State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Department of Endocrinology and Metabolism, Shanghai General Hospital Shanghai Jiao Tong University, Shanghai 200080, China.
J Agric Food Chem. 2020 Dec 9;68(49):14555-14563. doi: 10.1021/acs.jafc.0c06544. Epub 2020 Nov 28.
Isoleucine dioxygenase (IDO)-catalyzed hydroxylation of isoleucine is a promising method for the synthesis of the diabetic drug (2,3,4)-4-hydroxyisoleucine [(2,3,4)-4-HIL]. However, the low activity of IDO significantly limits its practical application. In this work, a high-throughput screening method was developed and directed evolution was performed on the IDO from , resulting in a double mutant with improvements in specific activity, protein expression level, and fermentation titer of 3.2-, 2.8-, and 9.4-fold, respectively. l-Isoleucine (228 mM) was completely converted to (2,3,4)-4-HIL by the best variant with a space-time yield of up to 80.8 g L d, which is the highest record reported so far. With a further increase of the substrate loading to 1 M, a high conversion of 91% could also be achieved. At last, enzymatic synthesis of (2,3,4)-4-HIL was successfully carried out on a 3 L scale, indicating tremendous potential of the IDO variant I162T/T182N for green and efficient production of (2,3,4)-4-HIL.
异亮氨酸双加氧酶（IDO）催化异亮氨酸的羟化是合成糖尿病药物（2,3,4）-4-羟基异亮氨酸[(2,3,4)-4-HIL]的有前途的方法。然而，IDO 的低活性显著限制了其实际应用。在这项工作中，开发了一种高通量筛选方法，并对 来源的 IDO 进行了定向进化，得到了一个双突变体，其比活性、蛋白表达水平和发酵效价分别提高了 3.2、2.8 和 9.4 倍。最佳变体可将 l-异亮氨酸（228 mM）完全转化为（2,3,4）-4-HIL，时空产率高达 80.8 g L d，这是迄今为止报道的最高记录。进一步将底物负荷增加到 1 M，仍可实现 91%的高转化率。最后，在 3 L 规模上成功进行了（2,3,4）-4-HIL 的酶促合成，表明 IDO 变体 I162T/T182N 具有巨大的潜力，可用于（2,3,4）-4-HIL 的绿色、高效生产。
