Directed Evolution of Enantiocomplementary Malonate Decarboxylases.

目前，药品、农用化学品和其他精细化学品的制造严重依赖于合成化学方法，这些方法使用有害的溶剂、试剂和催化剂以及不可再生的石化前体，对环境有严重的不利影响。因此，人们寻求以生物技术为基础的替代工艺，以更经济和环境上可持续的方式制造对维持人类健康和生活质量至关重要的化学品。工业生物技术发展的核心是获得具有特定特性的新酶，这些酶可用于在无害环境的条件下催化将可再生前体转化为所需产品。迄今为止，酶的工业应用依赖于有限数量的已建立的酶，这些酶催化的转化范围很窄。然而，最近的基因组测序已经导致从天然来源发现了更广泛的酶。此外，新的定向进化技术允许酶的性质，甚至它们催化的反应被改变和优化特定的过程。最近，我们解决了第一个结构，并确定了脱羧酶（AMDase）的详细机制，该酶催化丙二酸衍生物的二氧化碳损失（脱羧）生成手性羧酸。在这个项目中，我们的目标是利用这些结构和机制的见解来开发更强大的脱羧酶，这些脱羧酶可以提供更广泛的结构多样化的羧酸，这是制药、农用化学品和其他产品生产中特别常见的中间体。新的脱羧酶也很有吸引力，因为底物可以由丙二酸产生，丙二酸是一种来自可再生资源（发酵）的天然前体。手性羧酸是单对映体（两种可能的非重叠镜像立体异构体中的一种），其可得性尤其对药品生产至关重要。通常，酶只产生两种可能的对映体中的一种，如果需要相反的对映体，这就有问题了。因此，我们将使用定向进化技术来开发对映互补酶。这样，一种酶可以产生一种对映体（左旋分子），而另一种酶可以产生相反的对映体（右旋分子）。我们将与全球最大的化学品制造商巴斯夫的工业合作伙伴一起，为生产关键的医药中间体定制新的对映互补脱羧酶。这包括用于制造非甾体抗炎药（来自布洛芬家族）的手性羧酸，抗血小板药物氯吡格雷（世界第二大畅销药物）和用于治疗心脏病的卡托普利。已知脱羧酶所属的酶家族是混杂的，并且可以催化比脱羧更广泛的反应，包括外消旋和异构化。我们的目标是进一步探索这种酶家族的滥交性，以期开发具有工业重要性的替代反应。

Currently, the manufacture of pharmaceuticals, agrochemicals and other fine chemicals relies heavily on synthetic chemical methods, which use deleterious solvents, reagents and catalysts as well as non-renewable petrochemical precursors, which have serious detrimental environmental impact. Consequently, alternative biotechnology based processes are sought for the more economic and environmentally sustainable manufacture of those chemicals that are essential to maintain human health and quality of life. Central to the development of industrial biotechnologies is the availability of new enzymes, with tailored properties, that can be used to catalyse the transformation of renewable precursors into the required products under environmentally benign conditions. To date, industrial applications of enzymes have relied on a limited number of established enzymes, which catalyse a narrow range of transformations. However, recent genome sequencing has led to the discovery of a wider range of enzymes from natural sources. In addition new directed evolution technologies allow the properties of enzymes and even the reactions they catalyse to be altered and optimised for specific processes. Recently we solved the first structure and determined the detailed mechanism of a decarboxylase enzyme (AMDase) that catalyses the loss of carbon dioxide (decarboxylation) from malonic acid derivatives to generate chiral carboxylic acids. In this project, we aim to use these structural and mechanistic insights to develop more powerful decarboxylase enzymes that can provide access to a much wider range of structurally diverse carboxylic acids, which are particularly common intermediates in production of pharmaceuticals, agrochemicals and other products. The new decarboxylase enzymes are also attractive because the substrates can be generated from malonic acid, a natural precursor derived from renewable sources (fermentation). The availability of chiral carboxylic acids, which are single enantiomers (one of two possible stereoisomers that are non-superimposable mirror images) is of critical importance particularly for pharmaceutical production. Typically, enzymes only produce one of the two possible enantiomers, which is problematic if the opposite enantiomer is required. We will therefore use directed evolution technologies to develop enzymes that are enantiocomplementary. In this way, one enzyme can be used to produce one enantiomer (left-handed molecule), whilst another enzyme can produce the opposite enantiomer (right-handed molecule). Along with our industrial partners at BASF, who are the world's largest chemical manufacturers, we will tailor the new enantiocomplementary decarboxylases for production of key pharmaceutical intermediates. This includes chiral carboxylic acids used to manufacture non-steroidal anti-inflammatory drugs (from the ibuprofen family), the antiplatelet agent clopidogrel (the world's second-best selling drug) and captopril which is used to treat cardiac conditions. The family of enzymes to which the decarboxylases belong are known to be promiscuous, and can catalyse a wider range of reactions than decarboxylations, including racemisations and isomerisations. We aim to further explore the promiscuity of this enzyme family, with a view to developing alternative reactions that would also be of industrial importance.