Synthesis Of S-adenosy-L-methionine (SAM) In Escherichia Coli

S-adenosy-L-methionine (SAM) is an important metabolic intermediate in all living cells,which is utilized by transmethylation, transsulfuration, and donation of aminopropyl groups. Incells, S-adenosy-L-methionine synthetase (SAMS) was used as biocatalyst to produce SAMfrom L-methionine (L-Met) and ATP by enzymatic synthesis. In clinical research, SAM has beenfound to have great potential as a chemotherapeutic agent in various diseases, such as alcoholicliver disease, depression, osteoarthritis, Alzheimer’s disease, et al. Reviews of clinical trialsindicate that stable SAM salts are absorbed and utilized well with a low incidence of side effectsand an excellent tolerability. However, SAM was highly expensive, which might be a limitationfor therapeutic applications. Therefore, it is of great importance to provide an efficient andlow-cost mean of SAM preparation for industrial applications.The gene encoding SAMS was amplified from Saccharomyces cereisiae chromosomal DNAusing PCR technique on the base of the specific primers from S. cereisiae in GenBank. TheSAMS gene was cloned into the expression vector pET-28a to create a recombinant expressionplasmid pET-28a-SAMS. The recombinant plasmid pET-28a-SAMS was transformed intocompetence E. coli BL21(DE3) strain to construct a genetically engineered E. coliBL21(pET-28a-SAMS). A protein band with molecular mass of approximately46kDa wasexpressed by inducing the engineered E. coli BL21(pET-28a-SAMS) using lactose (4mg/mL) asan inducer. Western blotting showed that the recombinant protein was the recombinant SAMS.By optimizing the expression conditions, the E. coli BL21(pET-28a-SAMS) was cultivatedin LB medium with50μg/mL kanamycin at37oC for about3.5h, induction of the engineered E.coli with4mg/mL lactose at log phase of OD600=1.5at37oC for10h. At the end, the yield ofSAM in the cells was21.5mg/L and that in the culture supernatant was47.8mg/L, which wouldgreatly simplify the purification process of SAM. The development of this method forfermentatie production of SAM has a distinct advantage over current methods.An ATP regeneration system was established by using the mixed culture of the two kinds ofmicroorganisms in suspension, which was composed of the SAMS in the recombinant E. coliBL21(pET-28a-SAMS) and the Embden-Meyerhof-Parnas (EMP) pathway in the Sacharomycescerevisiae JM-310. The system was used successfully in the biosynthesis of SAM. By optimizingcoupling conditions, the results showed that the ratio of the two kinds of microorganism cellswas4:1, coupling time was5h, coupling temperature was37°C, toluene direct treatment withthe final concentration of5%(v/v) was beneficial for the improvement of penentrability of cell membrane. The composition of coupling buffer include200mmol/L phosphate buffer,200mmol/L glucose,10mmol/L adenosine,30mmol/L Mg2+,30mmol/L L-Met. Finally, the SAMconcentration in the coupling system could reach1.7g/L, which was10times higher than thecontrol sample.SAM is separated and purified by weak cation exchange resin D113. The optimaladsorption condition was pH4~5. Using sulphuric acid to elute SAM and the suitable elutionconditionwas confirmed as0.5mol/L H2SO4. Under these conditions, the High PerformanceLiquid Chromatography (HPLC) purity of our product and recovery efficiency can reach93%and90%respectively. The purified SAM was identified by High Performance Liquid-tandemMass Spectrometry (LC-MS/MS).In order to obtain a higher stability of SAM salt products. The stability of Ademetionine1,4-Butanedisulfonate and SAM sulphate at4°C,30°C and45°C was investigated. It was foundthat the thermal stability of Ademetionine1,4-Butanedisulfonate was better than that of SAMsulphate. Therefore, the Ademetionine1,4-Butanedisulfonate was suitable for a commercialproduct.