Publications

The John Innes Centre Publications Repository contains details of all publications resulting from our researchers.

The repository also includes Open Access publications, which can be identified by the icons found on search results.

 Green open access publications are marked by the PDF icon. Click on the publication title, or the PDF icon, and read a pre-print PDF version of the publication.  Gold open access publications have the gold open padlock icon. You can read the full version of these papers on the publishing journal’s website without a subscription. 

The creation of this publications repository was funded by BBSRC.

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Huang A. C., Hong Y. J., Bond A. D., Tantillo D. J., Osbourn A. (2017)

Diverged Plant Terpene Synthases Reroute the Carbocation Cyclization Path towards the Formation of Unprecedented 6/11/5 and 6/6/7/5 Sesterterpene Scaffolds.

Angewandte Chemie (International ed. in English)

Publisher's version: 10.1002/anie.201711444

ID: 57911

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Abstract

Sesterterpenoids are a relatively rare class of plant terpenes. Sesterterpene synthase (STS)-mediated cyclization of the linear C25 isoprenoid precursor geranylfarnesyl diphosphate (GFPP) defines sesterterpene scaffolds. So far only a very limited number of STSs have been characterized. The discovery of three new plant STSs is reported that produce a suite of sesterterpenes with unprecedented 6/11/5 and 6/6/7/5 fused ring systems when transiently co-expressed with a GFPP synthase in Nicotiana benthamiana. Structural elucidation, feeding experiments, and quantum chemical calculations suggest that these STSs catalyze an unusual cyclization path involving reprotonation, intramolecular 1,6 proton transfer, and concerted but asynchronous bicyclization events. The cyclization is diverted from those catalyzed by the characterized plant STSs by forming unified 15/5 bicyclic sesterterpene intermediates. Mutagenesis further revealed a conserved amino acid residue implicated in reprotonation

Boobier S., Osbourn A., Mitchell J. B. O. (2017)

Can human experts predict solubility better than computers?

Journal of cheminformatics (9) 63

Publisher's version: 10.1186/s13321-017-0250-y

ID: 57890

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Abstract

In this study, we design and carry out a survey, asking human experts to predict the aqueous solubility of druglike organic compounds. We investigate whether these experts, drawn largely from the pharmaceutical industry and academia, can match or exceed the predictive power of algorithms. Alongside this, we implement 10 typical machine learning algorithms on the same dataset. The best algorithm, a variety of neural network known as a multi-layer perceptron, gave an RMSE of 0.985 log S units and an R2 of 0.706. We would not have predicted the relative success of this particular algorithm in advance. We found that the best individual human predictor generated an almost identical prediction quality with an RMSE of 0.942 log S units and an R2 of 0.723. The collection of algorithms contained a higher proportion of reasonably good predictors, nine out of ten compared with around half of the humans. We found that, for either humans or algorithms, combining individual predictions into a consensus predictor by taking their median generated excellent predictivity. While our consensus human predictor achieved very slightly better headline figures on various statistical measures, the difference between it and the consensus machine learning predictor was both small and statistically insignificant. We conclude that human experts can predict the aqueous solubility of druglike molecules essentially equally well as machine learning algorithms. We find that, for either humans or algorithms, combining individual predictions into a consensus predictor by taking their median is a powerful way of benefitting from the wisdom of crowds.

Huang A. C., Kautsar S. A., Hong Y. J., Medema M. H., Bond A. D., Tantillo D. J., Osbourn A. (2017)

Unearthing a sesterterpene biosynthetic repertoire in the Brassicaceae through genome mining reveals convergent evolution.

Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A. 2017 Jul 3. pii: 201705567. doi: 10.1073/pnas.1705567114. ) Epub ahead of print

