Studies On Preparation Of S-adenosylmethionine By Enzymatic Synthesis And Microbial Fermentation

S-adenosylmethionine (SAM) is an important metabolic intermediate in all living cells. SAM participates in a variety of important metabolic activities as a donor of methyl groups in transmethylation reactions and a precursor molecule in transulfuration and aminopropylation pathways. SAM also regulates the activities of various enzymes. In cells, the biosynthesis of SAM is from L-methionine (Met) and ATP. S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, EC 2.5.1.6, SAM synthetase) is the only enzyme known to catalyze this reaction. SAM has two kinds of isomers: (R, S)-SAM and (S, S)-SAM. Only (S, S)-SAM, also named (-)-SAM has biological activity.Dietary intake does not normally provide sufficient quantities of SAM, and the body relies on de novo synthesis to maintain the required concentrations of this essential metabolite. This synthesis is often seriously altered with the increase of age and in various pathologic states, and administration of supplementary SAM becomes crucial to the restoration of cellular function. A number of clinical studies support the effectiveness of stable SAM salts in treating a variety of disorders including depression, liver disease, osteoarthritis, dementia, and vacuolar myelopathy. Stable SAM salts are also senior health drugs for preventing cancer, cardiovascular disease and anti-aging. Reviews of clinical trials indicate that stable SAM salts are absorbed and utilized well with a low incidence of side effects and an excellent tolerability.However, SAM is highly expensive, that might be a limitation for therapeutic applications. Therefore, it is of great importance to provide an efficient and low-cost mean of SAM preparation for industrial applications.SAM is mainly produced by chemical coupling reactions, enzymatic synthesis or microbial fermentation. Chemical coupling reactions are not currently used because of purification difficulty and costly starting materials. In recent years, enzymatic synthesis for SAM preparation appears quite attractive. This method simulates the biosynthesis of SAM, and SAM synthetase catalyzes the synthesis (S,S)-SAM from Met and ATP in vitro. Enzymatic preparation of SAM presents many advantages including straightforward optimization, controllable reaction rates, and simplified purification. But it is difficult to obtain adequate SAM synthetase and the synthetase has not been extensively characterized, which increases production costs. Enzymatic synthesis is generally used for the isotope labeling of SAM. Microbial fermentation for SAM preparation has been the main method since 1960s. This method is the accumulation of higher concentration of intracellular SAM by fermention of yeast. Fermentation make the SAM content in the cells increase significantly on the basis of dry cell weight, but the volumetric yield of SAM is still low. Furthermore, the purification of SAM is complicated, since the cells have to be disrupted first and SAM has to be separated from all other cell components. These factors limited the production and the application of SAM.Considering the current problems in producing SAM, we propose new strategies to improve enzymatic synthesis and microbial fermentation for SAM preparation.1. Expression of secreted SAM synthetase in Pichia pastoris for SAM preparation by enzymatic synthesisThe key of enzymatic synthesis for SAM preparation is how to solve the SAM synthetase problem. In the past studies, whether by purification from the organisms or by recombinant expression, it was difficult to conveniently obtain a large number of SAM synthetase.Among the SAM synthetase isoenzymes prevalent in microorganisms and animal tissues, the SAM synthetase encoded by sam2 from Saccharomyces cerevisiae has some advantages. Though the sam1 gene from S. cerevisiae is 92% of the same as sam2, expression of the sam2 gene is induced by the presence of excess methionine in the growth medium, while expression of the sam1 gene is repressed, and the sam2 gene has no problem of product inhibition observed in use of the Escherichia coli SAM synthetase. The SAM synthetase encoded by sam2 is a good choice for larger-scale preparation.The methylotrophic yeast P. pastoris is a eukaryotes expression system for heterologous proteins. The feature of P. pastoris is particularly relevant for target proteins containing multiple disulphide bonds or requiring glycosylation, phosphorylation, the absence of an amino-terminal methionine or no oligomer