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.

生产药品、农药和燃料等基本化学品的传统方法效率低下、成本高昂，并对环境造成巨大负担。为了维持我们当前的生活水平并向全球人口提供必需的产品，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工艺。一项令人兴奋的技术已被主要化学公司广泛采用，称为生物催化，利用微生物（例如细菌和酵母）来生产大量酶（天然催化剂），随后将其用于环保工艺中以有效地转化原材料转化为所需产品和高价值中间体。重要的是，科学家现在能够通过称为定向进化的过程快速修改和优化酶的自然功能和特性，使其适合其所需的应用，该过程在实验室时间尺度上模仿达尔文进化论。原则上，通过定向进化，最终应该有可能用更环保、更有效的生物催化替代品来取代那些存在天然对应物的化学过程。不幸的是，为了生产我们的必需化学物质，我们严重依赖一系列非自然反应，而能够进行这些转化的酶根本不存在。这意味着，对于生产基本化学品所需的典型多步骤序列，现有技术可能只能让我们用清洁的酶促过程代替一两个步骤，其余步骤仍然依赖于危险的化学反应。我的研究旨在通过创造能够有效催化具有合成价值的非天然反应的酶来克服这些重大限制。大自然的酶是由仅二十种标准氨基酸结构单元的各种组合组成的，这些结构单元通常不适合促进非自然反应。自然反应。为了实现创造人工酶的宏伟目标，我们将为微生物提供必要的工具来生产生物催化剂，其中含有具有独特性能的新功能性催化氨基酸。这些残留物经过精心设计，可以廉价、清洁、高效地生产，并且具有必要的功能，可以进行目前使用危险化学试剂进行的多种重要非自然反应，而不是一种反应。与天然酶相比，最初产生的原始且混杂的酶预计表现出较低的活性，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。然而，定向进化提供了一种理想的方法来快速优化这些生物催化剂的活性，以生产适合在制造过程中使用的专门的、强大的酶。这些酶可用作独立催化剂来制造高价值中间体，也可用于多步骤生物催化途径来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，因此将可供全球人口广泛使用。

项目成果

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

设计一种用于 Morita-Baylis-Hillman 反应的高效且对映选择性酶。

• DOI: http://dx.10.1038/s41557-021-00833-9
• 发表时间: 2022
• 期刊: Nature chemistry
• 影响因子: 21.8
• 作者: Crawshaw R

A Non-Canonical Nucleophile Unlocks a New Mechanistic Pathway in a Designed Enzyme

非典型亲核试剂在设计的酶中解锁了新的机制途径

• DOI: http://dx.10.21203/rs.3.rs-2922796/v1
• 发表时间: 2023
• 作者: Crossley A

Biocatalysis

生物催化

• DOI: http://dx.10.1038/s43586-021-00044-z
• 发表时间: 2021
• 期刊: Nature Reviews Methods Primers
• 作者: Bell E

Directed evolution of an efficient and thermostable PET depolymerase

高效且热稳定的 PET 解聚酶的定向进化

• DOI: http://dx.10.1038/s41929-022-00821-3
• 发表时间: 2022
• 期刊: Nature Catalysis
• 影响因子: 37.8
• 作者: Bell E

Engineering enzyme activity using an expanded amino acid alphabet.

使用扩展的氨基酸字母表设计酶活性。

• DOI: http://dx.10.1093/protein/gzac013
• 发表时间: 2023
• 期刊: PEDS
• 作者: Birch