Periodic Reporting for period 1 - TRIGEM (Unlocking Triterpenoid Structural Diversity and Bioactivity through Genome Mining)
Okres sprawozdawczy: 2016-10-01 do 2018-09-30
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.
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.
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.