Publisher's version: 10.1073/pnas.1705567114

ID: 56890

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Abstract

Sesterterpenoids are a rare terpene class harboring untapped chemodiversity and bioactivities. Their structural diversity originates primarily from the scaffold-generating sesterterpene synthases (STSs). In fungi, all six known STSs are bifunctional, containing C-terminal trans-prenyltransferase (PT) and N-terminal terpene synthase (TPS) domains. In plants, two colocalized PT and TPS gene pairs from Arabidopsis thaliana were recently reported to synthesize sesterterpenes. However, the landscape of PT and TPS genes in plant genomes is unclear. Here, using a customized algorithm for systematically searching plant genomes, we reveal a suite of physically colocalized pairs of PT and TPS genes for the biosynthesis of a large sesterterpene repertoire in the wider Brassicaceae. Transient expression of seven TPSs from A. thaliana, Capsella rubella, and Brassica oleracea in Nicotiana benthamiana yielded fungal-type sesterterpenes with tri-, tetra-, and pentacyclic scaffolds, and notably (-)-ent-quiannulatene, an enantiomer of the fungal metabolite (+)-quiannulatene. Protein and structural modeling analysis identified an amino acid site implicated in structural diversification. Mutation of this site in one STS (AtTPS19) resulted in premature termination of carbocation intermediates and accumulation of bi-, tri-, and tetracyclic sesterterpenes, revealing the cyclization path for the pentacyclic sesterterpene (-)-retigeranin B. These structural and mechanistic insights, together with phylogenetic analysis, suggest convergent evolution of plant and fungal STSs, and also indicate that the colocalized PT-TPS gene pairs in the Brassicaceae may have originated from a common ancestral gene pair present before speciation. Our findings further provide opportunities for rapid discovery and production of sesterterpenes through metabolic and protein engineering.

Reed J., Stephenson M. J., Miettinen K., Brouwer B., Leveau A., Brett P., Goss R. J. M., Goossens A., O'Connell M. A., Osbourn A. (2017)

A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like molecules.

Metabolic Engineering (42) 185-193

Publisher's version: 10.1016/j.ymben.2017.06.012

ID: 56918

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Abstract

Plants are an excellent source of drug leads. However availability is limited by access to source species, low abundance and recalcitrance to chemical synthesis. Although plant genomics is yielding a wealth of genes for natural product biosynthesis, the translation of this genetic information into small molecules for evaluation as drug leads represents a major bottleneck. For example, the yeast platform for artemisinic acid production is estimated to have taken >150 person years to develop. Here we demonstrate the power of plant transient transfection technology for rapid, scalable biosynthesis and isolation of triterpenes, one of the largest and most structurally diverse families of plant natural products. Using pathway engineering and improved agro-infiltration methodology we are able to generate gram-scale quantities of purified triterpene in just a few weeks. In contrast to heterologous expression in microbes, this system does not depend on re-engineering of the host. We next exploit agro-infection for quick and easy combinatorial biosynthesis without the need for generation of multi-gene constructs, so affording an easy entrée to suites of molecules, some new-to-nature, that are recalcitrant to chemical synthesis. We use this platform to purify a suite of bespoke triterpene analogs and demonstrate differences in anti-proliferative and anti-inflammatory activity in bioassays, providing proof of concept of this system for accessing and evaluating medicinally important bioactives. Together with new genome mining algorithms for plant pathway discovery and advances in plant synthetic biology, this advance provides new routes to synthesize and access previously inaccessible natural products and analogs and has the potential to reinvigorate drug discovery pipelines.

Owen C., Patron N. J., Huang A., Osbourn A. (2017)

Harnessing plant metabolic diversity.

Current Opinion in Chemical Biology (40) 24-30

Publisher's version: 10.1016/j.cbpa.2017.04.015

ID: 56498

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Abstract

Advances in DNA sequencing and synthesis technologies in the twenty-first century are now making it possible to build large-scale pipelines for engineering plant natural product pathways into heterologous production species using synthetic biology approaches. The ability to decode the chemical potential of plants by sequencing their transcriptomes and/or genomes and to then use this information as an instruction manual to make drugs and other high-value chemicals is opening up new routes to harness the vast chemical diversity of the Plant Kingdom. Here we describe recent progress in methods for pathway discovery, DNA synthesis and assembly, and expression of engineered pathways in heterologous hosts. We also highlight the importance of standardization and the challenges associated with dataset integration in the drive to build a systematic framework for effective harnessing of plant metabolic diversity.

Kautsar S. A., Suarez Duran H. G., Blin K., Osbourn A., Medema M. H. (2017)

plantiSMASH: automated identification, annotation and expression analysis of plant biosynthetic gene clusters.

