文献类型：期刊文献

中文题名：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

英文题名：Optimization of L-Methionine Feeding Strategy for Improving S-Adenosyl-L-Methionine Production by Recombinant Pichia pastoris G12-CBS

作者：武晓乐[1];秦秀林[1];姚高峰[1];储炬[1];钱江潮[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2012

卷号：38

期号：5

起止页码：587

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;Scopus;北大核心:【北大核心2011】；CSCD:【CSCD2011_2012】；

基金：国家自然科学基金(20976050);国家高技术研究发展计划(2012AA021201)

语种：中文

中文关键词：毕赤酵母;S-腺苷甲硫氨酸;甲硫氨酸

外文关键词：Pichia pastoris ; S-adenosyl- L-methionine; L-methionine

摘要：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。
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-fl 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 enzyme activity indicated that the bottleneck for （L· h）） was the insufficient L-Met supply, but reduced with the L-Met feeding rate higher than SAM accumulation and cell growth inhibition. the original strain. The analyses of key metabolites and SAM production with the low L-Met feeding rate （0.2 g/ both the tricarboxylic acid cycle and nitrogen uptake were 0.4 g/（L· h）, which was probably the reason for the low SAM accumulation and cell growth inhibition.