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• 生物工程 •    下一篇

组成型过表达ido基因对4-羟基异亮氨酸合成的影响及利用响应面法优化其发酵培养基

何继龙,张成林   

  1. 天津科技大学
  • 收稿日期:2018-08-09 修回日期:2019-06-11 出版日期:2019-07-25 发布日期:2019-07-23
  • 通讯作者: 张成林 E-mail:zcl@tust.edu.cn
  • 基金资助:
    国家自然科学基金;中国博士后科学基金特别资助项目;中国博士后科学基金面上项目;天津市科技计划项目

Effect of constitutively overexpressing ido on 4-hydroxyisoleucine production and optimization of the fermentation medium by response surface methodology

1,   

  • Received:2018-08-09 Revised:2019-06-11 Online:2019-07-25 Published:2019-07-23

摘要: 4-羟基异亮氨酸具有葡萄糖浓度依赖的促进胰岛素分泌活性,是治疗糖尿病的潜在新药。异亮氨酸羟化酶 ((isoleucine dioxygenase,IDO)由ido编码,) 能够特异性催化L-异亮氨酸合成4-羟基异亮氨酸。为考察组成型过表达ido基因对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添加量为玉米浆34.10 mLg/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%。

关键词: 4-羟异亮氨酸, 异亮氨酸羟化酶, 谷氨酸棒杆菌, 组成型过表达, 响应面法

Abstract: 4-Hydroxyisoleucine exhibits a unique glucose-dependent insulinotropic activity, which is a promising candidate for the treatment of diabetes. Isoleucine dioxygenase (IDO, encoded by ido) can specifically catalyze the L-isoleucine conversion to 4-hydroxyisoleucine. To detect the effect of constitutively overexpressing ido on 4-hydroxyisoleucine production, the constitutive expression plasmid pXM01-ido and strain HIL017 harbouring the plasmid were constructed. The transcription level of ido and activity of IDO in HIL017 were significantly increased as compared with those in HIL016. Moreover, the 4-hydroxyisoleucine production by HIL017 was increased by 19.4%.To further improve 4-hydroxyisoleucine production by HIL017, the main factors in the fermentation medium was proved to be corn steep liquor, glutamic acid and FeSO4?7H2O by the Plackett-Burman test. Following, the steepest ascent method and response surface method were then used, which determined the optimal values of the three factors: corn steep liquor 34.10 gmL/L, glutamic acid 2.98 g/L, and FeSO4?7H2O 0.016 7 g/L, and the theoretical production was 5.57 g/L. With the optimized culture, 4-hydroxyisoleucine production was increased by 19.7%, up to 5.53 g/L.

Key words: 4-hydroxyisoleucine, isoleucine dioxygenase, Corynebacterium glutamicum, constitutively overexpression, response surface methodology

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