SAMs synthesis

To automate the process of SAM synthesis in the microchannels for the generation of sensing arrays, a multichannel chip suitable for continuous flow of reagents was designed. A microchannel chip (4 x 2.2 cm2) with five parallel channels confined in an area of 200 pm was fabricated. Each channel can be individually addressed and... 

Interference with SAM synthesis has been reported with a number of compounds (L6). The most active of these and also several compounds that block methylation of histones, e.g., adenosine, spermidine, histidine, have all been shown to be ulcerogenic (Mil). 

S -adenosyl-L-homocysteine Fig. 16.2 The simplified pathway for SAM synthesis and conversion... 

A half century ago, MAT was purihed from animal liver and employed to catalyze SAM synthesis in vitro (Catoni 1953). However, MAT cannot be extracted efficiently at low cost, thus there have been few advances using this method (Gross et al. 1983 Matos et al. 1987 Schlenk and Depalma 1957). Recently, Luo et al. (2008) constructed a recombinant Pichia pastoris KM71, which secreted MAT by exploiting an a-factor secretion signal. After cultivation at flask scale, MAT was harvested at a content about 200 mg/L and a specific activity of 23.84 U/mg. 

Compared to the in vitro method, SAM synthesis in vivo anploying microorganism is more competitive. Because the in vivo methods are more efficient, convenient... 

P pastoris has a unique advantage that it is capable of growing on minimal media to achieve a relatively high biomass (Cereghino and Cregg 2000). Furthermore, the excessive SAM can be sequestered in the vacuole of yeast cell, so yeast (such as P. pastoris and S. cerevisiae) can be regarded as an optimal host strain for SAM synthesis in vivo (Chan and Appling 2003 Shobayashi etal. 2007). Many reports have been published to document improvements of SAM production by P. pastoris (Li et al. 2002 Yu et al. 2003). 

To investigate the effect of MAT activity on SAM synthesis, and then to optimize MAT expression for SAM synthesis, Qin et al. (2011) firstly constructed a library of Pg p with the aid of error-prone PCR. The P mutants were then characterized and seven P p mutants were selected to drive MAT expression. From this an optimal Pq p mutant for MAT and SAM production was identified. 

The industrial production of SAM has been achieved in bioreactors with working volumes usually greater than 100 L. In order to enhance SAM synthesis while depress its conversion, strain modification is not sufficient and optimization of the process is also required. 

The rate of ATP synthesis is closely related to the type of carbon source. As a strategy to boost SAM synthesis, Hu et al. (2007) report a novel supplement strategy for inducible Pichia. The classical induction period was divided into several 10-h subintervals, and methanol (initial 6 h) and glycerol (subsequent 4 h) were fed alternately during every subinterval. Compared to the typical feeding mode, the novel scheme elevated growth and MAT volumetric activity, while decreased the specific MAT activity. The rate of SAM synthesis within Pichia cells, however, was still improved, mainly due to the enhancement of ATP supply. The switch from methanol to glycerol was invariably associated with inaeased ATP synthesis. SAM synthesis was also favored by an extension of the production period such that SAM content was reputed to be elevated to 13.24 g/L at 5-L bioreactor scale. 

Fig. 17.5 Effect of nitric oxide on the synthesis of methionine and S-adenosylmethionine and methylation reactions. NO inhibits methyltetrahydrofolate reductase (MTR). This results in a decrease in tetrahydrofolate (FH4) and methionine. Additional reduction in the FH4 level may occur by the NO-induced oxidation of ferritin, a compound that inhibits the proteasomal degradation of FH4. NO affects SAM synthesis not only by inducing a decrease in methionine synthesis but also by directly inhibiting the liver-specific methyl-thioadenosyltransferase I/III (MATI/III) isozymes. The fall in SAM level cannot be fully compensated by an increase in the extrahepatic isozyme MATH, since this enzyme is inhibited by its reaction product. The reduction in homocysteine utilization for methionine synthesis may result in homocysteine accumulation. This probably does not lead to a consistent rise in cystathionine and reduced glutathione synthesis, dne to a reduced stabilization of cystathionine P-synthase (CBS) by SAM. Consequently, an inciea.se in SAH, associated with a decrease in the SAM/SAH ratio, inhibits methyltransferases (MT) and DNA methylation. The reduction in SAM level may decrease IicBa activation, thus favoring NF-kB activity...

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