Simultaneous Directed Evolution of Coupled Enzymes for Efficient Asymmetric Synthesis of l-Phosphinothricin
Simultaneous Directed Evolution of Coupled Enzymes for Efficient Asymmetric Synthesis of l-Phosphinothricin
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DOI：
10.1128/aem.02563-20
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发表时间：
2020-12
影响因子：
4.4
通讯作者：
Feng Cheng;Qing-hua Li;Hua-Yue Zhang;Lan Wei;Jia-min Zhang;Ju-Mou Li;Yaping Xue;Yuguo Zheng
中科院分区：
文献类型：
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作者：
Feng Cheng;Qing-hua Li;Hua-Yue Zhang;Lan Wei;Jia-min Zhang;Ju-Mou Li;Yaping Xue;Yuguo Zheng
传统的提高多酶催化反应效率的策略可能会导致单酶催化效率的提高，但也可能导致酶之间协调性的丧失。我们描述了一个定向进化策略，整个耦合酶系统，同时提高酶的协调和催化效率。摘要提高整个偶联酶系统效率的传统策略依赖于参与其各自酶促反应的酶的进化的单独方向。这种策略可以导致增强的单酶催化效率，但也可能导致酶之间的协调损失。本研究旨在通过对一个组合组中的多个酶执行定向进化策略来克服这些缺点，该组合组催化l-膦丝菌素的不对称生物合成。来自莫氏假单胞菌的谷氨酸脱氢酶（PmGluDH）和来自西伯利亚古杆菌的葡萄糖脱氢酶（EsGDH）的基因与其他基因部分（启动子、核糖体结合位点[RBS]和终止子）一起沿着同时进化。PmGluDH的催化效率通过引入有益突变A164 G（从1.29 s-1 mM-1提高到183.52 s-1 mM-1）而提高，并且EsGDH表达水平通过优化RBS和gdh起始密码子之间的接头长度而提高。生物反应的总转换数从115（GluDH_WTNANADPH [NADPH依赖性野生型GluDH]）增加到5，846（A164 GNANADPH与低表达EsGDH偶联）和33，950（A164 GNANADPH与高表达EsGDH偶联）。偶联效率从约30%（GDH低表达的GluDH_WT）增加到83.3%（GDH高表达的GluDH_A164G）。在使用全细胞催化的l-膦丝菌素的批量生产中，最强的生物催化反应表现出高时空产率（6，410 g ·l-1 · day-1）和严格的立体选择性（>99%对映体过量）。传统的提高多酶催化反应效率的策略可能会导致单酶催化效率的提高，但也可能导致酶之间协调性的丧失。我们描述了一个定向进化策略，整个耦合酶系统，同时提高酶的协调和催化效率。将进化策略应用于多酶催化不对称合成l-膦丝菌素的反应中，不仅提高了GluDH的催化效率，而且改善了GluDH与GDH之间的配位作用。由于该方法不依赖于酶，因此也适用于其它偶联酶体系的手性化学合成。
The traditional strategy to improve multienzyme-catalyzed reaction efficiencies may lead to enhanced single-enzyme catalytic efficiencies but may also result in the loss of coordination among enzymes. We describe a directed evolution strategy for an entire coupled-enzyme system to simultaneously enhance enzyme coordination and catalytic efficiency. ABSTRACT The traditional strategy to improve the efficiency of an entire coupled-enzyme system relies on the separate direction of the evolution of enzymes involved in their respective enzymatic reactions. This strategy can lead to enhanced single-enzyme catalytic efficiency but may also lead to a loss of coordination among enzymes. This study aimed to overcome such shortcomings by executing a directed evolution strategy on multiple enzymes in one combined group that catalyzes the asymmetric biosynthesis of l-phosphinothricin. The genes of a glutamate dehydrogenase from Pseudomonas moorei (PmGluDH) and a glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), along with other gene parts (promoters, ribosomal binding sites [RBSs], and terminators), were simultaneously evolved. The catalytic efficiency of PmGluDH was boosted by introducing the beneficial mutation A164G (from 1.29 s−1 mM−1 to 183.52 s−1 mM−1), and the EsGDH expression level was improved by optimizing the linker length between the RBS and the start codon of gdh. The total turnover numbers of the bioreaction increased from 115 (GluDH_WTNADPH [NADPH-dependent wild-type GluDH]) to 5,846 (A164GNADPH coupled with low expression of EsGDH) and to 33,950 (A164GNADPH coupled with high expression of EsGDH). The coupling efficiency was increased from ∼30% (GluDH_WT with low expression of GDH) to 83.3% (GluDH_A164G with high expression of GDH). In the batch production of l-phosphinothricin utilizing whole-cell catalysis, the strongest biocatalytic reaction exhibited a high space-time yield (6,410 g · liter−1 · day−1) with strict stereoselectivity (>99% enantiomeric excess). IMPORTANCE The traditional strategy to improve multienzyme-catalyzed reaction efficiencies may lead to enhanced single-enzyme catalytic efficiencies but may also result in the loss of coordination among enzymes. We describe a directed evolution strategy for an entire coupled-enzyme system to simultaneously enhance enzyme coordination and catalytic efficiency. The simultaneous-evolution strategy was applied to a multienzyme-catalyzed reaction for the asymmetric synthesis of l-phosphinothricin, which not only enhanced the catalytic efficiency of GluDH but also improved the coordination between GluDH and GDH. Since this strategy is enzyme independent, it may be applicable to other coupled-enzyme systems for chiral chemical synthesis.