Nucleic Acids Research (epub ) epub

Publisher's version: 10.1093/nar/gkx305

ID: 56210

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Abstract

Plant specialized metabolites are chemically highly diverse, play key roles in host-microbe interactions, have important nutritional value in crops and are frequently applied as medicines. It has recently become clear that plant biosynthetic pathway-encoding genes are sometimes densely clustered in specific genomic loci: biosynthetic gene clusters (BGCs). Here, we introduce plantiSMASH, a versatile online analysis platform that automates the identification of candidate plant BGCs. Moreover, it allows integration of transcriptomic data to prioritize candidate BGCs based on the coexpression patterns of predicted biosynthetic enzyme-coding genes, and facilitates comparative genomic analysis to study the evolutionary conservation of each cluster. Applied on 48 high-quality plant genomes, plantiSMASH identifies a rich diversity of candidate plant BGCs. These results will guide further experimental exploration of the nature and dynamics of gene clustering in plant metabolism. Moreover, spurred by the continuing decrease in costs of plant genome sequencing, they will allow genome mining technologies to be applied to plant natural product discovery. The plantiSMASH web server, precalculated results and source code are freely available from http://plantismash.secondarymetabolites.org.

Zhou Y., Ma Y., Zeng J., Duan L., Xue X., Wang H., Lin T., Liu Z., Zeng K., Zhong Y., Zhang S., Hu Q., Liu M., Zhang H., Reed J., Moses T., Liu X., Huang P., Qing Z., Liu X., Tu P., Kuang H., Zhang Z., Osbourn A., Ro D. K., Shang Y., Huang S. (2016)

Convergence and divergence of bitterness biosynthesis and regulation in Cucurbitaceae.

Nature plants (2) 16183

Publisher's version: 10.1038/nplants.2016.183

ID: 55347

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Abstract

Differentiation of secondary metabolite profiles in closely related plant species provides clues for unravelling biosynthetic pathways and regulatory circuits, an area that is still underinvestigated. Cucurbitacins, a group of bitter and highly oxygenated tetracyclic triterpenes, are mainly produced by the plant family Cucurbitaceae. These compounds have similar structures, but differ in their antitumour activities and ecophysiological roles. By comparative analyses of the genomes of cucumber, melon and watermelon, we uncovered conserved syntenic loci encoding metabolic genes for distinct cucurbitacins. Characterization of the cytochrome P450s (CYPs) identified from these loci enabled us to unveil a novel multi-oxidation CYP for the tailoring of the cucurbitacin core skeleton as well as two other CYPs responsible for the key structural variations among cucurbitacins C, B and E. We also discovered a syntenic gene cluster of transcription factors that regulates the tissue-specific biosynthesis of cucurbitacins and may confer the loss of bitterness phenotypes associated with convergent domestication of wild cucurbits. This study illustrates the potential to exploit comparative genomics to identify enzymes and transcription factors that control the biosynthesis of structurally related yet unique natural products.

Salmon M., Thimmappa R., Minto R., Melton R., Hughes R., O'Maille P., Hemmings A. M., Osbourn A. (2016)

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases.

Proceedings of the National Academy of Sciences of the United States of America (113(30)) E4407-E4414

Publisher's version: 10.1073/pnas.1605509113

ID: 53714

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Abstract

Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply.

Abstract

The last decade has seen the first major discoveries regarding the genomic basis of plant natural product biosynthetic pathways. Four key computationally driven strategies have been developed to identify such pathways, which make use of physical clustering, co-expression, evolutionary co-occurrence and epigenomic co-regulation of the genes involved in producing a plant natural product. Here, we discuss how these approaches can be used for the discovery of plant biosynthetic pathways encoded by both chromosomally clustered and non-clustered genes. Additionally, we will discuss opportunities to prioritize plant gene clusters for experimental characterization, and end with a forward-looking perspective on how synthetic biology technologies will allow effective functional reconstitution of candidate pathways using a variety of genetic systems

Nuetzmann H. W., Huang A., Osbourn A. (2016)

