Centre for Plant Molecular Biology, Osmania University, Hyderabad 500 007, India.
National Centre for Mass Spectroscopy, Indian Institute of Chemical Technology, Hyderabad 500 007, India.
AMB Express. 2013 Jul 27;3:40. doi: 10.1186/2191-0855-3-40. eCollection 2013.
A genetically engineered strain of Pichia pastoris expressing S-adenosylmethionine synthetase gene from Saccharomyces cerevisiae under the control of AOX 1 promoter was developed. Induction of recombinant strain with 1% methanol resulted in the expression of SAM2 protein of ~ 42 kDa, whereas control GS115 showed no such band. Further, the recombinant strain showed 17-fold higher enzyme activity over control. Shake flask cultivation of engineered P. pastoris in BMGY medium supplemented with 1% L-methionine yielded 28 g/L wet cell weight and 0.6 g/L S-adenosylmethionine, whereas control (transformants with vector alone) with similar wet cell weight under identical conditions accumulated 0.018 g/L. The clone cultured in the bioreactor containing enriched methionine medium showed increased WCW (117 g/L) as compared to shake flask cultures and yielded 2.4 g/L S-adenosylmethionine. In spite of expression of SAM 2 gene up to 90 h, S-adenosylmethionine accumulation tended to plateau after 72 h, presumably because of the limited ATP available in the cells at stationery phase. The recombinant P pastoris seems promising as potential source for industrial production of S-adenosylmethionine.
利用基因工程酵母毕赤酵母，在 AOX1 启动子的控制下表达来自酿酒酵母的 S-腺苷甲硫氨酸合成酶基因。用 1%甲醇诱导重组菌株，可表达约 42kDa 的 SAM2 蛋白，而对照 GS115 则没有这种带。此外，重组菌株的酶活比对照高 17 倍。在补充了 1% L-蛋氨酸的 BMGY 培养基中摇瓶培养工程化的毕赤酵母，可得到 28g/L 的湿细胞重量和 0.6g/L 的 S-腺苷甲硫氨酸，而在相同条件下具有相似湿细胞重量的对照（仅含有载体的转化体）仅积累 0.018g/L。与摇瓶培养相比，在含有富集蛋氨酸培养基的生物反应器中培养的克隆提高了 WCW（117g/L），并产生了 2.4g/L 的 S-腺苷甲硫氨酸。尽管 SAM2 基因的表达高达 90h，但 S-腺苷甲硫氨酸的积累在 72h 后趋于平稳，这可能是由于在静止期细胞中可用的 ATP 有限。重组毕赤酵母似乎是工业生产 S-腺苷甲硫氨酸的有前途的潜在来源。
