Rational molecular engineering of L-amino acid deaminase for production of α-ketoisovaleric acid from L-valine by Escherichia coli
Rational molecular engineering of L-amino acid deaminase for production of α-ketoisovaleric acid from L-valine by Escherichia coli
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DOI：
10.1039/c6ra26972a
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发表时间：
2017-01-01
期刊：
影响因子：
3.9
通讯作者：
Liu, Long
中科院分区：
文献类型：
--
作者：
Li, Ruoxi;Sakir, Hossain Gazi;Liu, Long
酶促效率的靶向修饰可以驱动所需代谢物的产量增加。α-酮基异戊酸（KIV）是用于制药和食品工业的候选材料。在本研究中，我们的目的是提高生物转化效率的L-氨基酸脱氨酶（L-aad），从L-缬氨酸提高KIV的生产。首先，在大肠杆菌BL 21（DE 3）中表达L-aad。我们采用转化的E.大肠杆菌细胞作为全细胞生物催化剂系统，并优化其生物转化L-缬氨酸的生化特性。然后，基于已知的粘杆菌L-aad的三维结构模型和与L-缬氨酸对接的模拟结果，确定了4个氨基酸残基（N100、Q276、R316和F318）为潜在的诱变靶位点。接下来，我们进行了定点饱和诱变，以提高生物转化效率。在最佳条件下，单突变株（F318 T）和双突变株（F318 T和N100 H）的L-缬氨酸转化效率分别为4.474和8.197 g L-1，而野生型菌株的转化效率为2.014 g L-1。综上所述，我们开发了一种通过在大肠杆菌中表达P. myxofaciens L-aad生产KIV的一步法。coli BL 21（DE 3）中，通过L-aad的定点饱和突变提高了KIV的产量。
The targeted modification of enzymatic efficiency can drive an increased production of desired metabolites. alpha-Ketoisovaleric acid (KIV) is a candidate material for use in the pharmaceutical and food industries. In the present study, we aimed to enhance the biotransformation efficiency of L-amino acid deaminase (L-aad) from Proteus myxofaciens ATCC 19692 to improve the production of KIV from L-valine. First, L-aad was expressed in Escherichia coli BL21(DE3). We employed transformed E. coli cells as a whole-cell biocatalyst system and optimized their biochemical characteristics for the biotransformation of L-valine. Then, based on the known 3D structural model of L-aad from P. myxofaciens and the simulation results for docking with L-valine, four amino acid residues (N100, Q276, R316, and F318) were identified as potential target sites for mutagenesis. Next, we performed site-directed saturation mutagenesis to improve the biotransformation efficiency. With 11.3 g L-1 L-valine, the bioconversion efficiencies of a single-mutant strain (F318T) and a double-mutant strain (F318T and N100H) were 4.474 and 8.197 g L-1, respectively, whereas that of the wild-type strain was 2.014 g L-1 under optimal conditions. In summary, we developed a one-step process for KIV production via expressing P. myxofaciens L-aad in E. coli BL21(DE3) and enhanced the yield of KIV by site-directed saturation mutagenesis of L-aad.