优化甲硫氨酸补料策略提高重组毕赤酵母G12-CBS合成S-腺苷甲硫氨酸

重组毕赤酵母G12-CBS经代谢工程改造,可高效表达重组S-腺苷甲硫氨酸(SAM)合成酶,并弱化了SAM转化途径的关键酶β-胱硫醚合成酶,是一株优良的SAM高产菌。为了利用G12-CBS高效合成SAM,需对前体(甲硫氨酸)的补料策略进行优化。首先在摇瓶中考察了不同甲硫氨酸(L-Methionine,L-Met)添加量对于G12-CBS的影响,发现每24hL-Met补加量超过3mg/mL时,会影响重组菌生长和SAM合成。在15L发酵罐中优化L-Met的补料速率,当外源补料速率为0.4g/(L·h)时SAM产量最高,分别比补料速率为0.2g/(L·h)和0.6g/(L·h)时提高22.2%和31.8%,达到13.01g/L,比出发菌最高产量提高54%。代谢物及酶活测定的结果表明,较低的L-Met补料速率(0.2g/(L·h))导致前体供应不足而影响SAM合成,过高的补料速率(0.6g/(L·h))可能会抑制三羧酸循环,并且影响氮源的摄取和利用,从而导致菌体生长和产物合成受到抑制。

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.