Generation of Alkaloid Diversity Using Biocatalysts

环境中的有机体--植物、细菌和真菌--会产生各种各样的被称为天然产物的复杂分子。这些生物使用生物催化剂(酶)从简单的起始材料构建复杂的分子。广义地说，通过了解酶是如何工作的，我们可以提高我们操纵和重新设计生物催化剂的能力，以便我们可以将它们用于我们自己的目的。生物催化剂已被证明是一种非常环保的策略，可以在实验室和工业规模上生产分子，但只被用于非常小的一组酶反应。我们设想，我们提议的工作将使生物催化方法更容易合成某些生物碱，即含有四氢-β-卡宾的吲哚。此外，我们提出的实验的成功和失败将使我们团队和其他人了解成功的酶工程背后的原理。最后，从根本的角度来看，这项建议中描述的工作将提供对酶和相应的化学转化的反应机理的洞察。在这项建议的目标1中，我们描述了修改某些酶的底物范围以产生更广泛使用的生物催化剂的方法。我们首先关注一种名为Strectosidine Synthase的植物酶，它催化Pictet-Spengler缩合反应，该反应被广泛用于产生合成和天然产品，在医药和工业中具有广泛的用途。最终，我们计划生产几种具有一系列底物特异性和立体选择性的Pictet-Spengler催化酶。此外，我们有初步数据表明，这些实验将展示Pictet-Spengler酶反应如何使用独特的拆分机制来实现立体选择性。接下来，我们将重点放在可以衍生吲哚环的卤化酶上。虽然这些酶通常会卤化色氨酸，但我们提出了一些突变，这些突变将扩大催化剂的底物范围，使其能够得到更广泛的利用，特别是与提案前面描述的Pictet-Spengler催化剂结合使用。在提案的目标2中，我们描述了使用这些催化剂生成新型生物碱的策略。我们将把这些催化剂转化为通常产生单萜吲哚生物碱的植物组织，这样我们就可以探索通过改变立体化学和卤化模式来产生复杂的“非天然”吲哚生物碱的前景。其次，我们探索了表达这些酶的酵母菌株如何有效地将简单的非手性起始物质大规模转化为光学活性的氯化四氢-β-咔啉生物碱。

Organisms in the environment - plants, bacteria and fungi - make a wide variety of complex molecules known as natural products. These organisms use biocatalysts (enzymes) to construct complicated molecules from simple starting materials. Broadly speaking, by understanding how enzymes work, we can improve our ability to manipulate and "re-engineer" biocatalysts so that we can use them for our own purposes. Biocatalysts have proven to be an exceptionally environmentally friendly strategy to produce molecules on both laboratory and industrial scales, but have only been utilised for a very small group of enzymatic reactions. We envision that our proposed work will make biocatalytic approaches to synthesizing certain alkaloids, namely the indole containing tetrahdro-beta-carbolines, more accessible. Additionally, the successes and failures of our proposed experiments will inform our group and others about the principles behind successful enzyme engineering. Finally, from a fundamental perspective, the work described in this proposal will provide insight into the reaction mechanism of the enzymatic and corresponding chemical transformations.In Objective 1 of this proposal we describe approaches to modify the substrate scope of certain enzymes to generate more broadly utilisable biocatalysts. We first focus on a plant enzyme called strictosidine synthase, which catalyzes the Pictet-Spengler condensation, a reaction that is widely used to generate both synthetic and natural products with a wide range of uses in medicine and industry. Ultimately we plan to generate several Pictet-Spengler catalyzing enzymes that have a range of substrate specificities and stereoselectivities. Moreover, we have preliminary data that suggest that these experiments will demonstrate how the Pictet-Spengler enzymatic reaction uses a unique resolution mechanism to achieve stereoselectivity. We next focus on halogenating enzymes that can derivatize the indole ring. While these enzymes normally halogenate tryptophan, we propose mutations that will expand the substrate scope of the catalysts so that they can be more broadly utilized, particularly in combination with the Pictet-Spengler catalysts described earlier in the proposal.In Objective 2 of the proposal, we describe strategies to use these catalysts to generate novel alkaloids. We will transform these catalysts into plant tissue that normally produces monoterpene indole alkaloids, so that we can explore the prospects of generating complex "unnatural" indole alkaloids with altered stereochemistry and halogenation patterns. Secondly, we explore how effectively yeast strains expressing these enzymes can convert simple achiral starting materials into optically active chlorinated tetrahydro-beta-carboline alkaloids on a large scale.