Chen Huaxin, Chu Ju, Zhang Siliang, Zhuang Yingping, Qian Jiangchao, Wang Yonghong, Hu Xiaoqing
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 200237 Shanghai, People's Republic of China.
Appl Microbiol Biotechnol. 2007 Apr;74(6):1205-12. doi: 10.1007/s00253-006-0705-y. Epub 2007 Feb 28.
To develop an efficient way to produce S-adenosylmethionine (SAM), methionine adenosyltransferase gene (mat) from Streptomyces spectabilis and Vitreoscilla hemoglobin gene (vgb) were coexpressed intracellularly in Pichia pastoris, both under control of methanol-inducible promoter. Expression of mat in P. pastoris resulted in about 27 times higher specific activity of methionine adenosyltransferase (SMAT) and about 19 times higher SAM production relative to their respective control, suggesting that overexpression of mat could be used as an efficient method for constructing SAM-accumulating strain. Under induction concentration of 0.8 and 2.4% methanol, coexpression of vgb improved, though to different extent, cell growth, SAM production, and respiratory rate. However, the effects of VHb on SAM content (specific yield of SAM production) and SMAT seemed to be methanol concentration-dependent. When cells were induced with 0.8% methanol, no significant effects of VHb expression on SAM content and specific SMAT could be detected. When the cells were induced with 2.4% methanol, vgb expression increased SAM content significantly and depressed SMAT remarkably. We suggested that under our experimental scheme, the presence of VHb might improve ATP synthesis rate and thus improve cell growth and SAM production in the recombinant P. pastoris.
为开发一种高效生产S-腺苷甲硫氨酸（SAM）的方法，将壮观链霉菌的甲硫氨酸腺苷转移酶基因（mat）和透明颤菌血红蛋白基因（vgb）在甲醇诱导型启动子的控制下于毕赤酵母细胞内共表达。mat在毕赤酵母中的表达导致甲硫氨酸腺苷转移酶（SMAT）的比活性提高约27倍，SAM产量提高约19倍，相对于各自的对照而言，这表明mat的过表达可作为构建SAM积累菌株的有效方法。在0.8%和2.4%甲醇的诱导浓度下，vgb的共表达在不同程度上改善了细胞生长、SAM产量和呼吸速率。然而，VHb对SAM含量（SAM生产的比产量）和SMAT的影响似乎取决于甲醇浓度。当用0.8%甲醇诱导细胞时，未检测到VHb表达对SAM含量和比SMAT有显著影响。当用2.4%甲醇诱导细胞时，vgb表达显著增加了SAM含量并显著降低了SMAT。我们认为，在我们的实验方案下，VHb的存在可能提高ATP合成速率，从而改善重组毕赤酵母中的细胞生长和SAM生产。
