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

生物科尔贝-施密特反应的理解和应用：芳香族 C-H 活化与 CO2 固定相结合。

• 批准号: BB/W016745/1
• 负责人: David Leys
• 金额: $ 114.28万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW016745%2F1
• 关键词: Understanding; application; biological; Kolbe; Schmitt

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.

羧化酶能够将二氧化碳与其他分子连接起来，从而将其用作易于获取且廉价的单碳结构单元。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一种关键的羧化酶的光合作用，这种酶能够从二氧化碳中产生生物量。因此，羧化酶可以用来应对减少大气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，这些酶的工程和应用往往进展缓慢。相比之下，脱羧酶（通常进行相反的反应，即从分子中分离二氧化碳）在相反的方向上取得了一些成功。传统上，实现羧化要么需要大量的二氧化碳（将反应推向正确的方向），要么需要额外的酶，这些酶能够高效地快速转化羧酸产物（将反应推向正确的方向）。第三种选择是将脱羧酶反应直接耦合到一个有利的反应中，这样脱羧酶在环境CO2下有效地反向运行，并以牺牲耦合反应中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，该系统涉及细菌苯酚降解。这种酶催化的生物反应相当于工业上的科尔比-施密特反应，它使用高压二氧化碳和超过100摄氏度的温度来羧酸化苯酚。相比之下，这种有趣的酶系统在环境条件下运行，并使用ATP（天然的“能量货币”）驱动苯酚+ CO2产生产品对羟基苯甲酸。我们试图确定苯酚羧化酶如何实现两种反应的耦合（即羧化和ATP消耗），以确定自然工程原理，并将这些原理应用于开发新的途径，这些途径包括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成分的结构，并将其与详细的计算和解决方案研究相结合，以呈现支撑活性的分子编排的完整画面。我们将利用所产生的见解作为实验室指导该系统进化的蓝图，将底物库扩展到非酚醛芳香醇。进化的羧化酶将被用来产生新的和可再生的途径，用于生产关键的化学商品，如FDCA（呋喃二甲酸）或对苯二甲酸酯。