Tuning amino acid dehydrogenases with featured sequences for L-phosphinothricin synthesis by reductive amination
Tuning amino acid dehydrogenases with featured sequences for L-phosphinothricin synthesis by reductive amination
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DOI：
10.1016/j.jbiotec.2020.03.001
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发表时间：
2020-03-20
影响因子：
4.1
通讯作者：
Zheng, Yu-Guo
中科院分区：
文献类型：
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作者：
Cheng, Feng;Li, Heng;Zheng, Yu-Guo
由于氨基酸脱氢酶（AADH）的还原胺化活性有限，生物合成非天然手性氨基酸具有挑战性。为了从2-氧代-4-[（羟基）（甲基）膦酰基]丁酸（PPO）不对称合成L-膦丝菌素，从蒙氏假单胞菌（Pseudomonasmonteilii）中选择谷氨酸脱氢酶基因（命名为GluDH 3），克隆并在大肠杆菌（Escherichiacoli）中表达。大肠杆菌）。为了提高其活性，开发了一种基于“两步”的计算方法，并应用于选择GluDH 3上潜在的有益氨基酸位置。以D-葡萄糖脱氢酶（EsGDH）为催化剂，通过不对称氨基化反应合成了L-膦丝菌素。使用冻干E.在共表达GluDH3_V375S和EsGDH的大肠杆菌中，高达89.04 g L-1的PPO负载量在35 ℃下在30 min内完全转化为L-膦丝菌素，时空产率高达4.752 kg.L-1.d（-1）。在K90、T193和底物PPO之间具有增加的极性相互作用的有益取代V375 S表现出168.2倍的催化效率（k（cat）/K-M）和344.8倍的比活性增强。在其它GluDH的特定位置引入丝氨酸残基后，40个工程GluDH表现出“草铵膦脱氢酶”对PPO的催化功能。
Biosynthesizing unnatural chiral amino acids is challenging due to the limited reductive amination activity of amino acid dehydrogenase (AADH). Here, for the asymmetric synthesis of L-phosphinothricin from 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO), a glutamate dehydrogenase gene (named GluDH3) from Pseudomonas monteilii was selected, cloned and expressed in Escherichia coli (E. coli). To boost its activity, a "two-step"-based computational approach was developed and applied to select the potential beneficial amino acid positions on GluDH3. L-phosphinothricin was synthesized by GluDH-catalyzed asymmetric amination using the D-glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH) for NADPH regeneration. Using lyophilized E. coli cells that co-expressed GluDH3_V375S and EsGDH, up to 89.04 g L-1 PPO loading was completely converted to L-phosphinothricin within 30 min at 35 degrees C with a space-time yield of up to 4.752 kg.L-1.d(-1). The beneficial substitution V375S with increased polar interactions between K90, T193, and substrate PPO exhibited 168.2-fold improved catalytic efficiency (k(cat)/K-M) and 344.8-fold enhanced specific activity. After the introduction of serine residues into other GluDHs at specific positions, forty engineered GluDHs exhibited the catalytic functions of "glufosinate dehydrogenase" towards PPO.