Biocatalysis using P450 Monooxygenases for the Synthesis of Novel Biodegradable Fragrances

使用 P450 单加氧酶的生物催化合成新型可生物降解香料

• 批准号: 2749601
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 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=studentship-2749601
• 关键词: Biocatalysis; using; P450; Monooxygenases; Synthesis

Flavour and fragrance (F&F) oils are widely used in the food, pharmaceutical, perfume, cosmetic and detergent industries. However, many F&F compounds that are widely used across many industries are not readily biodegraded in the environment and hence accumulate over time. The poor biodegradation of such materials will make them untenable F&F products in the future; and certainly, from the perspective of Unilever, the industrial partner of this project, will not be used in any products by 2030. Further, materials used as ingredients in perfumes, cosmetics and household products, and flavourings in oral care products must be registered under the European REACH regulation, and so information on biodegradation and bioaccumulation is required. F&F molecules with high performance in many home and personal care products can bioaccumulate because of their hydrophobicity and chemical inertness. The aim of this project is to insert oxygen in the carbon skeletons of problematic F&F molecules that, upon oxidation, could afford F&F molecules for the future. The application of designed cytochrome P450 monooxygenases may render the new molecules more water soluble, bioavailable and biodegradable compared to the parent molecule. The approach has good precedence from nature as metabolically diverse microorganisms contain oxidative enzymes such as P450s to catalyse C-H bond oxidation to activate chemically inert compounds in metabolic and catabolic processes. The project involves screening of F&F molecules with an existing library of P450 enzyme variants known to oxidise a wide range of organic compounds to diverse products, characterisation of products isolated from preparative scale (0.1g-1g) reactions, followed by the design of new variant enzymes via docking-guided mutagenesis of the most selective variant to increase substrate conversion, scalability and selectivity for target products. In collaboration with Unilever scientists, the oxidation products will be studied for skin sensitisation and by odorant-binding assays for receptor activation to demonstrate that they have the potential to provide a sensory modality. Their biodegradability will be assessed using BioWin biodegradation and QSAR methods and experimental biodegradation assays. Current biodegradation models can provide poor fits due to having no data on a particular structure; hence, the project will use bioinformatics techniques to establish structure-function relations for the new molecules designed with respect to their bioavailability and biodegradation within an aqueous media. The project aligns perfectly with the BBSRC Strategic Priority Area 2: Tackling Strategic Challenges - Bioscience for Renewable Resources and Clean Growth. Specifically, the application of enzymatic processes to create new high value industrially relevant materials, developing higher performing bio-based processes, and the use of Techno Economic Analysis to provide an impact analysis will be at the heart of this project. The Clean Growth potential is realised by the application of biotechnology to tackle the most difficult step in creating new materials, that of C-H bond oxidation, which would otherwise require high energy and polluting chemical processes. The converted products include those from renewable sources, components of which will be converted to more degradable products with desirable properties. This positive downstream impact will be delivered by enzyme engineering and evolution methodology that is adaptable to generate the product with the most desirable combination of properties. The combination of library screening and computationally-aided design is transformative because it delivers initial diversity for discovery purposes followed by selectivity enhancements to produce the desired new entries.

香精油广泛应用于食品、医药、香水、化妆品和洗涤剂等行业。然而，在许多行业中广泛使用的许多F&F化合物在环境中不易生物降解，因此随着时间的推移而积累。这些材料的生物降解能力差，将使它们在未来无法成为F&F产品；当然，从联合利华的角度来看，这个项目的工业合作伙伴，到2030年将不会在任何产品中使用。此外，香水、化妆品和家用产品中使用的成分以及口腔护理产品中的调味料必须根据欧洲REACH法规进行注册，因此需要提供有关生物降解和生物积累的信息。由于其疏水性和化学惰性，在许多家庭和个人护理产品中具有高性能的F&F分子可以进行生物积累。这个项目的目的是在有问题的F&F分子的碳骨架中插入氧气，氧化后，可以为未来提供F&F分子。设计的细胞色素P450单加氧酶的应用可能使新分子比母分子更具水溶性、生物可利用性和可生物降解性。该方法具有良好的自然优先性，因为代谢多样化的微生物含有氧化酶，如p450，以催化C-H键氧化，以激活代谢和分解代谢过程中的化学惰性化合物。该项目包括使用现有的P450酶变体库筛选F&F分子，已知P450酶变体库将广泛的有机化合物氧化为多种产品，从制备规模（0.1g-1g）反应中分离出的产品进行表征，然后通过对接引导的最具选择性的变体诱变设计新的变体酶，以增加底物转化，可扩展性和目标产品的选择性。与联合利华的科学家合作，将研究氧化产品的皮肤致敏性，并通过气味结合试验来激活受体，以证明它们具有提供感官模式的潜力。它们的生物降解性将使用BioWin生物降解和QSAR方法以及实验生物降解分析进行评估。由于没有特定结构的数据，目前的生物降解模型可能提供较差的拟合；因此，该项目将使用生物信息学技术，根据其在水介质中的生物利用度和生物降解，为设计的新分子建立结构-功能关系。该项目完全符合BBSRC战略重点领域2：应对战略挑战-可再生资源和清洁增长的生物科学。具体来说，应用酶促工艺来创造新的高价值工业相关材料，开发更高性能的生物基工艺，以及使用技术经济分析来提供影响分析将是这个项目的核心。清洁增长的潜力是通过应用生物技术来解决创造新材料中最困难的步骤，即C-H键氧化，否则将需要高能量和污染的化学过程。转化的产品包括来自可再生资源的产品，其成分将转化为具有理想性能的更易降解的产品。这种积极的下游影响将通过酶工程和进化方法来实现，该方法可适应生成具有最理想特性组合的产品。图书馆筛选和计算辅助设计的结合是革命性的，因为它为发现目的提供了最初的多样性，然后增强了选择性，以产生所需的新条目。