Improving The Production Of S-adenosyl-L-methionine By Recombinant Pichia Pastoris

S-Adenosyl-L-methionine (SAM) is an important molecule for normal cell function and survival. It participates in a number of biochemical reactions including transmethylation, transsulphuration and polyamine synthesis. SAM is also closely related to proteins, nucleic acids, neurotransmitters, phospholipids and vitamin synthesis.In recent years, considerable interest in SAM production has arisen for it has good effect on liver disease, disorders, osteoarthritis and migraine. What's more, it also can treat Alzheimer's disease, epilepsy, Parkinson's disease, etc. Based on SAM's physiological functions and wide application, it is pressed for solutions to prepare high-yield SAM on a large scale.Currently, there are four methods to produce SAM. That's chemical synthesis, enzymatic synthesis, microbial fermentation and whole cell catalysis. The main way in industry is by fermentation using recombinant Yeast added with precursor L-methionine.By knocking in S-Adenosyl-L-methionine synthase and/or knocking out Cystanthionine β-synthase from Pichia pastor is GS115, we got four strains, GS115, ZJGSU1, ZJGSU2and ZJGSU3. Their SAM yield reached0.010g/L,0.804g/L,0.131g/L and1.152g/L in shake flask, respectively.The optimal conditions for production of SAM by engineered ZJGSU3were investigated in this study. With SAM yield as the target, the concentration of several important factors, L-met, methanol, pH, inoculum volume, medium volume, oleic acid, PTM1, were optimized by2-level Factorial and Box-Behnken design. The optimal culture medium components obtained were as follows:1.00%yeast extract,2.00%tryptone,1.34%YNB,1.0%L-met,100mM potassium phosphate,1.40%methanol,0.10%oleic acid,0.4%PTM1and4.00×10-5%biotin; the optimum conditions were:pH5.0, inoculum volume5%, medium volume30mL in500mL flask. Under the optimum conditions, the highest yield of SAM was obtained at84h with yield of3.23g/LFinally, the recombinant strain ZJGSU3was cultured for SAM accumulation in a5L fermentor. The whole fermentation period included four stages, that was preincubation, glycerin fed, carbon starvation and induction. The highest SAM yield and biomass were obtained at4.37g/L and247g/L at120h, respectively, indicating that it could be used for industrial fermentation to produce large scale of SAM.