The clustering of biosynthetic enzymes is used in nature to channel reaction products and increase the yield of compounds produced by multiple reaction steps. The coupling of multiple enzymes has been shown to increase the biosynthetic product yield. Different clustering strategies have particular advantages as the spatial organization of multiple enzymes creates biocatalytic cascades with a higher efficiency of biochemical reaction. However, there are also some drawbacks, such as misfolding and the variable stability of interaction domains, which may differ between particular biosynthetic reactions and the host organism. Here, we compared different protein-based clustering strategies, including direct fusion, fusion mediated by intein, and noncovalent interactions mediated through small coiled-coil dimer-forming domains. The clustering of enzymes through orthogonally designed coiled-coil interaction domains increased the production of resveratrol in Escherichia coli more than the intein-mediated fusion of biosynthetic enzymes. The improvement of resveratrol production correlated with the stability of the coiled-coil dimers. The coiled-coil fusion-based approach also increased mevalonate production in Saccharomyces cerevisiae, thus demonstrating the wider applicability of this strategy.

中文翻译：

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盘绕线圈的代谢酶簇聚改善了白藜芦醇和甲羟戊酸酯的生物合成。

自然界中使用生物合成酶的簇来引导反应产物并增加通过多个反应步骤产生的化合物的产率。已显示多种酶的偶联增加了生物合成产物的产量。不同的聚类策略具有特殊的优势，因为多种酶的空间组织以更高的生化反应效率产生了生物催化级联。但是，也存在一些缺陷，例如错折叠和相互作用域的可变稳定性，这可能在特定的生物合成反应和宿主生物之间有所不同。在这里，我们比较了不同的基于蛋白质的聚类策略，包括直接融合，intein介导的融合以及通过小的卷曲螺旋二聚体形成域介导的非共价相互作用。通过正交设计的卷曲螺旋相互作用域进行酶的聚集比在蛋白质介导的生物合成酶融合中增加了白藜芦醇在大肠杆菌中的产量。白藜芦醇产量的提高与卷曲螺旋二聚体的稳定性有关。基于盘绕线圈融合的方法还增加了酿酒酵母中甲羟戊酸的产生，从而证明了该策略的广泛适用性。