Biocatalytic Asymmetric Reduction Animation Using Reductive Aminases (RedAms)

该项目与制药和精细化工行业保持一致。挑战在于开发用于酮和醛与胺的对映选择性还原胺化的稳健的生物催化剂，从而能够直接一步合成生物学上重要的靶分子。本计画的目的是开发还原胺酶（RedAm）生物催化剂应用于有机合成。具体而言，我们将工程化现有的野生型RedAm，使它们具有扩大的底物范围和改善的稳定性和活性。该项目福尔斯完全属于BBSRC的战略重点工业生物技术范围。酮的还原胺化是有机合成中最强大和最常用的反应之一，可以使各种酮与1 o和2 o胺共轭。鉴于产物通常是手性的事实，人们越来越希望开发该反应的不对称变体，特别是涉及催化而不是化学计量使用试剂。在生物催化领域中，目前存在两种可能的方法来催化该反应;（i）使用工程化章鱼碱还原酶（OctDH）和（ii）使用工程化亚胺还原酶（IRED）。Codexis/Merck已部分解决了使用OctDH的前一种方法，尽管迄今为止仅出现了专利。已经报道了基于IRED的替代方法，尽管这些反应涉及过量的IRED酶和高的胺与酮的比率（>20：1）以实现适度的转化。IRED用于还原胺化的低效率是由于它们催化亚胺还原但不催化亚胺形成的事实。最近曼彻斯特和合作者发现了一个新的IRED分支，称为还原胺酶（RedAms），其催化具有高活性（kcat高达300 min-1）和周转数（TTN高达200，000）的对映选择性还原胺化。稳态动力学已经揭示了底物（NADPH;酮;胺）的有序结合，并且重要地确定RedAms催化亚胺形成以及亚胺还原。通过一系列的配体：蛋白质结构，曼彻斯特还开发了催化机制的工作模型，并确定了诱变的关键目标。学生的目标是利用这些初步的令人兴奋的结果，（i）使用原型曲霉RedAm鉴定新型RedAm（ii）使用一组确定的胺和酮表征和比较RedAm，以确定底物范围以及对映选择性和转化率的水平;（iii）在配体的存在下确定新型RedAm的结构，以识别用于突变研究的活性位点氨基酸，以及（iv）进行基于速率的工艺优化并探索RedAm催化反应的工艺强化，以确定限制RedAm催化还原胺化的前景更大规模。

This project aligns with the pharmaceutical and fine chemical sector. The challenge is to develop robust biocatalysts for the enantioselective reduction amination of ketones and aldehydes with amines, thereby enabling the direct one-step synthesis of biologically important target molecules. The aim of this project is to develop reductive aminase (RedAm) biocatalysts for applications in organic synthesis. Specifically we shall engineer existing wild-type RedAms such that they possess expanded substrate scope and improved stability and activity. This project falls squarely within the Industrial Biotechnology remit which is a strategic priority of BBSRC.The reductive amination of ketones is one of the most powerful and frequently employed reactions in organic synthesis enabling a wide range of ketones to be conjugated to 1o and 2o amines. Given the fact that the products are often chiral there is an increasing desire to develop asymmetric variants of this reaction, particularly involving catalysis rather than stoichiometric use of reagents. In the field of biocatalysis there are currently two possible approaches to catalysing this reaction; (i) using engineered octopine dehydrogenases (OctDHs) and (ii) using engineered imine reductases (IREDs). The former approach employing OctDHs has been partly addressed by Codexis/Merck although to date only a patent has appeared. The alternative approach based upon IREDs has been reported although these reactions involve excessively large quantities of IRED enzyme and high ratios of amine to ketone (>20:1) in order to achieve moderate conversions. The low efficiency of IREDs for reductive amination is due to the fact that they catalyse imine reduction but not imine formation. Recently Manchester and collaborators have discovered a new sub-clade of IREDs termed reductive aminases (RedAms) which catalyse enantioselective reductive amination with high activity (kcat up to 300 min-1) and turnover number (TTN up to 200,000). Steady-state kinetics have revealed ordered binding of substrates (NADPH;ketone;amine) and importantly established that RedAms catalyse imine formation, as well as imine reduction. Through a series of ligand:protein structures Manchester have also developed a working model for the catalytic mechanism and have identified key targets for mutagenesis. The student shall aim to capitalize on these preliminary exciting results by (i) identifying novel RedAms using the prototype Aspergillus oryzae RedAm (AspRedAm) and the putitative RedAms from Pfizer as a guide sequence; (ii) characterizing and comparing the RedAms using a defined panel of amines and ketones in order to determine substrate scope together with levels of enantioselectivity and conversion; (iii) determining structures of novel RedAms, in the presence of ligands, to identify active-site amino acids for mutagenesis studies and (iv) carrying out rate-based process optimization and exploring process intensification of RedAm catalyzed reactions to determine limit the prospects for carrying out RedAm catalyzed reductive aminations on a larger scale.