Optimization of L-Methionine Feeding Strategy for Improving SAdenosylL-Methionine Production by Recombinant Pichia pastoris G12-CBS

As the second most widely used enzyme substrate after ATP, SadenosylLmethionine (SAM) has wide applications in clinic and health care. The recombinant Pichia pastoris G12CBS is a SAM producing strain, which was engineered to overexpress recombinant methionine adenosyltransferase and to downregulate the cystathionineβ synthase to reduce SAM transformation. In order to enhance SAM production, the feeding strategy of Lmethionine (LMet, the substrate for SAM synthesis) should be optimized. In shake flasks it was found that both cell growth and SAM production decreased when LMet 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 LMet 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 LMet feeding rate (0.2 g/(L·h)) was the insufficient LMet supply, but both the tricarboxylic acid cycle and nitrogen uptake were reduced with the LMet feeding rate higher than 0.4 g/(L·h), which was probably the reason for the low SAM accumulation and cell growth inhibition.