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Unlocking Triterpenoid Structural Diversity and Bioactivity through Genome Mining

Periodic Reporting for period 1 - TRIGEM (Unlocking Triterpenoid Structural Diversity and Bioactivity through Genome Mining)

Reporting period: 2016-10-01 to 2018-09-30

Plants produce myriads of bio-active small molecules/ natural products (NPs) (e.g. terpenoids), yet the diversity and bio-activity of plant NPs are still largely untapped and access to bio-active NPs remains challenging.
The triterpenoids are one of the largest and most structurally complex plant natural products (NPs). They are widespread in the Plant Kingdom and have a huge array of structures and numerous associated biological activities. They have important roles in plant defence and signalling. They are also exploited by humans as food supplements, drugs and cosmetics across various sectors.2 In the past the discovery and production of triterpenoids has relied mainly on isolation from extracts of natural resources and subsequent structural elucidation and chemical synthesis. These methods suffer from low efficiency and high cost, and are not environmentally sustainable. We undertake a greener and more sustainable but as yet largely unexplored synthetic biology-based approach that involves genome mining and metabolic engineering to synthesise structural variants of triterpenoids, with a view to discovering novel structures with biological activities for various potential applications in a rapid manner. This project will lead to discovery of novel bioactive triterpenoids that can potentially be developed into commercial products to benefit the society. It will shed new light on the biosynthetic pathways of triterpenoids by uncovering new genes and enzymes, opening up opportunities for production of important triterpenoids via further metabolic manipulation in plant-based ‘green factories’ or in microbes.

The overall objectives of TRIGEM are as follows:

1. To mine for triterpene genes encoding novel TCC, CYP and other tailoring enzymes. The 15 new TTC/CYP genes referred to above are already available for use in this project. We will also mine the sequenced genomes of other plants that produce bioactive triterpenoids to augment this resource, focusing on genes that are physically
clustered and so likely to be functionally connected.
2. To functionally analyse the selected coding sequences with predicted functions in triterpene synthesis by expressing them in yeast and N. benthamiana.
3. To identify new compounds generated by heterologous expression of these selected genes by GC-MS or LC-MS as appropriate. Novel compounds will be isolated by chromatography and their structures determined by NMR.
4. To evaluate the bioactivities of isolated compounds using readily available assay models such as cytotoxicity and standard antifungal assays and other commercially available models.
We have carried out bioinformatics analysis on the publicly available high-quality plant genomes using two recently developed algorithms including the plantiSMASH (1) and PlantClusterFinder (2) algorithm to mine for candidate triterpene gene clusters that could potentially encode new natural products. We also used coexpression analysis to check if the genes in the candidate clusters are coexpressed in order to select top candidates. We identified four highly coexpressed triterpene gene clusters in Arabidopsis thaliana. We used agrobacterium-mediated transient expression in Nicotiana benthamiana leaves to investigate the functions of the gene clusters. We combinatorially coexpressed the cluster genes in Nicotiana benthamiana and found that the thalianol cluster genes are fully functional and one of the nearby gene ACT2 could act on other gene clusters, forming a metabolic network. We further found three other coexpressed but not clustered genes in the A. thaliana genome that completed the biosynthesis of arabidiol and thalianol pathway leading to formation of previously unkonwn triterpenoids in A. thaliana roots. These non-clustered genes encode promiscuous enzymes that act on different triterpene cluster products and further expand the chemical diversity of this triterpene metabolic network. This biosynthetic network has the capacity of synthesising over 50 previously unknown metabolites and over 15 have been isolated and fully characterised following scale up expression of the corresponding genes in N. benthamiana in the project.

The discovery of this biosynthetic network has allowed us to investigate its potential ecological functions. We found that A. thaliana mutants affected in the biosynthesis of metabolites from the metabolic network have altered root microbiota compared to the wild type, indicating that metabolites can have impacts on selecting root bacteria. In vitro bioassays with purified compounds reveal diverse interaction modes of the metabolites with root microbiota members. Selective growth-promoting and inhibitory activities of root metabolites against diverse bacterial isolates and biochemical transformations and utilization of metabolites by bacteria was observed. Taken together the evidences of sequencing and in vitro bioassays, we have demonstrated that plant specialised metabolites can selectively modulate Arabidopsis root microbiota members and shape the root microbiota. Our findings pave way for further investigation of the functions of the novel triterpenoids and the mechanisms that underpin their interactions with bacteria. This work has been written for submission to a high impact journal and will be published in due course.
1. This project has generated a synthetic biology-based approach for the rapid discovery of previously unknown terpenoids directly from plant genomes. This approach combines bioinformatics, heterologous expression and natural product chemistry techniques and proved to be very effective and rapid for natural product discovery. Such an approach will be of great value to the field of natural product research and greatly accelerate the discovery and utilisation of plant NPs. The effectiveness of this pipeline is demonstrated by the discovery of a biosynthetic repertoire of sesterterpenes (a rare subclass of plant terpenes) in the course of the project.

2. The discovery of a triterpene biosynthetic network for the synthesis of unknown triterpenes has demonstrated the chemical diversity plants have evolved to harbour and represented an examplar for biosynthetic pathway evolution.

3. The discovery that the triterpene biosynthetic network has a profound impact on the establishment of A. thaliana root microbiota builds foundation for engineering plant microbiota by engineering root specialised metabolism. Their activities of the pathway metabolites on selective modulation of bacterial growth indicates that these compounds may have great application potential (i.e. as antibiotics or proliferation agents).

4. The diverse modulation patterns of this metabolic network on root bacteria have also highlighted the significant interactions of root bacteria with hydrophobic triterpenes, opening questions for the mechanisms that underline such interactions.

5. Our findings have built solid foundation for further investigation of the biological functions of this metabolic network and bring a step forward towards understanding causation of microbiota establishment.