Tansley Review: Plant metabolic clusters - from genetics to genomics

New Phytologist (211) 771789

Publisher's version: 10.1111/nph.13981

ID: 53072

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Abstract

Plant natural products are of great value for agriculture, medicine and a wide range of other industrial applications. The discovery of new plant natural product pathways is currently being revolutionized by two key developments. First, breakthroughs in sequencing technology and reduced cost of sequencing are accelerating the ability to find enzymes and pathways for the biosynthesis of new natural products by identifying the underlying genes. Second, there are now multiple examples in which the genes encoding certain natural product pathways have been found to be grouped together in biosynthetic gene clusters within plant genomes. These advances are now making it possible to develop strategies for systematically mining multiple plant genomes for the discovery of new enzymes, pathways and chemistries. Increased knowledge of the features of plant metabolic gene clusters – architecture, regulation and assembly – will be instrumental in expediting natural product discovery. This review summarizes progress in this area.

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., Osbourn A. (2016)

Delineation of metabolic gene clusters in plant genomes by chromatin signatures.

Nucleic Acids Research (44) 1-11

Publisher's version: 10.1093/nar/gkw100

ID: 52626

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Abstract

Plants are a tremendous source of diverse chemicals, including many natural product-derived drugs. It has recently become apparent that the genes for the biosynthesis of numerous different types of plant natural products are organized as metabolic gene clusters, thereby unveiling a highly unusual form of plant genome architecture and offering novel avenues for discovery and exploitation of plant specialized metabolism. Here we show that these clustered pathways are characterized by distinct chromatin signatures of histone 3 lysine trimethylation (H3K27me3) and histone 2 variant H2A.Z, associated with cluster repression and activation, respectively, and represent discrete windows of co-regulation in the genome. We further demonstrate that knowledge of these chromatin signatures along with chromatin mutants can be used to mine genomes for cluster discovery. The roles of H3K27me3 and H2A.Z in repression and activation of single genes in plants are well known. However, our discovery of highly localized operon-like co-regulated regions of chromatin modification is unprecedented in plants. Our findings raise intriguing parallels with groups of physically linked multi-gene complexes in animals and with clustered pathways for specialized metabolism in filamentous fungi.

Osbourn A., Morgan J. (2016)

Editorial overview: Plant biotechnology

Current Opinion in Biotechnology (37) 153154

Publisher's version: 10.1016/j.copbio.2015.12.006

ID: 52572

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Abstract

In summary, we believe that the articles in this issue give timely and inspiring insights into advances, challenges and opportunities in some of the key aspects of plant biotechnology. Plants are globally cultivated at extremely low cost, harvested on the giga-tonne scale, and routinely used to produce the widest range of products from fibers, wood, oils, sugar, fine chemicals, drugs to food. They represent an enormous and largely untapped source of enzymes and biochemicals for food, health, and industrial biotechnology applications. They can also be used as workhorses for the heterologous production of vaccines and other high value products. The continued development of ever more sophisticated tools and technologies for investigating, engineering, and improving plants coupled with the array of potential applications of plant biotechnology makes this a very exciting field to be in.

Osbourn A., Morgan J. (2016)

Editorial overview: Plant biotechnology

Current Opinion in Biotechnology (37) 153-154

Publisher's version: 10.1016/j.copbio.2015.12.006

ID: 52432

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Abstract

In summary, we believe that the articles in this issue give timely and inspiring insights into advances, challenges and opportunities in some of the key aspects of plant biotechnology. Plants are globally cultivated at extremely low cost, harvested on the giga-tonne scale, and routinely used to produce the widest range of products from fibers, wood, oils, sugar, fine chemicals, drugs to food. They represent an enormous and largely untapped source of enzymes and biochemicals for food, health, and industrial biotechnology applications. They can also be used as workhorses for the heterologous production of vaccines and other high value products. The continued development of ever more sophisticated tools and technologies for investigating, engineering, and improving plants coupled with the array of potential applications of plant biotechnology makes this a very exciting field to be in.

Alagna F., Geu-Flores F., Kries H., Panara F., Baldoni L., O'Connor S. E., Osbourn A. (2015)

Identification and characterization of the iridoid synthase involved in oleuropein biosynthesis in olive (Olea europaea) fruits.

