In Vivo Assembly of a Genetically Encoded Artificial Metalloenzyme for Hydrogen Production
In Vivo Assembly of a Genetically Encoded Artificial Metalloenzyme for Hydrogen Production
复制标题
用于制氢的基因编码人工金属酶的体内组装
DOI：
10.1021/acssynbio.1c00177
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发表时间：
2021
影响因子：
4.7
通讯作者：
Shafaat, Hannah S.
中科院分区：
文献类型：
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作者：
Naughton, Kassandra J.;Treviño, Regina E.;Moore, Peter J.;Wertz, Ashlee E.;Dickson, J. Alex;Shafaat, Hannah S.
相对于传统的分子催化剂优化，人工酶的遗传编码代表了实质性的优势，因为基于实验室的定向进化结合高通量筛选方法可以提供酶文库的快速开发和功能表征。然而，由于需要体外辅因子金属化，这些技术在人工金属酶领域的实用性有限。在这里，我们报告的方法的发展forin体内生产镍取代rubredoxin，人工金属酶，是一种结构，功能和机械模拟的[NiFe]氢化酶。细胞裂解物上的直接伏安法为电化学筛选的发展建立了先例。该技术将广泛适用于需要非天然金属辅因子的人工金属酶的体内生成，提供了一种快速酶优化的途径，并为人工金属酶整合到不同宿主的生化途径中奠定了基础。
The genetic encoding of artificial enzymes represents a substantial advantage relative to traditional molecular catalyst optimization, as laboratory-based directed evolution coupled with high-throughput screening methods can provide rapid development and functional characterization of enzyme libraries. However, these techniques have been of limited utility in the field of artificial metalloenzymes due to the need forin vitrocofactor metalation. Here, we report the development of methodology forin vivoproduction of nickel-substituted rubredoxin, an artificial metalloenzyme that is a structural, functional, and mechanistic mimic of the [NiFe] hydrogenases. Direct voltammetry on cell lysate establishes precedent for the development of an electrochemical screen. This technique will be broadly applicable to thein vivogeneration of artificial metalloenzymes that require a non-native metal cofactor, offering a route for rapid enzyme optimization and setting the stage for integration of artificial metalloenzymes into biochemical pathways within diverse hosts.