Development of oxidases for synthesis of bioplastics intermediates

由于环境压力，迫切需要从生物可再生原料中开发新型、高性能、可生物降解和可持续的塑料。植物木质纤维素衍生的2，5-呋喃二甲酸（FDCA）具有在各种塑料聚合物（例如聚对苯二甲酸乙二醇酯（PET））中替代石油衍生的化学品的巨大潜力。正在开发掺入FDCA的生物衍生的和生物可降解的塑料，例如聚（呋喃二甲酸乙二醇酯）（PEF）和聚（己二酸丁二醇酯-共-呋喃二甲酸丁二醇酯）（PBAF）。然而，目前还没有大规模的FDCA单体生产商来支持这些潜在的高价值聚合物的大规模生产。该项目旨在开发一种新的工艺，用于从生物质中生产FDCA，并将其输送到需求不断增长的生物塑料生产管道中。酶促转化比传统的工业化学路线具有优势，因为它们需要更少的能源，并且在更温和的条件下进行。酶通常允许高度选择性地生产所需的目标化合物，并提供更高水平的纯度。这在生产单体如FDCA时尤其重要，因为杂质会干扰聚合过程。最近，利物浦与Biome Bioplastics和利兹大学合作进行的研究表明，使用4种酶的组合来生产FDCA的工艺的概念验证，其中一些酶难以大规模生产。在利物浦，我们发现了一种新的酶（E56），它可以取代这些酶中的3种。虽然这种酶在这种能力上是独一无二的，但目前它的效率很低。我们还发现了另一种新的酶（E5），具有更高的活性，但它对部分途径起作用，可以与其他新酶结合使用。该项目旨在采用先进的合成生物学技术和最先进的设备，通过定向进化来提高酶的能力。该技术允许在实验室中进化酶催化活性，并且该方法将涉及产生和筛选非常大量的遗传变体。在确定了新的酶之后，我们将测试它们的活性和是否适合作为以前使用的酶的替代品。项目成果将包括开发可用于生产生物塑料前体的改良酶，以及建立高效的超高通量筛选平台，可应用于其他需要改进的酶。

Due to environmental pressure, there is an urgent need to develop novel, high-performance, biodegradable and sustainable plastics from bio-renewable feedstocks. Plant lignocellulose-derived 2,5-furan dicarboxylic acid (FDCA) has great potential for replacing petroleum-derived chemicals in various plastic polymers, for instance in polyethylene terephthalate (PET). Bio-derived and biodegradable plastics incorporating FDCA, such as poly(ethylene furanoate) (PEF) and poly(butylene adipate-co-butylene furandicarboxylate) (PBAF) are being developed. However, currently there are no large-scale producers of the FDCA monomer which is needed to support large scale production of these potentially high-value polymers. This project addresses the need to a develop novel process for the production of FDCA from biomass which will feed into bioplastic manufacturing pipelines for which there is growing demand.Enzymatic conversions confer advantages over traditional industrial chemical routes as they require less energy and are carried out in milder conditions. Enzymes often permit highly selective production of desired target compounds and deliver a higher level of purity. This is particularly important when producing monomers such as FDCA since impurities can interfere with the polymerisation process. Recent research at Liverpool in collaboration with Biome Bioplastics and Leeds University showed proof-of-concept for a process that uses a combination of 4 enzymes to produce FDCA, some of which were difficult to produce on scale. At Liverpool we identified a single new enzyme (E56) which could replace 3 of these enzymes. While the enzyme is unique in this ability, its efficiency is currently low. We also discovered another new enzyme (E5), with higher activity, but that works for part of the pathway and could be used as a combination with the other new enzyme. This project seeks to employ advanced synthetic biology techniques and state-of-the art equipment to improve the enzymes' capabilities through directed evolution. This technique allows evolution of enzyme catalytic activity in the laboratory and the approach will involve generating and screening a very large number of genetic variants. Having identified new enzymes, we will test their activity and suitability as a drop-in replacement for the previously used enzymes.The project outcomes will include development of improved enzymes that can be used in the production of bioplastic precursors, and establishment of an efficient ultra-high throughput screening platform that can be applied to other enzymes in need of improvement.