Dynamic modulation of enzyme activity by synthetic CRISPR–Cas6 endonucleases
Dynamic modulation of enzyme activity by synthetic CRISPR–Cas6 endonucleases
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DOI：
10.1038/s41589-022-01005-7
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发表时间：
2022-04
影响因子：
14.8
通讯作者：
Alexander A Mitkas;M. Valverde;Wilfred Chen
中科院分区：
文献类型：
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作者：
Alexander A Mitkas;M. Valverde;Wilfred Chen
在自然界中，酶之间的动态相互作用在定义细胞代谢中起着至关重要的作用。通过控制这些称为代谢的超分子复合物的空间和时间组织，可以以高度动态的方式调整自然代谢。在这里，我们将 CRISPR-Cas6 家族蛋白重新用作一种合成策略，通过结合 RNA 加工的简易性和蛋白质组装的 RNA 杂交的可预测性来创建动态代谢。通过使用立足点介导的链置换反应扰乱 RNA-RNA 网络，使用合成 RNA 触发器和 mCherry 信使 RNA 实现按需组装和拆卸。色氨酸-2-单加氧酶和吲哚乙酰胺水解酶途径酶的直接组装和“启动”组装均可将吲哚-3-乙酸产量提高九倍。甚至可以组装多聚酶以将苹果酸产量提高三倍。通过与内源 mRNA 连接，可以构建更复杂的代谢物，从而产生能够适应不断变化的细胞需求的自我响应代谢机制。
In nature, dynamic interactions between enzymes play a crucial role in defining cellular metabolism. By controlling the spatial and temporal organization of these supramolecular complexes called metabolons, natural metabolism can be tuned in a highly dynamic manner. Here, we repurpose the CRISPR–Cas6 family proteins as a synthetic strategy to create dynamic metabolons by combining the ease of RNA processing and the predictability of RNA hybridization for protein assembly. By disturbing RNA–RNA networks using toehold-mediated strand displacement reactions, on-demand assembly and disassembly are achieved using both synthetic RNA triggers andmCherrymessenger RNA. Both direct and ‘Turn-On’ assembly of the pathway enzymes tryptophan-2-monooxygenase and indoleacetamide hydrolase can enhance indole-3-acetic acid production by up to ninefold. Even multimeric enzymes can be assembled to improve malate production by threefold. By interfacing with endogenous mRNAs, more complex metabolons may be constructed, resulting in a self-responsive metabolic machinery capable of adapting to changing cellular demand.