From Cell-Free Protein Synthesis to Whole-Cell Biotransformation: Screening and Identification of Novel α-Ketoglutarate-Dependent Dioxygenases for Preparative-Scale Synthesis of Hydroxy-l-Lysine
From Cell-Free Protein Synthesis to Whole-Cell Biotransformation: Screening and Identification of Novel α-Ketoglutarate-Dependent Dioxygenases for Preparative-Scale Synthesis of Hydroxy-l-Lysine
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DOI：
10.3390/catal11091038
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发表时间：
2021-08
期刊：
影响因子：
3.9
通讯作者：
J. Rolf;Philipp Nerke;Annette Britner;S. Krick;S. Lütz;Katrin Rosenthal
中科院分区：
文献类型：
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作者：
J. Rolf;Philipp Nerke;Annette Britner;S. Krick;S. Lütz;Katrin Rosenthal
非活性C-H键的选择性羟基化反应仍然是化学中具有挑战性的反应。非血红素Fe ~（2+）/α-酮戊二酸双加氧酶是一种重要的C-H键活化催化剂，主要催化羟基化反应。因此，发现具有生物技术应用的合适反应性的新的Fe 2 +/α-酮戊二酸依赖性双加氧酶对于扩展迄今为止描述的有限的酶范围是高度相关的。在这项研究中，我们进行了蛋白质BLAST，以确定同源酶已经描述的赖氨酸双加氧酶（KDO）。六个新的，但未表征的蛋白质的选择和无细胞蛋白质合成（CFPS）的合成。随后对所选同系物的体外筛选揭示了对L-赖氨酸（Lys）羟基化为羟基-1-赖氨酸（Hyl）的活性，羟基-1-赖氨酸是一种通用的手性结构单元。在生物技术应用方面，开发了大肠杆菌全细胞生物催化剂，并在小规模生物转化中进行了表征。由于表达编码来自发光杆菌的KDO的基因的全细胞生物催化剂显示出8.6 ± 0.6 U gCDW−1的最高比活性，因此选择其用于制备性合成Hyl。实现了多克规模的产物浓度，为Hyl生产的进一步生物工艺开发提供了良好的起点。建立了一种系统的方法来筛选和鉴定新的Fe 2 +/α-酮戊二酸依赖性双加氧酶，涵盖了从基因到产物的整个途径，这有助于加速生物过程的发展，以生产增值化学品。
The selective hydroxylation of non-activated C-H bonds is still a challenging reaction in chemistry. Non-heme Fe2+/α-ketoglutarate-dependent dioxygenases are remarkable biocatalysts for the activation of C-H-bonds, catalyzing mainly hydroxylations. The discovery of new Fe2+/α-ketoglutarate-dependent dioxygenases with suitable reactivity for biotechnological applications is therefore highly relevant to expand the limited range of enzymes described so far. In this study, we performed a protein BLAST to identify homologous enzymes to already described lysine dioxygenases (KDOs). Six novel and yet uncharacterized proteins were selected and synthesized by cell-free protein synthesis (CFPS). The subsequent in vitro screening of the selected homologs revealed activity towards the hydroxylation of l-lysine (Lys) into hydroxy-l-lysine (Hyl), which is a versatile chiral building block. With respect to biotechnological application, Escherichia coli whole-cell biocatalysts were developed and characterized in small-scale biotransformations. As the whole-cell biocatalyst expressing the gene coding for the KDO from Photorhabdus luminescens showed the highest specific activity of 8.6 ± 0.6 U gCDW−1, it was selected for the preparative synthesis of Hyl. Multi-gram scale product concentrations were achieved providing a good starting point for further bioprocess development for Hyl production. A systematic approach was established to screen and identify novel Fe2+/α-ketoglutarate-dependent dioxygenases, covering the entire pathway from gene to product, which contributes to accelerating the development of bioprocesses for the production of value-added chemicals.