Encapsulin Nanocontainers as Versatile Scaffolds for the Development of Artificial Metabolons
Encapsulin Nanocontainers as Versatile Scaffolds for the Development of Artificial Metabolons
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DOI：
10.1021/acssynbio.0c00636
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发表时间：
2021-03-26
影响因子：
4.7
通讯作者：
Lutz, Stefan
中科院分区：
文献类型：
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作者：
Jenkins, Matthew C.;Lutz, Stefan
非天然生物合成途径的构建代表了用于生产有价值的合成子和精细化学品的强大的模块化策略。因此，人工将催化连续反应的酶固定到 DNA、蛋白质或合成支架上已被证明是产生具有新颖功能和卓越效率的从头代谢的有效途径。近年来，被称为封装蛋白的纳米级微生物隔室已成为一类坚固且高度工程化的蛋白质容器，在生物技术和合成生物学中具有多种应用。在此，我们报告了来自海栖热袍菌的封装蛋白的同时表面功能化和内部包装，以产生具有催化能力的双酶代谢物。封装蛋白被设计为使用 SpyCatcher 生物共轭系统在其外表面上共价隔离多达 60 个二氢叶酸还原酶 (DHFR) 酶变体，而其内腔则使用从封装蛋白天然蛋白货物中提取的短肽亲和标签，用四氢叶酸依赖性脱甲基酶包装。两种共定位酶之间的成功串扰被证实，因为外部束缚的 DHFR 产生的四氢叶酸能够通过包装的去甲基化酶驱动木质素衍生的芳基底物去甲基化，尽管速度很慢。随后在封装蛋白壳中引入先前报道的扩大孔的缺失被证明可以增强代谢物交换，使得基于封装蛋白的代谢物以与自由分散在溶液中的两种酶相当的速度发挥作用。因此，我们的工作进一步强调了封装蛋白的可工程性及其作为生物催化应用的柔性支架的潜在用途。
The construction of non-native biosynthetic pathways represents a powerful, modular strategy for the production of valuable synthons and fine chemicals. Accordingly, artificially affixing enzymes that catalyze sequential reactions onto DNAs, proteins, or synthetic scaffolds has proven to be an effective route for generating de novo metabolons with novel functionalities and superior efficiency. In recent years, nanoscale microbial compartments known as encapsulins have emerged as a class of robust and highly engineerable proteinaceous containers with myriad applications in biotechnology and synthetic biology. Herein we report the concurrent surface functionalization and internal packaging of encapsulins from Thermotoga maritima to generate a catalytically competent two-enzyme metabolon. Encapsulins were engineered to covalently sequester up to 60 copies of a dihydrofolate reductase (DHFR) enzyme variant on their exterior surfaces using the SpyCatcher bioconjugation system, while their lumens were packaged with a tetrahydrofolate-dependent demethylase enzyme using short peptide affinity tags abstracted from the encapsulin's native protein cargo. Successful cross-talk between the two colocalized enzymes was confirmed as tetrahydrofolate produced by externally tethered DHFR was capable of driving the demethylation of a lignin-derived aryl substrate by packaged demethylases, albeit slowly. The subsequent introduction of a previously reported pore-enlarging deletion in the encapsulin shell was shown to enhance metabolite exchange such that the encapsulin-based metabolon functioned at speeds equivalent to those of the two enzymes freely dispersed in solution. Our work thus further emphasizes the engineerability of encapsulins and their potential use as flexile scaffolds for biocatalytic applications.