Shi Feng, Fang Huimin, Niu Tengfei, Lu Zhengke
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Enzyme Microb Technol. 2016 Jun;87-88:79-85. doi: 10.1016/j.enzmictec.2016.04.008. Epub 2016 Apr 21.
4-hydroxyisoleucine (4-HIL) exhibits unique insulinotropic and insulin-sensitizing activities and is an attractive candidate for the treatment of type II and type I diabetes. In our previous study, l-isoleucine dioxygenase gene (ido) was cloned and overexpressed in an l-isoleucine-producing strain, Corynebacterium glutamicum ssp. lactofermentum SN01, and 4-HIL was produced from the endogenous l-isoleucine (Ile). In this study, ppc and lysC were co-expressed with ido to increase the supply of Ile, the direct precursor of 4-HIL, and to further improve the 4-HIL yield. After 144h of fermentation, the ido-ppc-expressing strain produced 95.72±1.52mM 4-HIL, 29% higher than the ido-expressing strain. The co-expression of lysC and ppc with ido resulted in a further 35% increment of carbon flux to l-aspartate family amino acids biosynthesis pathway. However, the conversion ratio of Ile to 4-HIL and the 4-HIL yield decreased to 0.31mol/mol and 30.16±2.01mM, respectively, likely due to the decreased IDO activity caused by lower pH and higher intracellular Ile concentration. Therefore, co-expression of ido and ppc was benefit for 4-HIL de novo biosynthesis, while co-expression of lysC with ido and ppc decreased the conversion ratio of Ile to 4-HIL.
4-羟基异亮氨酸（4-HIL）具有独特的促胰岛素分泌和胰岛素增敏活性，是治疗II型和I型糖尿病的极具吸引力的候选药物。在我们之前的研究中，l-异亮氨酸双加氧酶基因（ido）被克隆并在产l-异亮氨酸的菌株谷氨酸棒杆菌亚种乳发酵短杆菌SN01中过表达，4-HIL由内源性l-异亮氨酸（Ile）产生。在本研究中，ppc和lysC与ido共表达以增加4-HIL的直接前体Ile的供应，并进一步提高4-HIL产量。发酵144小时后，表达ido-ppc的菌株产生了95.72±1.52mM的4-HIL，比表达ido的菌株高29%。lysC和ppc与ido的共表达使流向l-天冬氨酸家族氨基酸生物合成途径的碳通量进一步增加了35%。然而，Ile向4-HIL的转化率和4-HIL产量分别降至0.31mol/mol和30.16±2.01mM，这可能是由于较低的pH值和较高的细胞内Ile浓度导致IDO活性降低所致。因此，ido和ppc的共表达有利于4-HIL的从头生物合成，而lysC与ido和ppc的共表达降低了Ile向4-HIL的转化率。
