Unlocking the chemical diversity of plant natural product pathways: Accessing the limonoids

总体而言，植物可以生物合成大量的天然产物。其中许多是由特定植物物种或谱系产生的专门代谢物。这些代谢物可能发挥重要的生态功能，例如，通过提供保护免受害虫和病原体的侵袭。植物代谢的多样化很可能是对不同生态位生存适应的反映。在这一提议中，我们特别对一大类结构复杂的植物天然产物柠檬苦素类化合物感兴趣。柠檬苦素类化合物是由十字花科(柑橘属)和梅花科(红木属)家族成员产生的。芸香科柠檬苦素类化合物对柑橘果实的苦味有贡献，也具有药用潜力，而苦瓜科柠檬苦素类化合物(如沙兰宁、印木素)因其抗昆虫活性而引起人们的兴趣。印木素(从印楝树中分离出来)因其强大的昆虫拒食活性和环境友好特性(全身摄取、可降解性、对哺乳动物、鸟类、鱼类和有益昆虫的低毒)而闻名。印度芒果提取物(含有大量印楝素)在作物保护方面有着悠久的传统和商业使用历史(例如，NeemAzal-T/S、Trifolio-M GmbH)。尽管2007年报道了印楝素的全化学合成，但这一过程涉及71个步骤，总收率为0.00015%。因此，化学合成印木素在工业规模上是不可行的。同样，化学合成芸香科柠檬苦素类化合物，如柠檬苦素(从香叶醇经过35步得到)也不太可能在商业上可行。因此，目前利用梧桐科柠檬苦素进行作物保护主要依赖于对其种子的提取。同样，芸香科柠檬苦素类化合物的潜在健康益处仍然局限于饮食消费。柠檬苦素类化合物属于被称为三萜的主要天然产品类别。然而，这些化合物是非正则的，因为它们的结构不同寻常。三萜类化合物通常有一个30碳的支架。相比之下，基本的柠檬苦素支架只有26个碳，据信是由一个30碳的“原柠檬苦素”前体通过丢失4个碳和支架重排形成的，机制尚不清楚。26个碳柠檬苦素类支架含氧量很高，可以作为简单的环完整结构存在，也可以作为环结构被破坏的高度修饰的衍生物存在。虽然在经典的30碳三萜生物合成的基因和酶的表征方面已经取得了相当大的进展，但柠檬苦素的生物合成途径在很大程度上仍然不清楚，直到我们最近于2019年发表的关于阐明形成原柠檬苦素的早期途径的论文，还没有关于柠檬苦素合成的生物合成基因的报道。确定柠檬苦素生物合成所需的生物合成基因将使我们能够理解前所未有的生物化学，这种生物化学创造了在这一重要的植物天然产物家族中发现的化学多样性。代谢工程为培育具有更强抗虫性的作物提供了机会，也提供了通过在异源宿主中表达来生产高价值柠檬苦素(例如，用于制药)的机会。然而，要实现这一点，必须首先对负责柠檬苦素生物合成和多样化的酶进行表征。在这项提案中，我们描述了我们将如何发现植物如何合成和多样化结构复杂的柠檬苦素。我们将获得催化这些过程的酶，并将它们部署到我们的瞬时植物表达平台中，以支持柠檬苦素支架的多样化。然后我们将研究这些分子的特征，这些分子决定了它们的抗昆虫活性。

Collectively, plants biosynthesise a vast array of natural products. Many of these are specialized metabolites that are produced by particular plant species or lineages. These metabolites likely perform important ecological functions, for example by providing protection against attach by pests and pathogens. Plant metabolic diversification is likely to be a reflection of adaptation to survival in different ecological niches.Within this proposal, we are especially interested in a large and structurally complex group of plant natural products known as limonoids. Limonoids are produced by members of the Rutaceae (Citrus) and Meliaceae (Mahogany) families. Rutaceae limonoids contribute to bitterness in citrus fruit and also have pharmaceutical potential, while Meliaceae limonoids (e.g. salannin, azadirachtin) are of interest because of their anti-insect activity. Azadirachtin (isolated from the neem tree, Azadirachta indica) is particularly well known for its potent insect antifeedant activity and environmentally friendly properties (systemic uptake, degradability, low toxicity to mammals, birds, fish, and beneficial insects). Extracts from A. indica seeds (which contain high quantities of azadirachtin) have a long history of traditional and commercial (e.g., NeemAzal-T/S, Trifolio-M GmbH) use in crop protection. Although the total chemical synthesis of azadirachtin was reported in 2007, this involved 71 steps and gave 0.00015% total yield. Chemical synthesis of azadirachtin is therefore not practical at industrial scale. Similarly, chemical synthesis of Rutaceae limonoids such as limonin (achieved in 35 steps from geraniol) is also unlikely to be commercially viable. Therefore, at present the use of Meliaceae limonoids for crop protection relies on extraction of A. indica seeds. Similarly, the potential health benefits of Rutaceae limonoids remain restricted to dietary consumption.Limonoids belong to the major class of natural products known as triterpenes. However, these compounds are non-canonical because of their unusual structures. Triterpenes typically have a 30-carbon scaffold. In contrast, the basic limonoid scaffold has only 26 carbons, which is believed to be formed from a 30-carbon 'protolimonoid' precursor by loss of four carbons and scaffold rearrangement by as yet unknown mechanisms. The 26 carbon limonoid scaffolds are heavily oxygenated and can exist as simple ring-intact structures or a highly modified derivatives in which the ring structure is broken. While considerable advances have been made in characterisation of the genes and enzymes for the biosynthesis of classical 30-carbon triterpenes, the routes to the biosynthesis of limonoids remain largely unknown, and until our recent publication in 2019 on the elucidation of the early pathway up to protolimonoids, no biosynthetic genes for limonoid production had been reported. Identifying the biosynthetic genes required for limonoid biosynthesis will enable us to understand the unprecedented biochemistry that creates the chemical diversity found within this important family of plant natural products. Metabolic engineering offers opportunities to generate crop plants with enhanced insect resistance and also to produce high-value limonoids (e.g., for pharmaceutical use) by expression in heterologous hosts. However, to achieve this the enzymes responsible for limonoid biosynthesis and diversification must first be characterized. In this proposal, we describe how we will discover how plants synthesise and diversify structurally complex limonoids. We will garner the enzymes that catalyse these processes and deploy them into our transient plant expression platform to support limonoid scaffold diversification. We will then investigate the features of these molecules that determine their anti-insect activities.