Understanding and application of a biological Kolbe-Schmitt reaction: aromatic C-H activation coupled to CO2 fixation.

羧化酶能够将CO2与其他分子连接起来，从而将其用作容易获得且廉价的单碳构建块。此外，羧化酶活性有助于降低全球二氧化碳水平。事实上，地球上的大多数生命直接或间接地依赖于能够从CO2产生生物质的关键羧化酶的光合作用驱动作用。因此，羧化酶可以用来应对降低大气中二氧化碳水平和创造更可持续经济的挑战。然而，由于这些蛋白质的复杂性和/或对活性的严格要求，这些酶的工程化和应用通常进展缓慢。相反，脱羧酶（通常实现相反的反应，即从分子中裂解CO2）在相反方向上取得了一些成功。实现羧化传统上需要大量的CO2（推动反应朝着正确的方向）或额外的酶，这些酶在快速转化羧酸盐产物方面非常有效（推动反应朝着正确的方向）。第三种选择是将脱羧酶反应直接偶联到有利的反应，使得脱羧酶在环境CO2下有效地反向运行，并以来自偶联反应的试剂为代价产生羧化产物。大自然以苯酚羧化酶系统的形式实现了这一壮举，该酶系统参与细菌苯酚降解。这种酶催化工业Kolbe-Schmitt反应的生物等效物，该反应使用高压CO2和超过100摄氏度的温度来羧酸苯酚。相比之下，这种有趣的酶系统在环境条件下运行，并使用ATP（天然“能量货币”）驱动苯酚+ CO2产生产物对羟基苯甲酸。我们试图确定苯酚羧化酶如何实现两个反应（即羧化和ATP消耗）的耦合，以确定天然工程原理，并将其应用于开发新的途径，将ATP依赖性羧化酶，如苯酚羧化酶。我们将利用蛋白质晶体学来确定这种酶的各种组分的结构，并将其与详细的计算和解决方案研究相结合，以呈现支持活性的分子编排的完整图像。我们将使用所产生的见解作为该系统的实验室指导进化的蓝图，以将底物库扩展到非酚类芳香醇。进化的羧化酶将用于产生新的和可再生的途径，用于从生物质生产关键化学商品，如FDCA（呋喃二羧酸）或对苯二甲酸。

Carboxylase enzymes are able to link CO2 to other molecules, thereby using it as a readily accessible and cheap one-carbon building block. Furthermore, carboxylase activity contributes to the reduction of global CO2 levels. Indeed, most life on the planet directly or indirectly depends on the photosynthesis driven action of a key carboxylase able to generate biomass from CO2. Thus, carboxylases could serve to meet the challenge of reducing atmospheric CO2 levels and creating a more sustainable economy. However, engineering and application of these enzymes has often met with slow progress due to the complexity and/or strict requirements of these proteins for activity. In contrast, decarboxylase enzymes (that normally achieve the opposite reaction, i.e. cleaving a CO2 from a molecule) in the reverse direction has met with some success. Achieving carboxylation traditionally requires either large amounts of CO2 (pushing the reaction in the right direction) or additional enzymes that are highly efficient in rapidly converting the carboxylate product (pulling the reaction in the right direction). A third option is to couple the decarboxylase reaction directly to a favourable reaction such that the decarboxylase effectively runs in reverse under ambient CO2 and yields the carboxylated product at the expense of the reagents from the coupled reaction. Nature has achieved this feat in the form of the phenol carboxylase enzyme system, which is involved in bacterial phenol degradation. This enzyme catalyses the biological equivalent of the industrial Kolbe-Schmitt reaction, which uses high pressure CO2 and temperatures exceeding 100 degrees Celsius to carboxylate phenol. In contrast, this intriguing enzyme system operates and ambient conditions, and uses ATP (the natural "energy currency") to drive phenol + CO2 yielding the product para-hydroxybenzoic acid. We seek to determine how the phenol carboxylase achieves the coupling of both reactions (i.e. carboxylation and ATP consumption) to determine the natural engineering principles and apply these to development of new pathways that incorporate ATP-dependent carboxylase enzymes such as the phenol carboxylase. We will make use of protein crystallography to determine the structure of the various components of this enzyme, and combine that with detailed computational and solution studies to present a complete picture of the molecular choreography that underpins activity. We will use the insights generated as a blueprint for the laboratory guided evolution of this system to expand the substrate repertoire to non-phenolic aromatic alcohols. Evolved carboxylase enzymes will be used to generate new and renewable pathways for the production of key chemical commodities such as FDCA (furan dicarboxylic acid) or terephthalate from biomass.