Journal of Biological Chemistry (291) 55425554

Publisher's version: 10.1074/jbc.M115.701276

ID: 52575

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Abstract

The secoiridoids are the main class of specialized metabolites present in olive (Olea europaea L.) fruit. In particular, the secoiridoid oleuropein strongly influences olive oil quality due to its bitterness, which is a desirable trait. In addition, oleuropein possesses a wide range of pharmacological properties, including antioxidant, antiinflammatory, and anti-cancer activities. In accordance, obtaining high-oleuropein varieties is a main goal of molecular breeding programs. Here we use a transcriptomic approach to identify candidate genes belonging to the secoiridoid pathway in olive. From these candidates, we have functionally characterized the olive homologue of iridoid synthase (OeISY), an unusual terpene cyclase that couples an NAD(P)H-dependent 1,4-reduction step with a subsequent cyclization, and we provide evidence that OeISY likely generates the monoterpene scaffold of oleuropein in olive fruits. OeISY, the first pathway gene characterised for this type of secoiridoid, is a potential target for breeding programs in a high value secoiridoid-accumulating species.

Osbourn A. (2015)

Anne Osbourn.

New Phytologist (208) 23-5

Publisher's version: 10.1111/nph.13616

ID: 51848

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Abstract

Anne Osbourn is a Project Leader at the John Innes Centre and Director of the Norwich Research Park Industrial Biotechnology and Bioenergy Alliance. She is also a Trustee of the New Phytologist Trust and an Editor of New Phytologist. Her research focuses on plant-derived natural products – function, synthesis, mechanisms of metabolic diversification and metabolic engineering. An important advance from the Osbourn laboratory has been the discovery that genes for specialized metabolic pathways are organized in ‘operon-like’ clusters in plant genomes, a finding that has opened up new opportunities for elucidation of new pathways and chemistries through genome mining. Anne has developed and coordinates the Science, Art and Writing (SAW) Initiative, a cross-curricular science education outreach programme (www.sawtrust.org).

Patron N. J., Orzaez D., Marillonnet S., Warzecha H., Matthewman C., Youles M., Raitskin O., Leveau A., Farré G., Rogers C., Smith A., Hibberd J., Webb A. A., Locke J., Schornack S., Ajioka J., Baulcombe D. C., Zipfel C., Kamoun S., Jones J. D., Kuhn H., Robatzek S., Van Esse H. P., Sanders D., Oldroyd G., Martin C., Field R., O'Connor S., Fox S., Wulff B., Miller B., Breakspear A., Radhakrishnan G., Delaux P. M., Loqué D., Granell A., Tissier A., Shih P., Brutnell T. P., Quick W. P., Rischer H., Fraser P. D., Aharoni A., Raines C., South P. F., Ané J. M., Hamberger B. R., Langdale J., Stougaard J., Bouwmeester H., Udvardi M., Murray J. A., Ntoukakis V., Schäfer P., Denby K., Edwards K. J., Osbourn A., Haseloff J. (2015)

Standards for plant synthetic biology: a common syntax for exchange of DNA parts.

New Phytologist (208) 13-9

Publisher's version: 10.1111/nph.13532

ID: 55372

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Abstract

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.

