Highly Efficient Synthesis S-Adenosyl-L-Methionine By Recombinant Escherichia Coli

S-adenosyl-L-methionine (SAM) also known as S-adenosylmethionine is widely existed in living organisms, which is next only to ATP as an important physiological active substance. It can transfer methyl, sulfenyl, aminopropyl etc., and it is closely related to the proteins, nucleic acids, neurotransmitters, phospholipids and vitamin synthetic. In aspect of pharmaceutical fields, it has great effect of liver disease, depression, arthritis and other diseases.The production methods of SAM mainly contain chemical synthesis method, enzymatic conversion method and fermentation method. The yield is low by chemical synthesis method, and the substrate is very expensive. In addition, it is difficult to isolate the active SAM from the product, so it is not easy to use this method. To some extent, the enzymatic conversion method can improve the enzyme activity of SAM synthase, but also has great purity of SAM, which makes it pollution-free. However, SAM synthase is low concentration in the body, which makes it not easy to extract. Meanwhile, the high cost of ATP becomes a limited factor. In recent years, people take the yeast fermentation as the principle method to get SAM, but the fermentation used in E.coli expression system is limited. So, we exploratory use the recombinant E.coli expression system to ferment, and we optimize the fermentation parameters.At present, the reason why SAM is expensive and can not apply in clinical widely, mainly depends on its instability and easy to lose activity. In addition, only the (S, S)-SAM, namely (-)-SAM just has biological activity. In our country, research for SAM is a rather late start, while the demand for patients with liver disease and arthritis is very large. Therefore, it’s very important for us to use the suitable method to improve the yield of SAM, meanwhile it’s very significantly to make it industrialized product.Firstly, we used the E.coli BL21 as the expression system, constructing two recombinants BL21-pET-28a-meetK and BL2\-pET-32a-metK, which makes sure they can clone the SAM synthase gene metK. Then, we testified the recombinant vectors by molecular and protein. Moreover, we contrasted the yield of them by HPLC, and we named after ZJGS-SAM for which yield is much higher.Secondly, we optimized the medium of ZJGS-SAM, picking six factors from medium compositions and fermentation conditions, using P-B experiment to get the three main factors:yeast extract, culture volume and pH. Then we took the method of steepest ascent path to get the concentration of the three central points. After that, we used the Box-Behnken Design to receive a group of datum, which can get a relatively high yield of SAM. The medium compositions are lactose 20 g/L, soybean 40 g/L, yeast extract 35 g/L, NaCl 10 g/L, MgSO41.2 g/L L-met 1 g/L. The optimized conditions contain that original medium pH is 7.5, induced temperature is 33 ℃, inoculation quantity is 1%, rotate speed is 220 rpm and culture volume is 30 mL(250 mL shake flask). Then, after fermenting 12 h on the basis of optimized conditions, the yield is enhanced 271.4% contrased with the unoptimized yield 35 mg/LFinally, we testified the yield on the fermentation tank. We optimized the pH and throughput in batch fermentation, making the yield reaches 87.88 mg/L. Then, we took the method of constant speed fed-bath fermentation using lactose as carbon source, improving the ultimate yield, which reaches the final concentration 300.9 mg/L. Moreover, we achieved a group of datum about suiting 5 L fermentation tank, which is the foundation for producting SAM in quantity.