Abstract：
Virus-like particles (VLPs) derived from bacteriophage P22 have been explored as biomimetic catalytic compartments. In vivo colocalization of enzymes within P22 VLPs uses sequential fusion to the scaffold protein, resulting in equimolar concentrations of enzyme monomers. However, control over enzyme stoichiometry, which has been shown to influence pathway flux, is key to realizing the full potential of P22 VLPs as artificial metabolons. We present a tunable strategy for stoichiometric control over in vivo co-encapsulation of P22 cargo proteins, verified for fluorescent protein cargo by Förster resonance energy transfer. This was then applied to a two-enzyme reaction cascade. l-homoalanine, an unnatural amino acid and chiral precursor to several drugs, can be synthesized from the readily available l-threonine by the sequential activity of threonine dehydratase and glutamate dehydrogenase. We found that the loading density of both enzymes influences their activity, with higher activity found at lower loading density implying an impact of molecular crowding on enzyme activity. Conversely, increasing overall loading density by increasing the amount of threonine dehydratase can increase activity from the rate-limiting glutamate dehydrogenase. This work demonstrates the in vivo colocalization of multiple heterologous cargo proteins in a P22-based nanoreactor and shows that controlled stoichiometry of individual enzymes in an enzymatic cascade is required for the optimal design of nanoscale biocatalytic compartments.
摘要：
来自噬菌体P22的病毒样颗粒（VLP）已被用作仿生催化区室。P22VLP内的酶的体内共定位使用与支架蛋白的顺序融合，导致等摩尔浓度的酶单体。然而,控制酶的化学计量，已经被证明会影响通路通量，是实现P22VLP作为人工代谢产物的全部潜力的关键。我们提出了一种可调节的策略，用于对P22货物蛋白的体内共封装进行化学计量控制，通过Förster共振能量转移验证了荧光蛋白货物。然后将其应用于双酶反应级联。l-高丙氨酸,一种非天然氨基酸和几种药物的手性前体，可以通过苏氨酸脱水酶和谷氨酸脱氢酶的顺序活性从容易获得的1-苏氨酸合成。我们发现两种酶的加载密度都会影响它们的活性，在较低的加载密度下发现较高的活性，这意味着分子拥挤对酶活性的影响。相反，通过增加苏氨酸脱水酶的量来增加总负载密度可以增加限速谷氨酸脱氢酶的活性。这项工作证明了多种异源货物蛋白在基于P22的纳米反应器中的体内共定位，并显示了酶级联中单个酶的受控化学计量是纳米级生物催化隔室的最佳设计所必需的。
