Research On The Metabolism And Gene Engineering Of S-adenosyl-L-methionine Synthetase

S-adenosyl-L-methionine is an important metabolic intermediate in organism, which is involved in many biochemical reactions. Its therapeuctic usage coμld be extensive and varied. Fermentation of microorganism and extracting SAM from the cell is the main industrial production process at present. SAM is always produced by fermentation of S.cerevisiae. By changing Pichia pastoris metabolic path rationally, a constitutive recombinant Pichia pastoris with riched SAM synthetase was obtained. The GAP gene promoter was amplified from P. pastoris GS115 and used to replace the AOX1 promoter (P_AOX1) on pPIC9K resulting in plasmid pGAP9K. The recombinant expression vector pGAP9K-AS was constructed by inserting the S-adenosyl-L-methionine synthetase gene (SAM2) into pGAP9K. pGAP9K-SAM was then transformed into P. pastoris GS115. The multi-copy integration transformant P. pastoris GS115 (pGAP9K-SAM) was used to investigate the constitutive expression of S-adenosyl-L-methionine synthetase in P. pastoris. The expression of S-adenosyl-L-methionine synthetase reached its peak after 4 d of culture in P. pastoris GS115 (pGAP9K-SAM). The S-adenosyl-L-methionine synthetase expression in inducible system reached the peak after 6d of induction but the expressed SAM was only 81% of that from constitutive system. The above data indicates that the constitutive promoter P_GAP can serve as an effective alternative to the inductive promoter P_AOX1 to express S-adenosyl-L-methionine synthetase and other proteins in P. pastoris.