Study On The Production Of S-Adenosylmethionine By Recombinant Pichia Pastoris

S-adenosyl-L-methionine(SAM), an important metabolic intermediate in the metabolic flux of sulphur is involved in many metabolic pathways. SAM has much physiological effection, and has clinical potential.A recombinant strain BIB2001, which knocking in S-Adenosylmethionine synthase and knocking out Cystanthionine β-synthase from Pichia pastoris GS115, can produce high level of SAM.There are three ways to produce SAM, including chemical synthesis, microbial fermentation and enzymatic synthesis, and microbial fermentation is the main way.The fermentation conditions, such as pH, medium capacity, concentration of methanol and L-Met, that effect the recombinant Pichia pastoris growth and the SAM yield in shake flask were studied. The culture conditions of the recombinant Pichia pastoris in fermentor (5L and 20L) were optimized, including the prescription of cultural medium, pH, dissolved oxygen, agitation speed , aeration , feeding strategy of glycerol, methanol and L-Met in different stages. And the SAM production of the recombinant Pichia pastoris reached 15g/L(5L) and 22g/L(20L) after fermentation for 5 days under the optimized conditions respectively.The separation and purification process of SAM was also established. Firstly, SAM was crudely crystallized in methnol after extraction with acetic ether (combined with sulfuric acid) and purification by cation-exchange resin; secondly, the obtained SAM sulfonic acid salt reacted with 15% p-toluenesulfonate/methanol solution to generate the SAM double salt; thirdly, the SAM double salt was precipitated by acetone, collected and freeze-dried to acquire the purified product. The recovery rate and purity of SAM reached 40% and 88% respectively.