Generation of Alkaloid Diversity Using Biocatalysts

环境中的生物——植物、细菌和真菌——制造各种各样的复杂分子，这些分子被称为天然产物。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。从广义上讲，通过了解酶的工作原理，我们可以提高操纵和“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，但只用于非常小的酶促反应。我们设想，我们提出的工作将使合成某些生物碱的生物催化方法更容易获得，即含有四氢- β -碳碱的吲哚。此外，我们提出的实验的成功和失败将告诉我们的团队和其他人成功的酶工程背后的原理。最后，从基础的角度来看，本提案中描述的工作将为酶和相应的化学转化的反应机制提供见解。在本提案的目标1中，我们描述了修改某些酶的底物范围以产生更广泛利用的生物催化剂的方法。我们首先关注一种叫做strictosidine synthase的植物酶，它可以催化Pictet-Spengler缩合反应，这种反应被广泛用于生成合成和天然产物，在医学和工业中有着广泛的用途。最终，我们计划生成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，我们有初步的数据表明，这些实验将证明Pictet-Spengler酶促反应如何使用独特的分辨机制来实现立体选择性。我们接下来的重点是卤化酶，可以衍生吲哚环。虽然这些酶通常卤化色氨酸，但我们建议突变将扩大催化剂的底物范围，以便它们可以更广泛地使用，特别是与提案中前面描述的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产生具有改变立体化学和卤化模式的复杂“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 -碳碱生物碱。

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.