Transglutaminase-mediated assembly of multi-enzyme pathway onto TMV brush surfaces for synthesis of bacterial autoinducer-2
Transglutaminase-mediated assembly of multi-enzyme pathway onto TMV brush surfaces for synthesis of bacterial autoinducer-2
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DOI：
10.1088/1758-5090/ab9e7a
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发表时间：
2020-10-01
期刊：
影响因子：
9
通讯作者：
Bentley, William E.
中科院分区：
文献类型：
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作者：
Bhokisham, Narendranath;Liu, Yi;Bentley, William E.
生物电子微器件，具有空间排列的生物合成机械，可以通过编程以受控的方式将原材料转化为高价值产品。能够在纳米和中观尺度上精确控制生物组分组装的通用生物功能化方法对于使这些系统的范围和能力多样化是必要的。本研究利用烟草花叶病毒（TMV）衍生的病毒样颗粒（vlp）作为三维界面支架，将生物合成酶组装到金电极上。通过半胱氨酸修饰衣壳蛋白并利用众所周知的金/半胱氨酸亲和力，TMV衣壳排列成垂直刷状结构。这种排列使高表面密度和生物合成酶-酶接近。通过N端和c端添加赖氨酸富集的组装域，酶与衣壳表面暴露的谷氨酰胺残基发生反应，共价连接到TMV的衣壳蛋白上；赖氨酸/谷氨酰胺键是由微生物谷氨酰胺转酶（mTG）介导的。我们展示了mtg介导的三酶生物合成途径的灵活组装，该途径将s -腺苷甲硫氨酸（SAM）转化为自诱导剂2 (AI-2)，这是一种介导群体感应行为的细菌信号分子。我们提出，我们基于VLP和mTG的制造方法将有助于将生物组件模块化组装到微电子设备上，并且这些将在许多应用中找到实用性，包括传感和芯片上的实验室设备。
Bioelectronic microdevices, with spatially arranged biosynthetic machinery, can be programmed to convert raw materials to high-value products in a controlled manner. Generic methods for biofunctionalization that enable precise control over biocomponent assembly at the nano and meso scales are necessary to diversify the range and capabilities of these systems. Here, we used tobacco mosaic virus (TMV) derived virus like particles (VLPs) as 3D interfacial scaffolds for the assembly of biosynthetic enzymes onto gold electrodes. The TMV capsids are aligned in a vertical brush configuration by cysteine modifications to the capsid protein and by taking advantage of the well-known gold/cysteine affinity. This alignment enables high surface density and biosynthetic enzyme-enzyme proximity. Enzymes are covalently tethered to the capsid protein of TMV by the N- and C-terminal addition of lysine-rich assembly domains which react with surface exposed glutamine residues on the capsid surfaces; the lysine/glutamine linkages are mediated by a microbial transglutaminase (mTG). We demonstrate flexible mTG-mediated assembly of a three-enzyme biosynthetic pathway that converts S-adenosylmethionine (SAM) to autoinducer-2 (AI-2), a bacterial signal molecule that mediates quorum sensing behavior. We propose that our VLP and mTG based fabrication approach will help in the modular assembly of biological components onto microelectronic devices and that these will find utility in many applications including sensing and lab on chip devices.