formation for the correct assembly of the mature protein. P. pastoris contributes to the protein expression with biological activity.We have successfully expressed a soluble form of SAM synthetase encoded by sam2 from S. cerevisiae in the recombinant P. pastoris. The secreted yield of the recombinant SAM synthetase was approximately 200 mg/L that was 200 times that of the wild strain. A 6-His tag was added into the recombinant SAM synthetase at the N terminal, and only a few own protein was secreted to the culture supernatant by host bacteria, so the secreted SAM synthetase was purified in a single chromatography step with a yield of approximately 82%. The purified protein appeared as a single band on the silver-stained-gels.The catalysis conditions for the recombinant SAM synthetase were investigated. SAM synthetase activity was assayed at 35oC for 1 h in the reaction buffer containing 20 mmol/L L-Met, 26 mmol/L ATP, 52 mmol/L MgCl2, 300 mmol/L KCl, 8 mmol/L reduced glutathione, 100 mmol/L Tris. The pH of the reaction solution was adjusted to 8.5 using 1 M KOH. The specific activity of SAM synthetase was 23.84 U/mg. This value was twice the activity of the endogenous S. cerevisiae SAM synthetase.In the kinetics researches, we proved that the recombinant SAM synthetase could be a kind of allosteric enzymes with negative regulation. We characterized the recombinant SAM synthetase. This synthetase was relatively stable at pH values from 6.5 to 8.5. Apart from Fe²⁺, Cu²⁺, Zn²⁺, the activity of the recombinant synthetase was not significantly affected by common ions.The thermal stability of the recombinant synthetase was not good. KCl could significantly protect the recombinant synthetase from thermal inactivation. Additional experiments were performed in which the recombinant SAM synthetase was purified and immobilized in one step using immobilized metal-chelate affinity chromatography. The stability of the immobilized synthetase was better than the free enzyme. The immobilized synthetase was found to be 40.4% of the free enzyme activity in catalyzing the synthesis of SAM.Racemic DL-Met is much cheaper than L-Met. In microbial fermentation, DL-Met could not replace L-Met as the substrate for the synthesis of SAM, but in enzymatic synthesis, the immobilized SAM synthetase catalyzed the synthesis of (S,S)-SAM from DL-Met and ATP, which greatly reduce the cost of production.These research results demonstrated that stable and high-level expression of SAM synthetase could be carried out in P. pastoris, laying the foundation for the application of enzymatic synthesis. Through the detailed studies of the enzyme reaction conditions, characterization, purification and immobilization, the theoretical understanding of SAM synthetase was deepened. In addition, important information was provided for taking full advantage of the SAM synthetase, increasing the enzyme utilization and improving the enzyme application.2. Microbial fermentation for SAM preparation in E. coliE. coli is the most widely used expression system for heterologous proteins. E. coli expression system presents many advantages over the yeast expression system used in SAM preparation including short growth time, low-cost culture, easy operation, clear genetics background and mature metabolic control method.We cloned sam2 from S. cerevisiae into E. coli to express SAM synthetase. The recombinant synthetase catalyzed the synthesis SAM in cell. There is no known transport system for SAM in E. coli, and passive diffusion to the outside in the case of a large and also charged molecule such as SAM appears impossible. But we found that SAM was not only in the cells, but also in the culture supernatant.After the optimization on fermentation conditions, the recombinant E. coli was cultured in LB containing 1 g/L L-Met. When OD600 of the cell density was 0.7, the strain was induced by 1 mmol/L IPTG for 7 h. The yield of SAM in the cells was 27 mg/L and that in the culture supernatant was 39.2 mg/L.In addition, we found that the yield of secreted SAM was seriously affected by cell state. The cell density at the beginning of the induction and the time of induction were decisive factors for cell state.The selective secretion of SAM into the culture supernatant can make purification method simple and production time short. The development of this method for fermentative production of SAM has a distinct advantage over current methods. The metabolism of E. coli was well studied, which is conducive to increase of the yield of SAM by metabolic regulation.