Abstract：
Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient in vitro hybrid biocatalytic system by assembling three enzymes that can convert styrene to (S)-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed. To assemble them, we fused two orthogonal peptide-protein pairs (i.e., SpyTag/SpyCatcher and SnoopTag/SnoopCatcher) to the three enzymes, allowing their spatial organization by covalent assembly. By doing this, we constructed a multienzyme complex, which could enhance the production of (S)-1-phenyl-1,2-ethanediol by 3 times compared to the free-floating enzyme system without assembly. After optimization of the reaction system, the final product yield reached 234.6 μM with a substrate conversion rate of 46.9% (based on 0.5 mM styrene). Taken together, our strategy integrates the merits of advanced biochemical engineering techniques, including cellular surface display, spatial enzyme organization, and cell-free expression, which offers a new solution for chemical biosynthesis by enzymatic cascade biotransformation. We, therefore, anticipate that our approach will hold great potential for designing and constructing highly efficient systems to synthesize chemicals of agricultural, industrial, and pharmaceutical significance.
摘要：
酶级联已经成为合成有价值的化学品和药物的绿色和可持续方法。使用顺序酶构建多酶复合物是增强生物合成途径整体性能的有效途径。在这里，我们报告了通过组装三种可以将苯乙烯转化为（S）-1-苯基-1,2-乙二醇的酶来设计有效的体外混合生物催化系统。具体来说,我们以不同的方式制备了三种酶，它们是细胞表面展示的，纯化,无细胞表达。为了组装它们，我们融合了两个正交的肽-蛋白质对(即，SpyTag/SpyCatcher和SnoopTag/SnoopCatcher）对三种酶，通过共价组装允许它们的空间组织。通过这样做，我们构建了一个多酶复合物，与不组装的自由漂浮酶系统相比，可以将（S）-1-苯基-1,2-乙二醇的产量提高3倍。优化后的反应体系，最终产物产率达到234.6μM,底物转化率为46.9%(基于0.5mM苯乙烯)。一起来看,我们的战略整合了先进生化工程技术的优点，包括细胞表面显示，空间酶组织，和无细胞表达，通过酶促级联生物转化为化学生物合成提供了新的解决方案。我们,因此,预计我们的方法将在设计和构建高效的农业化学品合成系统方面具有巨大潜力，工业,和药学意义。
