Plant natural products

Workpackage H: Tools for engineering plant natural products

Plants produce a rich and diverse array of natural products. These compounds have important ecological functions, providing protection against pests, diseases, ultraviolet-B damage and other environmental stresses. They are also exploited as pharmaceutical drugs, agrochemicals, within the food and drink industry, and for a wide variety of other industrial biotechnology applications. Although plants are potentially a tremendous source of diverse and valuable natural products, identifying the pathways for the synthesis of these compounds is more complicated than in microbes because the genomes are larger and more complex. However advances in sequencing technology coupled with the recent discovery that the genes for natural products pathways are in many cases organised in operon-like clusters within plant genomes; now makes it possible to access the genes and enzymes of specialised metabolism in plants far more readily and so to harness and exploit metabolic diversity using synthetic biology approaches.

OpenPlant is generating genetic toolkits for synthesis and modification of plant natural products.

Pathway discovery and engineering

Glowing oat seedlings                                                     image NRP-229: Aymeric Leveau (JIC), Gemma Farré Martinez (UDL) & Andrew Davis (JIC), licenced under CC-BY 4.0

Glowing oat seedlings                                                     image NRP-229: Aymeric Leveau (JIC), Gemma Farré Martinez (UDL) & Andrew Davis (JIC), licenced under CC-BY 4.0

The Osbourn lab has developed strategies for discovery of new plant natural product pathways and chemistries based on genome mining for biosynthetic gene clusters (Nützmann et al. 2016; Medema & Osbourn 2016).  The HyperTrans system for rapid transient expression of genes of interest in tobacco leaves is enabling rapid functional characterisation of new enzymes and pathways. The Osbourn lab has been focussing on developing this system for triterpene metabolic engineering and has recently generated gram-scale quantities of purified triterpene analogs, levels that are ample for both structural determination by NMR and for screening for bioactivity.  The triterpenes have numerous potential medicinal, agricultural and industrial applications, and the ability to engineer ‘designer’ molecules optimised for particular purposes using this platform opens up unprecedented opportunities for production of high-value compounds.  Aymeric Leveau (Osbourn lab) and Christian Boehm (Haseloff lab) have also been awarded an OpenPlant Fund grant for a project entitled "Engineering Marchantia polymorpha chloroplasts for the production of high-value specialised terpenes”. More information coming soon.

Strictosidine from periwinkle                                                   image NRP111: Sarah O'Connor (JIC) & Andrew Davis(JIC) , licensed under CC-BY 4.0

Strictosidine from periwinkle                                                   image NRP111: Sarah O'Connor (JIC) & Andrew Davis(JIC) , licensed under CC-BY 4.0

The O’Connor lab recently engineered the pathway for the iridoid alkaloid strictosidine from Madagascan periwinkle into yeast (Brown et al. 2015). OpenPlant researcher, Benjamin Lichman (O’Connor lab), is currently discovering additional enzymes in this pathway to generate more “building blocks” for this work. He has generated a proteome database for trichomes of iridoid-producing plants and is searching this for new candidate pathway enzymes. In a hunt for novel iridoid biosynthesis enzymes, Benjamin is identifying enzymes involved in nepatalactone biosynthesis, the key bioactive ingredient in catnip and catmint (Nepeta sp.).  In work on a different class of plant natural products, Don Nguyen (previously O’Maille lab) has engineered enzymes from the Asteraceae family into yeast to generate oxygenated sesquiterpenes (Nguyen et al., 2016).

During his time as an OpenPlant post-doc in the Martin lab, Yang Zhang, now a Principal Investigator at Sichuan University College of Life Sciences, used the HyperTrans system in the characterization of a new pathway for synthesis of root-specific flavones in Scutellaria baicalensis (Zhao et al., 2016). S. baicalensis is used in Chinese traditional medicine to treat fever, lung and liver complaints. Recent evidence suggests that other specialised flavones are important tumor suppressors. 

Regulation of biosynthesis

image: Eugenio Butelli

image: Eugenio Butelli

The Martin lab have developed vectors for transient induction of gene expression in tomato fruit, and a reliable protocol for inoculation of fruit for optimised gene expression. The Golden Braid compatible vectors are based on the HyperTrans vectors, but have been modified to improve fruit expression using the E8 promoter as well as vectors for expression driven by the 35S promoter.

