Design and Evolution of Artificial Enzymes with Non-Canonical Organocatalytic Residues

具有非典型有机催化残基的人工酶的设计和进化

• 批准号: BB/M027023/1
• 负责人: Anthony Green
• 金额: $ 118.2万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM027023%2F1
• 关键词: Design; Evolution; Artificial; Enzymes; Non

Traditional methods of producing essential chemicals such as medicines, pesticides and fuels are inefficient, expensive and create a huge burden on the environment. In order to maintain our current standard of living and to make essential products available to the global population, we urgently need to develop clean, efficient and sustainable manufacturing technologies to replace traditional chemical processes. An exciting technology that is already being widely adopted by major chemical companies is called Biocatalysis, whereby microorganisms (e.g. bacteria and yeast) are used to produce large amounts of enzymes (nature's catalysts) which are subsequently used in environmentally friendly processes to efficiently convert starting materials into desired products and high-value intermediates. Importantly, scientists are now able to quickly modify and optimize the natural function and properties of an enzyme to make it suitable for its desired application through a process called directed evolution, which mimics Darwinian evolution on a laboratory timescale. In principal, through directed evolution it should ultimately be possible to replace those chemical processes for which there is a natural counterpart with greener, more efficient biocatalytic alternatives. Unfortunately, to produce our essential chemicals we rely heavily on a series of non-natural reactions, and enzymes capable of performing these transformations simply do not exist. This means that for a typical multi-step sequence required to produce an essential chemical, existing technology may only allow us to replace one or two steps with a clean enzymatic process, with the remaining steps still reliant on hazardous chemical reactions. My research aims to overcome these significant limitations by creating enzymes which are able to efficiently catalyze synthetically valuable, non-natural reactions.Nature's enzymes are made up from various combinations of only twenty standard amino acid building blocks which are generally not suitable to promote non-natural reactions. To achieve the ambitious goal of creating artificial enzymes, we will supply microorganisms with the necessary tools to produce biocatalysts which contain new functional, catalytic amino acids with unique properties. These residues are carefully designed so that they can be produced cheaply, cleanly and efficiently and have the necessary functionality to perform not one, but many important non-natural reactions which are currently carried out using hazardous chemical reagents. The primitive and promiscuous enzymes initially produced are expected to display low activity compared with natural enzymes, since they have not been subjected to millions of years of natural evolutionary processes to optimize their function. However, directed evolution offers an ideal method to rapidly optimize the activity of these biocatalysts to produce specialized, robust enzymes suitable for use in manufacturing processes. These enzymes can be used as standalone catalysts to make high-value intermediates or in multi-step biocatalytic pathways to produce new and existing medicines, pesticides and fuels. Since these essential products will be produced cheaply in an environmentally friendly manner, they will be widely accessible for use by the global population.

产生必需化学物质（例如药物，农药和燃料）的传统方法效率低下，昂贵，并给环境带来了巨大负担。为了维持我们当前的生活水平并使全球人口可用的必需品可用，我们迫切需要开发清洁，高效和可持续的制造技术来取代传统的化学过程。主要化学公司已经广泛采用的一种令人兴奋的技术称为生物催化，从而使用微生物（例如细菌和酵母）生产大量酶（自然的催化剂），随后在环境友好的过程中使用，以有效地将起始物质转化为所需的产品和高价值中间人。重要的是，科学家现在能够快速修改和优化酶的自然功能和特性，以使其适合其所需的应用程序，该过程称为“定向进化”，该过程模仿了达尔文的进化在实验室时间表上。本金中，通过定向进化，最终应该可以替代那些自然具有更绿色，更有效的生物催化替代方案的化学过程。不幸的是，为了产生我们的必需化学物质，我们在很大程度上依赖一系列的非天然反应，而能够执行这些转化的酶根本不存在。这意味着，对于产生必需化学物质所需的典型多步序列，现有技术可能只会使我们可以用干净的酶促过程替换一个或两个步骤，其余步骤仍然依赖有害化学反应。我的研究旨在通过创建能够有效催化合成有价值的非天然反应的酶来克服这些重大局限性。天气的酶是由仅二十种标准氨基酸构件的各种组合组成的，这些氨基酸构建块通常不适合促进非自然反应。为了实现创建人工酶的雄心勃勃的目标，我们将为微生物提供必要的工具，以生产具有具有独特特性的新功能性催化氨基酸的生物催化剂。这些残留物经过精心设计，以便可以廉价，清洁，有效地生产它们，并具有必要的功能，而不是执行一个，而是许多重要的非天然反应，这些反应目前是使用有害化学试剂进行的。与天然酶相比，最初产生的原始酶和混杂酶的活性低，因为它们没有经过数百万年的自然进化过程来优化其功能。但是，定向进化提供了一种理想的方法，可以快速优化这些生物催化剂的活性，以生产适合在制造工艺中使用的专业，健壮的酶。这些酶可以用作独立催化剂，以制造高价值中间体或在多步生物催化途径中生产新的和现有的药物，农药和燃料。由于这些基本产品将以环保的方式廉价生产，因此全球人口将广泛使用。

项目成果

Strategies for designing biocatalysts with new functions

设计具有新功能的生物催化剂的策略

• DOI: 10.1039/d3cs00972f
• 发表时间: 2024
• 期刊: Chemical Society Reviews
• 影响因子: 46.2
• 作者: Bell E

Biocatalysis

• DOI: 10.1038/s43586-021-00044-z
• 发表时间: 2021-06-24
• 期刊: NATURE REVIEWS METHODS PRIMERS
• 作者: Bell, Elizabeth L.;Finnigan, William;Flitsch, Sabine L.
• 通讯作者: Flitsch, Sabine L.

Engineering an efficient and enantioselective enzyme for the Morita-Baylis-Hillman reaction.

• DOI: 10.1038/s41557-021-00833-9
• 发表时间: 2022-03
• 期刊: Nature chemistry
• 影响因子: 21.8
• 作者: Crawshaw R;Crossley AE;Johannissen L;Burke AJ;Hay S;Levy C;Baker D;Lovelock SL;Green AP
• 通讯作者: Green AP

Engineering enzyme activity using an expanded amino acid alphabet.

• DOI: 10.1093/protein/gzac013
• 发表时间: 2023-01-21
• 期刊: PROTEIN ENGINEERING DESIGN & SELECTION
• 影响因子: 2.4
• 作者: Birch-Price, Zachary;Taylor, Christopher J.;Ortmayer, Mary;Green, Anthony P.
• 通讯作者: Green, Anthony P.

Directed Evolution of an Efficient and Thermostable PET Depolymerase

• DOI: 10.26434/chemrxiv-2021-mcjh6
• 发表时间: 2021-01-01
• 期刊: ChemRxiv
• 作者: Bell, E.;Smithson, R.;Day, P.
• 通讯作者: Day, P.