组成型过表达ido基因对4-羟基异亮氨酸合成的影响及其发酵培养基的响应面优化

Effect of Constitutively Overexpressed ido Gene on 4-Hydroxyisoleucine Production and Optimization of the Fermentation Medium by Response Surface Methodology

为考察组成型过表达异亮氨酸羟化酶(isoleucine dioxygenase,IDO)基因ido对4-羟基异亮氨酸合成的影响,构建ido组成型表达质粒pXM01-ido及菌株HIL017,其ido转录量及IDO活性较诱导型过表达菌株HIL016显著提升,4-羟基异亮氨酸产量较HIL016提高19.4%。为进一步提高4-羟基异亮氨酸产量,通过Plackett-Burman试验确定HIL017发酵培养基中玉米浆、谷氨酸和FeSO4·7H2O用量为主要影响因素,利用最陡爬坡试验和响应面法确定其最优用量为玉米浆34.1 mL/L、谷氨酸2.98 g/L、FeSO4·7H2O 0.016 7 g/L,此时4-羟基异亮氨酸理论产量为5.57 g/L。验证实验结果表明,最佳条件下4-羟基异亮氨酸产量为5.53 g/L,较优化前提高19.7%。

To determine the effect of constitutively overexpressed isoleucine dioxygenase gene(ido) on 4-hydroxyisoleucine production, an ido constitutively expressing plasmid pXM01-ido and a strain HIL017 harbouring the plasmid were constructed. The transcription level of ido and the activity of IDO in HIL017 were significantly increased as compared with those in HIL016, inductively expressing the gene. Moreover, 4-hydroxyisoleucine production by HIL017 was increased by 19.4%. To further improve 4-hydroxyisoleucine production, corn steep liquor, glutamic acid and FeSO4·7 H2O were recognized as main medium components using the Plackett-Burman design. By using he steepest ascent method and response surface methodology the optimal values of these factors were determined as follows: corn steep liquor 34.1 mL/L,glutamic acid 2.98 g/L, and FeSO4·7 H2O 0.016 7 g/L, giving a predicted yield of 5.57 g/L. Validation experiments showed that 4-hydroxyisoleucine production was 5.53 g/L, increasing by 19.7% compared with that before optimization.