Overproduction

The most common defect in oxypurine metabolism is that manifested by hypermicemia and hyperuricosuria. The many epidemiological investigations of uric acid levels have shown a distribution of serum uric acid values ranging from approximately 0.5 to 9.5 mg/100 ml of serum (E7, F6, N2). Many of the individuals with extreme levels are perfectly healthy, although there is a correlation between secondary complications and high uric acid values.  

Just as there is no clear line of differentiation between hyperuricemic gout and normo-uricemic nongout, so there is no clear indication of the mechanism of the overproduction of uric acid. Many investigators have shown, by the administration of various labeled precursors, that in hyperuricemic and hyperuricosuric individuals there is a greater incorporation of labeled precursors into uric acid than there is in normal controls. Actually these studies merely confirm the fact that more uric acid is made by these overexcretors. If more uric acid is made, then more of the precursor must be incorporated, and we should therefore be most surprised to find that there is no increase in the labeled precursors. 

These studies seem to indicate that the uric acid production found in cases of hyperuricemia and gout occurs by the usual biosynthetic pathways. 

The most fundamental observation regarding the biochemical nature of gout was in all probability that of Benedict et oZ. (B14), who showed that the uric acid pool size in gouty overproducers was considerably larger than that of normal individuals. In those cases where the urinary output was not greater than normal, there were greater extrarenal elimination and degradation of the uric acid. This was shown both by the fact that the slope of the activity curve (Section 4.1) indicated a replacement of much more uric acid than was actually found in the urine, and by the fact that only a small fraction of the injected uric acid was recovered as urate. The fraction of the injected uric acid recovered from the urine was equal to the fraction of the daily turnover of uric acid which appeared as lu-inary uric acid. 

A second aspect of the precipitation of acute attacks of gout is related to the inflammatory reaction produced by the injection of sodium urate crystals. The historical background for believing that such materials are related to the occurrence of acute attacks is reviewed by McCarty (MIO). Since synovial fluid from patients with acute attacks contains micro-crystalline sodium urate, it appeared reasonable to believe that the presence of these crystals would cause gouty attacks. A number of investigators showed that administration of a microcrystalline sodium urate resulted in attacks in normal human subjects, in dogs, and in gouty patients in a quiescent phase (FI, H12, M4). 

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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). 

Development of Resistance. One of the principal disadvantages of sulfonamide therapy is the emergence of dmg-resistant strains of bacteria. Resistance develops by several mechanisms overproduction of PABA (38) altered permeabiUty of the organisms to sulfonamides (39) and reduced affinity of dihydropteroate synthetase for sulfonamides while the affinity for PABA is retained (40). Sulfonamides also show cross-resistance to other sulfonamides but not to other antibacterials. In plasmodia, resistance may occur by means of a bypass mechanism in which the organisms can use preformed foHc acid (41). 

High test molasses (invert molasses) is produced from cane sugar when sucrose manufacture is restricted because of overproduction. The cane sugar at ca 55 wt % solids is en2ymatically converted to invert symp to prevent crystallisation and evaporated to a symp. The product is used in the same applications as blackstrap molasses. 

A number of the genes involved in the biosynthesis of thiamine in E. coli (89—92), i hium meliloti (93), B. suhtilis (94), and Schi saccharomycespomhe (95,96) have been mapped, cloned, sequenced, and associated with biosynthetic functions. Thiamine biosynthesis is tightly controlled by feedback and repression mechanisms limiting overproduction (97,98). A cost-effective bioprocess for production of thiamine will require significant additional progress. 

Situated as it is between glycolysis and the electron transport chain, the TCA cycle must be carefully controlled by the ceil. If the cycle were permitted to run unchecked, large amounts of metabolic energy could be wasted in overproduction of reduced coenzymes and ATP conversely, if it ran too slowly, ATP would not be produced rapidly enough to satisfy the needs of the cell. Also, as just seen, the TCA cycle is an important source of precursors for biosynthetic processes and must be able to provide them as needed. 

Hyperthyroidism, that is, the overproduction of thyroid hormones, is usually treated by surgical removal of the thyroid gland. Before such a procedure is undertaken, the hyperthyroidism is usually first brought under control by treatment with so-called antithyroid agents. 

Tantalum production has increased steadily and strongly since 1993. An optimistic forecast regarding the strongly increasing demand for tantalum capacitors caused excessive demand for tantalum powder in 2000, when the overproduction of capacitors led to a sharp shortage in tantalum powder. It is still difficult to predict when the electronics industry will return to balanced condition. 

In order to develop a rational approach to improving rates of metabolite production, it is necessary to consider the fate of the nutrients that are required for its synthesis. However, overcoming the major flux control points within a metabolic pathway may not lead to metabolite overproduction if the energetic consequences of the alteration are unfavourable to the organism. 

Normally amino add synthesis will just satisfy the metabolic demand. In some cases, when the amino add occurs in both biosynthetic and energy production pathways, overproduction of the amino add can take place. This is the case, espedally for L-glutamic add, with Corynebacterium, Brevibacterium, Microbacterium and Arthrobacter. 

The small overproduction of amino adds by wild type strains in culture media is the result of regulatory mechanisms in the biosynthetic pathway. These regulatory mechanisms are feedback inhibition and repression. 

It is obvious that in the case of overproduction of amino adds the above mentioned regulatory mechanisms are not wanted. One way to overcome these regulations is to make use of mutants. 

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. 

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 ... 

Regulatory mutants improve the rate of L-phenylalanine overproduction by... 

An E. cdi mutant, Try", is likely to enhance L-phenylalanine overproduction. 8.4.3 Methods of 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. 

Loss of genetic material (for example the constructed plasmid containing the genetic information necessary for overproduction of the amino add) can also occur. 

A Try mutant would not be subject to feedback inhibition by overproduction of tryptophan. Also, the mutation may allow more chorismate to proceed to prephenate via E3 (see Figure 8.4) and thus through to L-phenylalanine. 

Kaplan, H. B., and Greenberg, E. P. (1987). Overproduction and purification of the luxR gene product transcriptional activator of the Vibrio fischeri luminescence system. Proc. Natl. Acad. Sci. USA 84 6639-6643. 

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