Abstract:
As the second most widely used enzyme substrate after ATP, SadenosylLmethionine (SAM) has wide applications in clinic and health care. The recombinant Pichia pastoris G12CBS is a SAM producing strain, which was engineered to overexpress recombinant methionine adenosyltransferase and to downregulate the cystathionineβ synthase to reduce SAM transformation. In order to enhance SAM production, the feeding strategy of Lmethionine (LMet, the substrate for SAM synthesis) should be optimized. In shake flasks it was found that both cell growth and SAM production decreased when LMet addition amount was higher than 3 mg/mL in a day. In a 15 L bioreactor, the maximum SAM production (13.01 g/L) was reached at the LMet feeding rate of 0.4 g/(L·h), which was 22.2% and 31.8% higher than that with the feeding rate of 0.2 g/(L·h) and 0.6 g/(L·h) respectively, and was improved by 54% in comparison with the maximum production in the original strain. The analyses of key metabolites and enzyme activity indicated that the bottleneck for SAM production with the low LMet feeding rate (0.2 g/(L·h)) was the insufficient LMet supply, but both the tricarboxylic acid cycle and nitrogen uptake were reduced with the LMet feeding rate higher than 0.4 g/(L·h), which was probably the reason for the low SAM accumulation and cell growth inhibition.