亮氨酸脱氢酶C端Loop区域的理性设计及多酶级联高效合成L-2-氨基丁酸

Rational design of the C-terminal Loop region of leucine dehydrogenase and cascade biosynthesis L-2-aminobutyric acid

亮氨酸脱氢酶(Leucinedehydrogenase,LDH)是制备L-2-氨基丁酸的关键限速酶,针对该酶的Loop区域进行改造以提高关键酶的酶活及稳定性从而高效合成L-2-氨基丁酸。通过亮氨酸脱氢酶的分子动力学模拟分析均方根涨落(Root mean square fluctuation,RMSF)值,对其波动非常明显的Loop区域合理设计以得到比酶活提高的截短突变体EsLDHD2,其比酶活为野生型的123.2%;此外,由于L-2-氨基丁酸制备过程中苏氨酸脱氨酶催化L-苏氨酸制备2-酮丁酸的速率过快导致多酶催化不平衡,因此双拷贝亮氨酸脱氢酶及甲酸脱氢酶以平衡多酶催化速率,构建多酶级联催化的单细胞E.coliBL21/pACYCDuet-RM,其摩尔转化率相较于E.coliBL21/pACYCDuet-RO提高74.6%;对菌株E. coli BL21/pACYCDuet-RM的全细胞转化条件进行优化,其最适pH、温度、底物浓度分别为7.5、35℃和80 g/L,此时摩尔转化率大于99%;在1 L转化体系和最适转化条件下分批加入L-苏氨酸80 g和40 g,L-2-氨基丁酸的产量达97.2g。总之,该策略为L-2-氨基丁酸的制备提供了绿色、高效的合成方法,具有工业化制备药物前体的巨大潜力。

Leucine dehydrogenase(LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid(L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation(RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 ℃ and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.