Department of Life Science and Engineering, Harbin Institute of Technology, Harbin 150001, China.
Mol Biol Rep. 2011 Mar;38(3):1831-40. doi: 10.1007/s11033-010-0299-4. Epub 2010 Sep 16.
The 972 bp length of transaldolase gene tal was cloned from Pichia stipitis CICC1960, encoding a 323 amino acid protein with a calculated molecular mass of 35.36 kDa and isoelectric point of 5.20. Real time PCR analysis demonstrated that the mRNA transcript level of constitutive tal gene rise on xylose, glucose, fructose, mannose, galactose and sucrose as carbon source, respectively. Furthermore, the transcription of tal gene in P. stipitis on xylose was higher than on other carbon source, indicating that transaldolase plays a part in xylose utilization. To deeply study the tal gene biological function, it was expressed in Fusarium oxysporum CCTCC M209040. Recombinant transaldolase activity of transformant F. oxysporum M209040-Tal2 was about 0.52 U mg(-1) protein and was 1.57 times higher than that of the wild type F. oxysporum CCTCC M209040, indicating that the improvement of transaldolase activity in transformant was due to expression of the exogenous tal gene. Growth of transformant F. oxysporum M209040-Tal2 without selection pressure did not affect the level of hygromycin resistance of the transformants, suggesting that integrated tal gene was stable in mitosis. Fermentation trials of F. oxysporum M209040-Tal2 showed that the ethanol yield improved by 8.39 and 11.71% on glucose and xylose substrates, respectively, demonstrating that the expression of tal gene from P. stipitis CICC1960 in F. oxysporum CCTCC M209040 could improve ethanol production.
从毕赤酵母 CICC1960 中克隆得到了长为 972bp 的转醛醇酶基因 tal，编码一个由 323 个氨基酸组成的蛋白质，分子量为 35.36kDa，等电点为 5.20。实时定量 PCR 分析表明，组成型 tal 基因在木糖、葡萄糖、果糖、甘露糖、半乳糖和蔗糖作为碳源时，mRNA 转录水平均上调。此外，毕赤酵母在木糖上的 tal 基因转录水平高于其他碳源，表明转醛醇酶在木糖利用中起作用。为了深入研究 tal 基因的生物学功能，将其在尖孢镰刀菌 CCTCC M209040 中表达。转化体 F. oxysporum M209040-Tal2 的重组转醛醇酶活性约为 0.52Umg(-1)蛋白，比野生型 F. oxysporum CCTCC M209040 高 1.57 倍，表明转化体中转醛醇酶活性的提高是由于外源 tal 基因的表达。在没有选择压力的情况下，转化体 F. oxysporum M209040-Tal2 的生长并未影响转化体的潮霉素抗性水平，表明整合的 tal 基因在有丝分裂中是稳定的。F. oxysporum M209040-Tal2 的发酵试验表明，在葡萄糖和木糖底物上，乙醇产量分别提高了 8.39%和 11.71%，表明毕赤酵母 CICC1960 的 tal 基因在尖孢镰刀菌 CCTCC M209040 中的表达可以提高乙醇产量。
