Precursor pools

While metabolic engineers traditionally sought the rate-limiting enzyme to unlock flow through a pathway, now they understand that there may be many points of control and feedback with the metabolic network, and seek to empirically determine the dynamics of the interactions between rate controllers and other factors. For example, the sizes of metabolic precursor pools and the catabolism or sequestration of products affect accumulation as well as flux through the pathway. 

Adjunctive to flux control analysis, other components of metabolism that contribute to product accumulation are needed including (1) substrate/precursor pool sizes (metabolomics), (2) co-factor capacities (metabolomics), (3) gene expression profiles (transcriptomics and quanfifafive real-time PCR), (4) protein profiles (pro-... 

Transgenic E. coli accumulate comparatively low levels of carotenoids " compared to microbial algae, yeasts, and bacteria. Many efforts ° have focused on increasing accumulation by manipulation of factors affecting metabolic flux and metabolite accnmnlation (listed and discnssed in Sections 5.3.1.1 and 5.3.1.3 A) and have been reviewed." - " In bacterial systems, approaches to control can be categorized as either infrastructural (plasmids, enzymes, strains) or ultrastructural (media and feeding, enviromnent, precursor pools, substrate flux). 

Matthews, P.D. and Wurtzel, E.T., Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid precursor pool with expression of deoxyxylulose phosphate synthase, Appl. Microbiol. Biotechnol. 53, 396, 2000. 

More data are needed on the effects of organic practices on phytochemicals and their precursors. How does modification of precursor pools affect biosynthesis For example, see studies on the effects of lower levels of free amino acids caused by a reduction in nitrogen input. 

These cells are relatively undifferentiated and have a large nucleus, distinguishable nucleolus but few, if any, cytoplasmic granules. Myeloblasts arise from a precursor pool of stem cells, and both the rough endoplasmic reticulum and the Golgi apparatus stain for peroxidase, indicating that this enzyme is beginning to be synthesised. This cell type is capable of proliferation. 

The findings of Darrow et al. [1996] suggest that there might be a precursor pool for Cx43 whereas Cx45 may be synthesized de novo (see below). 

Phase I and II clinical trials indicated that acronycine reduced pain of the spine in some patients with multiple myeloma [280,282,283]. Acronycine has been reported to cause leukopoenia and to have CNS-depressant activity [284], Biochemically, acronycine inhibits incorporation of extracellular nucleosides into the RNA and DNA of leukaemia L-5178Y cell culture. There is, however, no evidence of interaction between acronycine and DNA or inhibition of template activity of DNA. This alkaloid does not inhibit nucleic acid synthesis in the cell, but rather inhibits the accumulation of extracellular uridine or thymidine, as nucleotides, in the intracellular precursor pool [285, 286], Acronycine, acting primarily on membranous organelles [287], seems to interfere with the structure, function and/or turnover of cell membrane components, thereby changing the fluidity of the plasma membrane [288]. 

In summary, then, the above short overview indicates that organisms from very early on probably minimized the threat of ammonia intoxication by metabolic mechanisms which serve to limit the amount of free ammonia in cell/body fluids formed in the first place. However, some formation and accumulation are inevitable. In mammals, and other animals that depend upon the urea cycle, this excess NH4+/NH3 together forms the precursor pool for urea synthesis. 

Fig. 3. The rate-limiting step of steroidogenesis under ACTH regulation. The transfer of cholesterol (C) from the outer to the inner mitochondrial membrane under ACTH regulation (step 3) makes cholesterol available to cytochrome /M50scc for conversion to pregnenolone (step 4), which diffuses out of the mitochondrion (step 5). Because of its insolubility in aqueous media, cholesterol must be transported to mitochondria, probably by SCP2, from a precursor pool (step 2). Here, cholesterol in the precursor pool is shown as being formed from cholesterol esters (CE) by cholesterol ester hydrolase (CEH) (step 1) other possible pathways are shown in Figs. 4 and 6. From Ref. 14.

Several actions of ACTH, which are unlikely to be rate-limiting for the immediate conversion of cholesterol to steroids, maintain the precursor pool of cholesterol and... 

Fig. 4. Potential sources of the immediate precursor pool of unesterified cholesterol available for mitochondrial steroidogenesis. ACAT, microsomal acyl coenzyme A cholesterol acyltransferase SEH, sterol ester hydrolase. From Ref. 14.

The basic terminology used in this review (Fig. 31.1 A) includes the precursor and product, where the precursor can either be labeled or unlabeled. When labeled, the precursor will also be referred to as the source since it supplies tracer to the progressively N enriched product pool. The process of isotope dilution refers to the addition of an unlabeled precursor to the source pool resulting in a decrease in the source isotopic ratio. The precursor could be known, such as for N03 isotope dilution where N02 is the only option, or unknown as with N02 isotope dilution where N03 or NH4+ could be the precursor. Isotopic equilibrium is achieved when the product pool has the same isotopic composition as the precursor pool and it is important to note that isotope dilution fosters this condition. At equihbrium, a rate can no longer be discerned by comparing the isotopic ratios of the pools. As suggested by the unidirectional arrows in Fig. 31.1, an assumption of the tracer method is that isotope recycfing does not occur. However, we know this... 

The universal precursors to terpenoids, the C5-compounds dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), originate from two pathways in plants (Fig. 1). The mevalonate (MEV) pathway is well described in many eukaryotic organisms. This pathway is present in the cy-tosol/endoplasmic reticulum of plants. More recently, another pathway has been described, the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway, which is found in the plastids of plants (19). The localization of the different pathways and the plastid-directing transit peptides found in hemi-TPS, mono-TPS, and di-TPS, but not in sesqui-TPS, result in the production of terpenoids from at least two different precursors pools. 

Eicosiq>entaaioic acid (20 5, (,>-3) The precursor for die series-3 eicosanoids. It replaces AA in membrane phospholipids. EPA is selectively incorporated into the eicosanoid precursor pool of phospholipids. Like AA, it is released from membrane phospholipids. It competes wtdi AA fiir oxygenases. 

Yeasts generally catabolize ethanol as follows ethanol —- acetaldehyde — acetic acid — acetyl-CoA —- other oxidative pathways [Figure 4]. It has been speculated that C. utilis forms ethyl acetate by the reaction of acetyl-CoA with ethanol (5). Thus if the flow of metabolites into the TCA cycle is inhibited or limited, as might occur under iron-limited conditions, acetyl-CoA would be expected to accumulate thus providing a precursor pool for esterification of ethanol. 

Whereas short-term drug exposure may result in rapid returns to baseline values, long-term drug exposure depletes both the response variable and the precursor pool, which requires significantly longer durations of time for the baseline response value to be achieved once drug is removed. 

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