Engineering Biocatalysts for the Next Generation of omega-Transaminase Processes

手性胺在天然产物中普遍存在，通常表现出强大的生物活性。这种手性胺基序也经常出现在药物化合物和化学构建块中，这意味着开发环境友好和可持续的途径来生产这些重要的基序是非常可取的。大自然通过高度特化的酶的作用合成了这些复杂而有价值的分子。这些天然催化剂能够从简单的起始材料进行极其高效的生物合成，并安装具有特殊选择性的功能基团。化学催化剂经常被设计成模仿酶的作用，并且常常能够达到令人印象深刻的选择性。然而，与酶不同，涉及这些催化剂的过程通常涉及高温、次优pH、有机溶剂和复杂的纯化方法。被称为ω -转氨酶（TAs）的酶催化将市售或容易获得的原料转化为高价值的胺。这些生物催化剂需要一个额外的供体分子来提供胺官能团。这个供体最终转化为副产物，形成所需的胺产物。然而，该反应是自由可逆的，除非该副产物从反应中去除，否则所需胺的低产量将被分离出来，因为酶将更容易催化逆反应以再生起始材料。一些优雅的方法已经报道了去除这种酮副产物，并允许获得相当数量的手性胺。这些策略包括添加昂贵的酶或使用极大量的胺供体，并结合技术上具有挑战性的酮副产物的去除。其中一种方法依赖于广泛修饰的TA，目前用于抗糖尿病药物西格列汀的工业合成。然而，这种方法远非有效，而且这种经过大量修饰的TA生物催化剂的开发极具挑战性，迫切需要更可持续的策略来执行这些生物转化和开发合适的酶催化剂。本研究将建立在我们实验室最近报告的工作基础上，该工作描述了迄今为止执行涉及TAs的最有效的生物转化方法。该方法的成功是由于副产品的自发沉淀，它不能再生起始材料。这种聚合物也是高度着色的，并且允许开发一种有效的高通量筛选策略，从而能够快速识别活性酶。我们现在的重点是进一步优化工艺，使其更适合工业应用。具体来说，将使用低成本的胺供体分子，以与我们之前报道的方法类似的方式自发地从反应中去除。我们还将应用一种简单的高通量筛选策略来协助天然酶的基因工程，以增加它们可以催化的反应范围，并使它们适合工业规模的合成。本研究开发的酶将为中小规模和工业规模生产一种最重要的化合物类别提供经济、可持续和环境中性的方法。

Chiral amines are prevalent in natural products, which often display potent biological activity. Such chiral amine motifs are also frequently found in pharmaceutical drug compounds and chemical building blocks meaning that the development of environmentally benign and sustainable routes to produce these important motifs is extremely desirable. Nature synthesizes these complex and valuable molecules through the action of highly specialized enzymes. These natural catalysts enable an extremely efficient biosynthesis from simple starting materials, installing functional groups with exceptional levels of selectivity. Chemical catalysts are frequently designed to mimic the action of enzymes and are often capable of achieving impressive selectivity. However, unlike enzymes, processes involving these catalysts usually involve high temperatures, sub-optimal pH, organic solvent and complex purification methods. Enzymes called omega-transaminases (TAs) catalyze the conversion of commercially available or easily accessible starting materials to high-value amines. These biocatalysts require an additional donor molecule to provide the amine functional group. This donor is ultimately converted to a by-product and the desired amine product is formed. However, the reaction is freely reversible and unless this by-product is removed from the reaction, low yields of the desired amine will be isolated, as the enzyme will more readily catalyse the reverse reaction to regenerate starting materials. A number of elegant approaches have been reported which remove this ketone by-product and allow access to appreciable quantities of the chiral amine. These strategies include the addition of expensive enzymes or the use of extremely large quantities of the amine donor in combination with the technically challenging removal of ketone by-products. One such approach, which relies on an extensively modified TA, is currently used for the industrial synthesis of the antidiabetic drug compound, sitagliptin. However, the approach is far from efficient and the development of this heavily modified TA biocatalyst was enormously challenging, highlighting an immediate need for more sustainable strategies for performing these biotransformations and for developing suitable enzyme catalysts. This research will build upon recent work reported in our laboratory that describes arguably the most efficient approach to date for performing biotransformations involving TAs. The success of the approach is due to spontaneous precipitation of the by-product, which cannot regenerate starting materials. This polymer is also highly colored and has allowed the development of an effective high-throughput screening strategy that enables the rapid identification of active enzymes. Our focus now is to optimize the process further and make it more suitable for industrial application. Specifically, low cost amine donor molecules will be used that are spontaneously removed from the reaction in a similar way to our previously reported method. We will also apply a simple high-throughput screening strategy to assist in the genetic engineering of natural enzymes in order to increase the scope of the reactions that they can catalyze and make them suitable for industrial scale synthesis. The enzymes developed in this study will enable cost-effective, sustainable and environmentally neutral methods for the small/medium and industrial scale production of one of the most important compound classes.