| S-adenosylmethionine (S-adenosyl methionine, SAM) is an important metabolic intermediate, and has been used for the treatment of a variety of clinical symptoms, and for health care. In order to get recombinant Pichia pastoris with high SAM accumulation, we explored the possibility of further enhance SAM production by knocking out 3 genes involved in the biotransformation of SAM in the SAM producer GS115/DS16 which contained a recombinant SAM synthase.With homologous recombination method, we knocked out S-adenosylmethionine decarboxylase gene spe2, S-adenosyl homocysteine hydrolase gene sah1 and L-methylthio-tRNA synthase gene msm1 to construct the corresponding recombinant strain GS115/Dspe,GS115/Dmsm and GS115/Dsah, respectively. Cell growth and SAM synthesis of the three knockout strains were investigated. In the shake flask, the growth of three knockout strains have no significant difference compared to the original strain. There were slight increases in SAM production in three kinds of recombinant strains. In comparison with the production of 0.11g/gDCW in the original strain GS115/DS16, SAM production reached 0.12, 0.13, and 0.12g/gDCW in GS115/Dspe, GS115/Dsah, and GS115/Dmsm, respectively.SAM transformation may be inhibited in knockout strains, and more methionine may be used as the substrate to synthesize SAM. So, the effect of methionine addition on the SAM synthesis in 3 different knockout strains was studied. No obvious effect could be observed in strain GS115/Dspe. In GS115/Dsah, SAM production increased by 15.4% in shake flask when methionine addition was decreased from 0.1% to 0.06% and reached 0.15 g/gDCW, which may be due to the low transmethylation and transsulfuration. In GS115/Dsah, SAM production increased by 8.3% when methionine addition was decreased from 0.1% to 0.06% and reached 0.13 g/gDCW, which may be caused by the inhibition of the polyamines formation. |
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