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 natural products in plants.

Enzyme building blocks

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). Use of the HyperTrans transient plant expression platform is enabling rapid functional characterisation of new candidate genes. The Osbourn lab has been focussing on developing the potential of this expression system for triterpene metabolic engineering and has recently been able to generate milligram/gram quantities of purified triterpene analogs, levels that are ample for both structural determination by NMR and for screening for bioactivity. This genome mining approach is also enabling the discovery of enzymes that make entirely new classes of plant natural products, including the first plant sesterterpene synthases.

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 produced the plant derived iridoid alkaloid strictosidine in 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 now searching this database for new candidate pathway enzymes. In a hunt for novel iridoid biosynthesis enzymes, Benjamin is identifying the enzymes involved in nepatalactone biosynthesis, the key bioactive ingredient in catnip and catmint (Nepeta sp.).

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 traditional Chinese medicine to treat fever and lunch and liver complaints. Recent evidence has suggested that specialised flavones, wogonin and baicalein, may also act as a tumor suppressors.

Optimised enzymes for terpene production

Barnadesia spinosa, Instituto_Humbolt on Flickr, licensed under CC BY-NC-ND 2.0

Barnadesia spinosa, Instituto_Humbolt on Flickr, licensed under CC BY-NC-ND 2.0

Don Nguyen (previously O’Maille lab) investigated the use of characterized cytochrome P450s in sesquiterpenoid metabolism to produce customized small molecule products. Using a plug-and-play approach based on the understanding of the involved enzymes, means the natural metabolic diversity can be used to produce desired and/or novel products. The work focused on a set of cytochrome P450s that oxidize sesquiterpenes in the Asteraceae family. Homology models of the P450s were generated and analysed. Libraries of mutants have been designed based on the enzymes’ differences, constructed and evaluated.

This work included investigation of the evolution of the Asteraceae family’s germacrene A synthase, the enzyme that produces the substrate for and may be evolutionarily coupled with the cytochrome P450s (Nguyen et al., 2016).

Control by transcription factors

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.

Gene clusters under the lens                                     image: Nützmann, Huang & Osbourn, 2016

Gene clusters under the lens                                     image: Nützmann, Huang & Osbourn, 2016

Hans-Wilhelm Nützmann (Osbourn lab) used yeast-one-hybrid assays to identify several transcription factors that bind to promoters of genes within a root-expressed triterpene metabolic gene cluster (the thalianol cluster) characterised in the Osbourn lab from Arabidopsis thaliana (Field and Osbourn, 2008). In collaboration with Alain Goossens lab (VIB), he was able to show that one of the identified candidate transcription factors activates promoters of metabolic gene clusters in transactivation assays.

Knock-out and overexpression lines of A. thaliana were used to investigate the roles of candidate transcription factors on cluster expression. Hans showed that the histone 2 variant H2A.Z is required for expression of metabolic gene clusters in A. thaliana (Nützmann and Osbourn, 2015). Wider investigations into chromatin regulation led to the identification of a chromatin mark that delineates silenced metabolic gene clusters on plant chromosomes. Knowledge about this mark was successfully applied to identify metabolic gene clusters in different plant species (Yu et al, 2016).

Synthetic metabolic clusters for crops

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 previously shown that the promoters for a specialized metabolite gene cluster from oat (the avenacin cluster) retain their characteristic expression patterns (in the epidermal cells of root meristems) when introduced into diverse plant species as promoter-reporter constructs (Kemen et al., 2014). Building on this, a synthetic gene cluster has been generated in which promoters from the avenacin pathway have been used to successfully drive the expression of a three-gene pathway for a plant defence compound (dhurrin) from sorghum in Arabidopsis thaliana roots. This proof of concept experiment will be the first step towards engineering synthetic clusters for the synthesis of other types of compound and for expression of other multigene traits (e.g. nitrogen fixation genes).

Aymeric Leveau (Osbourn lab) and Christian Boehm (Haseloff lab) have 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.

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.