Department of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain.
Biological Research Center, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Biochim Biophys Acta Proteins Proteom. 2020 Jan;1868(1):140293. doi: 10.1016/j.bbapap.2019.140293. Epub 2019 Oct 30.
Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-alcohol oxidase (AAO) and 5-hydroxymethylfurfural oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.
糠酸-2,5-二甲酸（FDCA）是一种可生物降解塑料的结构单元，可用于替代源自化石碳源的塑料。近年来，人们对通过级联酶反应从生物基 5-羟甲基糠醛（HMF）合成 FDCA 产生了浓厚的兴趣。芳醇氧化酶（AAO）和 5-羟甲基糠醛氧化酶（HMFO）是葡萄糖-甲醇-胆碱黄素酶，可通过三次连续氧化将 HMF 转化为 FDCA，而无需辅助酶的协助。这种具有挑战性的过程取决于原始醛基和形成的醛基的水合程度，限速步骤在于将中间 5-甲酰基糠酸（FFCA）氧化为 FDCA 的最后一步。虽然 HMFO 接受 FFCA 作为 HMF 反应途径的最终底物，但 AAO 实际上无法氧化它。在这里，我们通过与 HMFO 的结构对齐和定向进化，对 AAO 进行了工程设计，使其能够将 HMF 逐步氧化为 FDCA。该进化后的 AAO 对 HMF 的催化效率提高了 3 倍，总转化率提高了 6 倍，是进一步工程化以生成高效生物催化剂从 HMF 合成 FDCA 的有希望的起点。
