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

羧基酶能够将二氧化碳连接到其他分子，从而将其用作一种易于获得和廉价的单碳构件。此外，羧基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光合作用驱动的一种关键羧基酶的作用，该酶能够从二氧化碳中产生生物质。因此，羧化酶可以用来应对降低大气二氧化碳水平和创造更可持续的经济的挑战。然而，由于这些蛋白质的复杂性和/或对活性的严格要求，这些酶的工程和应用往往进展缓慢。相比之下，相反方向的脱羧酶(通常实现相反的反应，即从分子中分解出二氧化碳)已经取得了一些成功。要实现羧化，传统上需要大量的二氧化碳(将反应推向正确的方向)或额外的酶，这些酶能高效地快速转化羧酸盐产品(将反应拉向正确的方向)。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，并以牺牲偶联反应的试剂为代价产生羧化产物。自然界以苯酚羧化酶系统的形式实现了这一壮举，该酶系统参与了细菌对苯酚的降解。这种酶催化的生物等价物是工业Kolbe-Schmitt反应，该反应使用高压二氧化碳和超过100摄氏度的温度来羧化苯酚。相比之下，这个耐人寻味的酶系统的运作和环境条件，并使用三磷酸腺苷(天然的“能源货币”)，以推动苯酚+二氧化碳产生的产品对羟基苯甲酸。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，以确定自然工程原理，并将这些原理应用于开发新的结合ATP依赖的羧基酶的途径，如苯酚羧基酶。我们将利用蛋白质结晶学来确定这种酶的各种成分的结构，并将其与详细的计算和溶液研究相结合，以呈现支持活性的分子编排的完整图景。我们将使用产生的洞察力作为该系统实验室指导的进化的蓝图，将底物谱系扩展到非酚类芳香醇。进化出的羧基酶将被用来产生新的和可再生的途径，用于从生物质中生产关键的化学品，如呋喃二元酸(FDCA)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.