Two sets of tomato introgression lines have been developed from crosses of the cultivated tomato, Solanum lycopersicum, with a wild tomato relative, Solanum pennellii, and the wild nightshade Solanum lycopersicoides. RNA-seq data from these introgression lines are being screened for Expression Quantitative Trait Loci (eQTLs) affecting expression of genes encoding enzymes of monoterpenoid biosynthesis. Candidate transcription factors are being identified from those intervals showing strong eQTLs.

Jie Li (Martin lab) and Greg Reeves (Hibberd lab) have been awarded an OpenPlant Fund grant for a collaborative project called "Hot tomato: complementation of the capsaicin biosynthetic pathway to engineer spicy tomatoes". More information coming soon.

Several biosynthetic gene clusters for natural product pathways have been identified in thalecress (Arabidopsis thaliana ) (Field and Osbourn, 2008; Nützmann et al. 2016).  Physical clustering of genes may enable pathway regulation at the level of chromatin. Hans-Wilhem Nützmann (Osbourn lab) has used a suite of A. thaliana lines affected in chromatin modification to investigate this, and has shown that the histone 2 variant H2A.Z is required for cluster expression (Nützmann and Osbourn, 2015). Wider investigations into chromatin-level regulation of biosynthetic gene clusters in A. thaliana , maize, rice and oats has revealed a role for Polycomb in cluster repression.  Building on this, we have shown that the Polycomb repressive mark histone 3 lysine 27 trimethylation hallmark associated with plant biosynthetic gene clusters can be used for pathway discovery (Yu et al, 2016).

References

Brown, S., Clastre, M., Courdavault, V., O'Connor S.E., 2015. De novo production of the plant-derived alkaloid strictosidine in yeast. Proc Natl Acad Sci 112(11), p3205-3210. DOI: 10.1073/pnas.1423555112.

Field, B. and Osbourn, A.E., 2008. Metabolic diversification – independent assembly of operon-like gene clusters in different plants. Science 320, p543-547. DOI: 10.1126/science.1154990.

Kemen, A.C., Honkanen, S., Melton, R.E., Findlay, K.C., Mugford, S.T., Hayashi, K., Haralampidis, K., Rosser, S.J., and Osbourn, A., 2014. Investigation of triterpene synthesis and regulation in oats reveals a role for β-amyrin in determining root epidermal cell patterning. Proc Natl Acad Sci. 111(23), p8679-84. DOI: 10.1073/pnas.1401553111.

Medema, M.H. and Osbourn, A., 2016. Computational genomic identification and functional reconstitution of plant natural product biosynthetic pathways. Nat Prod Rep. 33(8), p951-962. DOI: 10.1039/c6np00035e.

Nguyen, T-D., Faraldos, J.A., Vardakou, M., Salmon, M., O'Maille, P.E. and Ro, D-K., 2016. Discovery of germacrene A synthases in Barnadesia spinosa: The first committed step in sesquiterpene lactone biosynthesis in the basal member of the Asteraceae. Biochem Biophys Res Commun 479(4), p622-627. DOI: 10.1016/j.bbrc.2016.09.165.

Nützmann, H.W., Huang, A., and Osbourn, A., 2016. Plant metabolic clusters - from genetics to genomics. New Phytol. 211(3), p771-789. DOI: 10.1111/nph.13981.

Nützmann, H.W. and Osbourn, A., 2015. Regulation of metabolic gene clusters in Arabidopsis thaliana. New Phytol. 205(2), p503-510. DOI: 10.1111/nph.13189.

Yu, N., Nützmann, H.W., MacDonald, J.T., Moore, B., Field, B., Berriri, S., Trick, M., Rosser, S.J., Kumar, S.V., Freemont, P.S., and Osbourn, A., 2016. Delineation of metabolic gene clusters in plant genomes by chromatin signatures. Nucleic Acids Res. 44(5), p2255-2265. DOI: 10.1093/nar/gkw100.

Zhao, Q., Zhang, Y., Wang, G., Hill, L., Weng, J.K., Chen, X.Y., Xue, H., and Martin, C., 2016. A specialized flavone biosynthetic pathway has evolved in the medicinal plant, Scutellaria baicalensis. Science Advances 2(4), e1501780. DOI: 10.1126/sciadv.1501780.