Medema M. H., Kottmann R., Yilmaz P., Cummings M., Biggins J. B., Blin K., de Bruijn I., Chooi Y. H., Claesen J., Coates R. C., Cruz-Morales P., Duddela S., Düsterhus S., Edwards D. J., Fewer D. P., Garg N., Geiger C., Gomez-Escribano J. P., Greule A., Hadjithomas M., Haines A. S., Helfrich E. J., Hillwig M. L., Ishida K., Jones A. C., Jones C. S., Jungmann K., Kegler C., Kim H. U., Kötter P., Krug D., Masschelein J., Melnik A. V., Mantovani S. M., Monroe E. A., Moore M., Moss N., Nützmann H. W., Pan G., Pati A., Petras D., Reen F. J., Rosconi F., Rui Z., Tian Z., Tobias N. J., Tsunematsu Y., Wiemann P., Wyckoff E., Yan X., Yim G., Yu F., Xie Y., Aigle B., Apel A. K., Balibar C. J., Balskus E. P., Barona-Gómez F., Bechthold A., Bode H. B., Borriss R., Brady S. F., Brakhage A. A., Caffrey P., Cheng Y. Q., Clardy J., Cox R. J., Donadio S., Donia M. S., van der Donk W. A., Dorrestein P. C., Doyle S., Driessen A. J., Ehling-Schulz M., Entian K. D., Fischbach M. A., Gerwick L., Gerwick W. H., Gross H., Gust B., Hertweck C., Höfte M., Jensen S. E., Ju J., Katz L., Kaysser L., Klassen J. L., Keller N. P., Kormanec J., Kuipers O. P., Kuzuyama T., Kyrpides N. C., Kwon H. J., Lautru S., Lavigne R., Lee C. Y., Linquan B., Liu X., Liu W., Luzhetskyy A., Mahmud T., Mast Y., Méndez C., Metsä-Ketelä M., Micklefield J., Mitchell D. A., Moore B. S., Moreira L. M., Müller R., Neilan B. A., Nett M., Nielsen J., O'Gara F., Oikawa H., Osbourn A., Osburne M. S., Ostash B., Payne S. M., Pernodet J. L., Petricek M., Piel J., Ploux O., Raaijmakers J. M., Salas J. A., Schmitt E. K., Scott B., Seipke R. F., Shen B., Sherman D. H., Sivonen K., Smanski M. J., Sosio M., Stegmann E., Süssmuth R. D., Tahlan K., Thomas C. M., Tang Y., Truman A. W., Viaud M., Walton J. D., Walsh C. T., Weber T., van Wezel G. P., Wilkinson B., Willey J. M., Wohlleben W., Wright G. D., Ziemert N., Zhang C., Zotchev S. B., Breitling R., Takano E., Glöckner F. O. (2015)

Minimum Information about a Biosynthetic Gene cluster

Nature Chemical Biology (11) 625-31

Publisher's version: 10.1038/nchembio.1890

ID: 51847

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Abstract

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters.  Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit.  To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.

Patron N. J., Orzaez D., Marillonnet S., Warzecha H., Matthewman C., Youles M., Raitskin O., Leveau A., Farré G., Rogers C., Smith A., Hibberd J., Webb A. A., Locke J., Schornack S., Ajioka J., Baulcombe D. C., Zipfel C., Kamoun S., Jones J. D., Kuhn H., Robatzek S., Van Esse H. P., Sanders D., Oldroyd G., Martin C., Field R., O'Connor S., Fox S., Wulff B., Miller B., Breakspear A., Radhakrishnan G., Delaux P. M., Loqué D., Granell A., Tissier A., Shih P., Brutnell T. P., Quick W. P., Rischer H., Fraser P. D., Aharoni A., Raines C., South P. F., Ané J. M., Hamberger B. R., Langdale J., Stougaard J., Bouwmeester H., Udvardi M., Murray J. A., Ntoukakis V., Schäfer P., Denby K., Edwards K. J., Osbourn A., Haseloff J. (2015)

Standards for plant synthetic biology: a common syntax for exchange of DNA parts.

New Phytologist (208) 1319

Publisher's version: 10.1111/nph.13532

ID: 51594

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Abstract

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.

Hao d. a. . C., Chen S. L., Osbourn A., Kontogianni V. G., Liu L. W., Jordán M. J. (2015)

Temporal transcriptome changes induced by methyl jasmonate in Salvia sclarea.

