Their synthesis is in equilibrium with metabolic requirement. This equilibrium position prevents their accumulation and hence the yield of these compounds is low. By changing the growth requirement (environmental stimulus) or by genetic manipulation, mutants could be found with limited or removed feedback inhibitors and repressors, e.g. auxotrophic and regulatory mutants 49). This needed a better understanding of biosynthesis and regulation of amino acid production. By selection of these mutants it became possible to alter microbial metabolism which led to the accumulation of the desired amino acids. 

For tryptophan production in E. coli, the natural regulation controlling production of tyrosine and phenylalanine was sufficient to keep carbon flowing specifically to tryptophan. This eliminated the need for addition of auxotrophic compounds to the growth medium. The major industrial producers of tryptophan are ADM, Kwoya Hakko, and Ajinomoto. 

Similar techniques were used by Shinohara et al (71) to develop hybrids with increased production of fusel alcohols and esters. Protoplast fusion techniques have been used to confer amylolytic activity to brewery yeasts (22) and ethanol tolerance to wine yeasts (70) Farris et al (72) used protoplast fusion to produce hybrids with killer factor that is, the ability to secrete proteinic toxins. Kunkee and coworkers (25) utilized a leucine auxotrophic mutant strain of S. cerevisiae (UCD Montrachet 522) to produce base wine for brandy production the mutant strain produces less isoamyl alcohol, reducing the quantity of fusel alcohols in the subsequent brandy. And Thornton (48) discussed the progress in utilizing plasmid vectors to introduce new genes into wine yeasts he cautioned, however, that until the yeast genome is better understood that direct gene manipulation techniques will be of limited value. 

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