Ultra-high throughput evolution of designer enzymes with extended amino acid alphabets

具有扩展氨基酸字母表的设计酶的超高通量进化

• 批准号: BB/X010724/1
• 负责人: Richard Obexer
• 金额: $ 51.71万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX010724%2F1
• 关键词: Ultra; throughput; evolution; designer; enzymes

With increasing societal and political acknowledgment of environment and climate issues, various industrial sectors are shifting their focus towards carbon neutral and environmentally benign technologies. As thus, biocatalysis is a rapidly expanding technology in chemical industry for the production of commodity chemicals and pharmaceuticals. In biocatalysis, enzymes, which are nature's catalysts, are repurposed for synthesizing chemicals in human devised processes. Enzymes can accelerate highly complex chemical reactions with speeds and specificities that are unrivalled by conventional chemical methods. In addition, reactions can be performed at low temperatures in aqueous solutions, unlike chemical processes that typically require high temperatures, toxic chemicals and large amounts of organic solvents. However, a major limitation for broad industrial exploitation of enzymes is that not for any desired reaction a suitable enzyme is available in nature's repertoire. As thus, enzymes that meet the specifications of organic chemists are urgently required. Rather than reengineering existing natural enzymes, bottom-up design of new enzymes is now becoming a feasible alternative. In fact, highly efficient artificial enzymes for simplistic reactions were successfully created through computation and experimental optimisation. The number of chemical mechanisms that can be designed is however inherently limited to natures repertoire of functional groups and amino acids. Through genetic code expansion, it is now possible to augment nature's amino acid alphabet with additional functionalities. In particular, through integration of amino acids that are fused to small molecule catalysts, which have been extensively explored by organic chemists, it is now possible to create enzymes with a whole new reaction scope that is unprecedented in nature. In order to design better enzymes with non-natural functionalities from scratch, it is essential to gain fundamental understanding of how artificial amino acids must be placed and further complemented within enzyme active sites. This project aims to address this by experimentally improving designer enzymes with non-natural amino acids, leading to a fundamental understanding of the true potential of augmenting nature with additional building blocks. This will be achieved through directed evolution, which is a mimic of Darwinian evolution on a laboratory time scale. Directed evolution allows for the discovery of mutations that improve enzyme activity but are rationally not predictable. Through iterative rounds of mutagenesis and selection, the activity levels of enzymes can be significantly improved. This is a laborious process as many enzyme variants have to be individually analysed, to identify rare mutations with beneficial effects. To accelerate this process, an ultra-high throughput assay will be implemented that utilises picolitre-sized droplets as reaction vessels that contain the enzyme variant and its coding gene. Droplets can be manipulated at high speeds of several thousand droplets per second and most importantly they can be sorted according to enzyme activity. With this technology at hand, it is now possible for the first time to explore the evolutionary limits of these designer enzymes for different unrelated reactions, including ester hydrolysis and an unnatural carbon-carbon bond forming reaction. In depth characterisation of the best performing enzymes will highlight functional and structural features that are essential for supporting catalysis by nonnatural amino acids. Recapitulating these findings by computational enzyme design will challenge our gained molecular understanding and give rise to new generations of improved designer enzymes. Overall, this research project will open up new avenues in development of highly active enzymes for abiological reactions with implications in biotechnology, biocatalysis and synthetic biology.

随着社会和政治对环境和气候问题的认识不断提高，各工业部门正在将重点转向碳中和和环境友好型技术。因此，生物催化是化学工业中一项迅速发展的技术，用于生产日用化学品和药品。在生物催化中，酶是自然界的催化剂，在人类设计的过程中被重新用于合成化学品。酶可以加速高度复杂的化学反应，其速度和特异性是传统化学方法无法比拟的。此外，反应可以在水溶液中在低温下进行，不像化学过程通常需要高温、有毒化学品和大量有机溶剂。然而，酶的广泛工业开发的主要限制是，对于任何期望的反应，合适的酶在自然界的库中不可用。因此，迫切需要满足有机化学家规格的酶。自下而上设计新的酶，而不是改造现有的天然酶，现在正成为一个可行的替代方案。事实上，通过计算和实验优化，成功地创造了用于简单反应的高效人工酶。然而，可以设计的化学机制的数量固有地限于官能团和氨基酸的性质库。通过遗传密码扩展，现在可以用额外的功能来增加自然界的氨基酸字母表。特别是，通过将氨基酸融合到小分子催化剂上，有机化学家已经广泛探索了这一点，现在有可能创造出具有自然界前所未有的全新反应范围的酶。为了从头开始设计具有非天然功能的更好的酶，必须对人工氨基酸必须如何放置并进一步补充酶活性位点获得基本的理解。该项目旨在通过实验改进具有非天然氨基酸的设计酶来解决这一问题，从而从根本上了解用额外的构建块增强自然的真正潜力。这将通过定向进化来实现，这是在实验室时间尺度上模仿达尔文进化论。定向进化允许发现提高酶活性但理性上不可预测的突变。通过反复的诱变和选择，可以显著提高酶的活性水平。这是一个费力的过程，因为必须单独分析许多酶变体，以识别具有有益效果的罕见突变。为了加速这一过程，将实施超高通量测定，其利用皮升大小的液滴作为含有酶变体及其编码基因的反应容器。液滴可以以每秒数千滴的高速操作，最重要的是，它们可以根据酶活性进行分选。有了这项技术，现在有可能第一次探索这些设计酶在不同的不相关反应中的进化极限，包括酯水解和非天然的碳-碳键形成反应。在深入表征的最佳表现酶将突出功能和结构特征，是支持催化的非天然氨基酸。通过计算酶设计来重述这些发现将挑战我们已经获得的分子理解，并产生新一代改进的设计酶。总的来说，该研究项目将为开发高活性酶的非生物反应开辟新的途径，并对生物技术，生物催化和合成生物学产生影响。

项目成果

Engineering Enzymes for Environmental Sustainability.

环境可持续性工程酶。

• DOI: 10.1002/anie.202309305
• 发表时间: 2023
• 期刊: Angewandte Chemie (International ed. in English)
• 作者: Radley E

Engineering Enzymes for Environmental Sustainability

环境可持续性工程酶

• DOI: 10.1002/ange.202309305
• 发表时间: 2023
• 期刊: Angewandte Chemie
• 作者: Radley E

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