Gene (558) 41-53

Publisher's version: 10.1016/j.gene.2014.12.043

ID: 49228

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Abstract

Salvia sclarea is a traditional medicinal and aromatic plant that grows in Europe and produces various economically important compounds, including phenylpropanoid derivatives and terpenoids. Methyl jasmonate (MeJA) is commonly used to elicit plant stress responses. However, how MeJA enhances production of secondary metabolites in S. sclarea is not well understood. We performed a genome-wide analysis of temporal gene expression in S. sclarea leaves and roots. The transcriptome profiles 0, 10 and 26 h after MeJA treatment were analyzed by Illumina RNA-Seq. A total of 16,142 isogenes (average length 866bp; N50 1035bp) were obtained by de novo assembly of 35,757,567 raw sequencing reads. When these sequencing reads were mapped onto the assembled Unigenes, 3236, 2792 and 798 Unigenes were found to be expressed differentially between 0 and 10h, 0 and 26 h, and 10 and 26h, respectively. These included many secondary metabolite biosynthesis, stress and defense-related genes. A qRT-PCR analysis confirmed the expression profiles of selected differentially expressed genes (DEGs) revealed by RNA-Seq data, and also extended our analysis of differential gene expression to 73 h. Our investigations revealed temporal differences in the responses of S. sclarea to MeJA treatment. MeJA treatment induced the expression of a large number of genes involved in phenylpropanoid biosynthesis, especially between 0 and 10h, and 0 and 26 h. Additionally, many genes encoding transcription factors, cytochrome P450s, glycosyltransferases, methyltransferases and transporters were shown to respond to MeJA elicitation. DEGs related to structural molecule activity and cell death showed a significant temporal variation. A chromatographic analysis of metabolites at 26h, 73h and six days after MeJA treatment indicated that these transcriptomic changes precede MeJA-induced changes in secondary metabolite content. This study sheds light on the molecular mechanisms of MeJA elicitation and is helpful in understanding how exogenous MeJA treatment mediates extensive plant transcriptome reprogramming/remodeling. Our results can be utilized to characterize genes related to secondary metabolism and their regulation, and in breeding S. sclarea for desirable chemotypes.

Ma P., Liu J., Osbourn A., Dong J., Liang Z. (2015)

Regulation and metabolic engineering of tanshinone biosynthesis

RSC Advances (5 ) 18137-18144

Publisher's version: 10.1039/C4RA13459A

ID: 49302

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Abstract

Salvia miltiorrhiza (Chinese name: dânshçn) is used in traditional Chinese medicine for the treatment of cardiovascular and cerebrovascular diseases. The tanshinones represent the most important biological active class of compounds present in dânshçn extracts. They are synthesized via either the cytoplasmic mevalonate or the plastidial 2-C-methyl-D-erythritol-4-phosphate pathway. Here, we summarize recent discoveries regarding the mechanisms underlying tanshinone biosynthesis and how the process is regulated. Tanshinone accumulation in planta is affected by a range of elicitors and by the composition of the culture medium. Its production in hairy root cultures can be enhanced by the over-expression of genes encoding 1-deoxy-D-xylulose 5-phosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, geranylgeranyl diphosphate synthase and allene oxide cyclase. The pathway leading to the biosynthesis of the tanshinone precursors miltiradiene and ferruginol, has been engineered in yeast.

Moses T., Pollier J., Shen Q., Soetaert S., Reed J., Erffelinck M., Van Nieuwerburgh F. C., Vanden Bossche R., Osbourn A., Thevelein J. M., Deforce D., Tang K., Goossens A. (2015)

OSC2 and CYP716A14v2 Catalyze the Biosynthesis of Triterpenoids for the Cuticle of Aerial Organs of Artemisia annua.

Plant Cell (27) 286-301

Publisher's version: 10.1105/tpc.114.134486

ID: 48914

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Abstract

Artemisia annua is widely studied for its ability to accumulate the antimalarial sesquiterpenoid artemisinin. In addition to producing a variety of sesquiterpenoids, A. annua also accumulates mono-, di-, and triterpenoids, the majority of which are produced in the glandular trichomes. A. annua also has filamentous trichomes on its aerial parts, but little is known of their biosynthesis potential. Here, through a comparative transcriptome analysis between glandular and filamentous trichomes, we identified two genes, OSC2 and CYP716A14v2, encoding enzymes involved in the biosynthesis of specialized triterpenoids in A. annua. By expressing these genes in Saccharomyces cerevisiae and Nicotiana benthamiana, we characterized the catalytic function of these proteins and could reconstitute the specialized triterpenoid spectrum of A. annua in these heterologous hosts. OSC2 is a multifunctional oxidosqualene cyclase that produces a-amyrin, ß-amyrin, and d-amyrin. CYP716A14v2 is a P450 belonging to the functionally diverse CYP716 family and catalyzes the oxidation of pentacyclic triterpenes, leading to triterpenes with a carbonyl group at position C-3, thereby providing an alternative biosynthesis pathway to 3-oxo triterpenes. Together, these enzymes produce specialized triterpenoids that are constituents of the wax layer of the cuticle covering the aerial parts of A. annua and likely function in the protection of the plant against biotic and abiotic stress.

