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

生产药品、杀虫剂和燃料等基本化学品的传统方法效率低下、价格昂贵，而且对环境造成巨大负担。为了维持我们目前的生活水平，并向全球人口提供必要的产品，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。生物催化是一项令人兴奋的技术，已被大型化工公司广泛采用，即利用微生物（如细菌和酵母）生产大量酶（自然界的催化剂），这些酶随后被用于环保工艺，有效地将原料转化为所需产品和高价值中间体。重要的是，科学家们现在能够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，通过一个称为定向进化的过程，在实验室时间尺度上模仿达尔文的进化。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取代那些有自然对应物的化学过程。不幸的是，为了生产我们必需的化学物质，我们严重依赖于一系列非自然反应，而能够进行这些转化的酶根本不存在。这意味着，对于生产一种基本化学物质所需的典型多步骤序列，现有技术可能只允许我们用清洁的酶促过程取代一两个步骤，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有合成价值的非自然反应的酶来克服这些重大限制。自然界的酶是由只有20种标准氨基酸组成的各种组合组成的，这些氨基酸通常不适合促进非自然反应。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，这些催化剂含有具有独特性质的新型功能催化氨基酸。这些残留物是经过精心设计的，因此它们可以廉价、清洁和高效地生产，并具有必要的功能，以执行不是一个，而是许多重要的非自然反应，这些反应目前使用危险的化学试剂进行。与天然酶相比，最初产生的原始和混杂酶的活性较低，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。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，以产生适合于制造过程中使用的专门的、健壮的酶。这些酶可以作为单独的催化剂来制造高价值的中间体，或者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无害环境的方式廉价生产，它们将广泛供全球人口使用。

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.