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，称为天然产物。这些生物体使用生物催化剂（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，但仅用于非常小的一组酶促反应。我们设想，我们提出的工作将使生物催化方法合成某些生物碱，即含有吲哚的四氢-β-咔啉，更容易。此外，我们提出的实验的成功和失败将告知我们的小组和其他人成功的酶工程背后的原则。最后，从一个基本的角度来看，本提案中所描述的工作将提供洞察酶和相应的化学transformation.In目标1的反应机制，本提案中，我们描述的方法来修改某些酶的底物范围，以产生更广泛的利用生物催化剂。我们首先关注一种名为strictosidine 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物，在医学和工业中具有广泛的用途。最终，我们计划产生几个Pictet-Spengler催化酶，具有一系列的底物特异性和立体选择性。此外，我们有初步的数据表明，这些实验将证明如何Pictet-Spengler酶促反应使用一个独特的决议机制，以实现立体选择性。我们接下来集中在卤化酶，可以衍生吲哚环。虽然这些酶通常卤化色氨酸，我们提出的突变，将扩大底物范围的催化剂，使它们可以更广泛地利用，特别是在结合Pictet-Spengler催化剂中描述的建议。在目标2的建议，我们描述的策略，使用这些催化剂，以产生新的生物碱。我们将把这些催化剂转化到通常产生单萜吲哚生物碱的植物组织中，这样我们就可以探索产生具有改变的立体化学和卤化模式的复杂的“非天然”吲哚生物碱的前景。其次，我们探讨如何有效地表达这些酶的酵母菌株可以转换成光学活性的氯化四氢-β-咔啉生物碱的大规模简单的非手性原料。

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.