Boutanaev A. M., Moses T., Zi J., Nelson D. R., Mugford S., Peters R., Osbourn A. (2015)

Investigation of terpene diversification across multiple sequenced plant genomes reveals non-random pairing of terpenoid synthases and cytochromes P450

Proceedings of the National Academy of Sciences of the United States of America (112) E81-E88

Publisher's version: 10.1073/pnas.1419547112

ID: 48826

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Abstract

The terpenes are the largest class of plant natural products.  This major class of compounds represents temendous chemical diversity of which only a realtively small fraction has so far been accessed and used by industry.  The primary drivers of terpene diversification are terpenoid synthases and cytochromes P450, which synthesize and modify terpene scaffolds.  Here, focusing on these two gene families, we investigate terpene synthesis and evolution across 17 sequenced plant genomes.  Our analyses shed light on the roots of terpene biosynthesis and diversification in plants.  They also reveal that different genomic mechanisms of pathway assembly pre-dominate in eudicots and monocots.

Liu J., Osbourn A., Ma P. (2015)

MYB transcription factors as regulators of phenylpropanoid metabolism in plants.

Molecular Plant (8) 689-708

Publisher's version: 10.1016/j.molp.2015.03.012

ID: 49462

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Abstract

Phenylpropanoid-derived compounds represent a diverse family of secondary metabolites that originate from phenylalanine. These compounds have roles in plant growth and development, and in defense against biotic and abiotic stress. Many of these compounds are also beneficial to human health and welfare. V-myb myeloblastosis viral oncogene homolog (MYB) proteins belong to a large family of transcription factors and are key regulators of the synthesis of phenylpropanoid-derived compounds. This review summarizes the current understanding of MYB proteins and their roles in the regulation of phenylpropanoid metabolism in plants

Abstract

Recent discoveries have revealed that the genes for the biosynthesis of a variety of plant specialized metabolites are organized in operon-like clusters within plant genomes. Here we identify a regulatory process that is required for normal expression of metabolic gene clusters in Arabidopsis thaliana. Comparative gene expression analysis of a representative clustered gene was performed in a set of chromatin mutant lines. Subsequently, metabolite levels were analysed by GC-MS and the local chromatin structure was investigated by chromatin immunoprecipitation and nucleosome positioning. We show that the transcript levels of genes within two metabolic clusters are coordinately reduced in an arp6 and h2a.z background. We demonstrate that H2A.Z enrichment in the clusters is positively correlated with active cluster expression. We further show that nucleosome stability within the cluster regions is higher in the arp6 background compared with the wild-type. These results implicate ARP6 and H2A.Z in the regulation of metabolic clusters in Arabidopsis thaliana through localized chromatin modifications that enable the coordinate expression of groups of contiguous genes. These findings shed light on the complex process of cluster regulation, an area that could in the future open up new opportunities for the discovery and manipulation of specialized metabolic pathways in plants.

Moses T., Papadopoulou K. K., Osbourn A. (2014)

Metabolic and functional diversity of saponins, biosynthetic intermediates and semi-synthetic derivatives

Critical Reviews In Biochemistry & Molecular Biology (49) 439-462

Publisher's version: 10.3109/10409238.2014.953628

ID: 48405

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Abstract

Saponins are widely distributed plant natural products with vast structural and functionaldiversity. They are typically composed of a hydrophobic aglycone, which is extensivelydecorated with functional groups prior to the addition of hydrophilic sugar moieties, to resultin surface-active amphipathic compounds. The saponins are broadly classified astriterpenoids, steroids, or steroidal glycoalkaloids, based on the aglycone structure fromwhich they are derived. The saponins and their biosynthetic intermediates display a variety ofbiological activities of interest to the pharmaceutical, cosmetic and food sectors. Althoughtheir relevance in industrial applications has long been recognised, their role in plants isunderexplored. Recent research on modulating native pathway flux in saponin biosynthesishas demonstrated the roles of saponins and their biosynthetic intermediates in plant growthand development. Here we review literature on the effects of these molecules on plantphysiology, which collectively implicate them in plant primary processes. The industrial usesand potential of saponins are discussed with respect to structure and activity, highlighting theundoubted value of these molecules as therapeutics.

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