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. This allows us to harness and exploit metabolic diversity using synthetic biology approaches, hence OpenPlant is generating genetic toolkits for synthesis and modification of plant natural products. The HyperTrans plant expression system (see Workpackage J) is being heavily used in this work programme. This platform supports the testing and investigation of metabolic pathways and the creation of new compounds. In turn, these projects inform and enable further optimisation of this powerful tool.

Pathway discovery and engineering

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 generated gram-scale quantities of purified triterpene analogs, levels that are ample for both structural determination by NMR and for screening for bioactivity (Reed et al., 2017).  They have used this approach to generate and 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 (Kautsar et al., 2017) 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.  Examples of our latest research can be found here: Hodgson et al. (2019), Huang et al. (2019), Huang and Osbourn (2019), Leveau et al. (2019), Liu et al. (2019), Orme et al. (2019), and Stephenson et al. (2019).

The O’Connor lab previously engineered the pathway for the iridoid alkaloid strictosidine from Madagascan periwinkle into yeast (Brown et al. 2015). As part of OpenPlant, the lab has discovered additional enzymes in this pathway to generate more “building blocks” for this work. The lab has generated a proteome database for trichomes of iridoid-producing plants and searched this for new candidate pathway enzymes. In a hunt for novel iridoid biosynthesis enzymes, enzymes involved in biosynthesis of nepatalactone, the key bioactive ingredient in catnip and catmint (Nepeta sp.), were identified (Sherden et al., 2017). 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

Optimized gene expression in tomato

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.

Anthocyanin production in tobacco cells

The Martin Lab has developed tobacco cell cultures for customisable anthocyanin production (Appelhagen et al., 2018): Anthocyanins are widely distributed, glycosylated, water-soluble plant pigments, which give many fruits and flowers their red, purple or blue colouration. Their beneficial effects in a dietary context have encouraged increasing use of anthocyanins as natural colourants in the food and cosmetic industries. However, the limited availability and diversity of anthocyanins commercially have initiated searches for alternative sources of these natural colourants. The tobacco cell cultures represent a customisable and sustainable alternative to conventional anthocyanin production platforms and have considerable potential for use in industrial and medical applications of anthocyanins (adapted from abstract Appelhagen et al., 2018).

Plant-sourced L-DOPA production

PDRA Noam Chayut worked on a project for plant-sourced L-DOPA production for Parkinson’s treatment. The current cost of L-DOPA makes it unavailable for deprived populations worldwide. In addition, there is a growing demand for ‘natural’ or plant-sourced pharmaceutical substances in the first world. L-DOPA, a product of tyrosine hydroxylation, is an intermediate metabolite in biosynthesis of violet and yellow betalain pigments, in Beta vulgaris (beetroot). Natural steady state levels of L-DOPA are very low, usually undetectable. The goal of this project is to block the turnover of L-DOPA in beetroot by CRISPR/Cas9-mediated gene silencing to allow its accumulation to levels that could enable low-tech accessible production in a plant system. L-DOPA accumulation was verified by high-pressure liquid chromatography and accumulated in μg quantities. The concept of agriculturally produced L-DOPA for pharmaceutical uses thus proves to be very promising.

Chromatin-level regulation of biosynthetic gene clusters

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).

Publications

Stephenson MJ and Osbourn A (2020). Making drugs out of sunlight and ‘thin air’: An emerging synergy of synthetic biology and natural product chemistry. The Biochemist 42: 34-39. https://doi.org/10.1042/BIO20200044

Nützmann HW, Doerr D, Ramírez-Colmenero A, Sotelo-Fonseca JE, Wegel E, Di Stefano M, Wingett SW, Fraser P, Hurst L, Fernandez-Valverde SL, Osbourn A (2020) Active and repressed biosynthetic gene clusters have spatially distinct chromosome states. PNAS 117 (24) 13800-13809; DOI: 10.1073/pnas.1920474117

Liu Z, Cheema J, Vigouroux M, Hill L, Reed J, Paajanen P, Yant L & Osbourn A. (2020). Formation and diversification of a paradigm biosynthetic gene cluster in plants. Nature Communications 11: 5354. https://doi.org/10.1038/s41467-020-19153-6

Lichman BR, Kamileen MO, Titchiner GR, Saalbach G, Stevenson CEM, Lawson DM, O’Connor SE. (2019) Uncoupled activation and cyclization in catmint reductive terpenoid biosynthesis. Nat Chem Biol. 1; 15(1): 71–79. doi: 10.1038/s41589-018-0185-2

Galdon-Armero J, Arce-Rodriguez ML, Martin C. (2019) A scanning electron microscopy-based screen of leaves of Solanum pennellii (ac. LA716) xSolanum lycopersicum (cv. M82) introgression lines provides a resource for identification of loci involved in epidermal development in tomato. bioRxiv 688077; doi: https://doi.org/10.1101/688077

Hodgson H, De La Pena R, Stephenson MJ, Thimmappa R, Vincent JL, Sattely E, Osbourn A. (2019) Identification of key enzymes responsible for protolimonoid biosynthesis in plants: Opening the door to azadirachtin production. PNAS 116:17096.

Huang A. C., Jiang T., Liu Y. X., Bai Y. C., Reed J., Qu B., Goossens A., Nützmann H. W., Bai Y., Osbourn A. (2019) A specialized metabolic network selectively modulates Arabidopsis root microbiota. Science 364(6440). pii: eaau6389. doi: 10.1126/science.aau6389.

Huang AC, Osbourn A. (2019) Plant terpenes that mediate below-ground interactions: prospects for bioengineering terpenoids for plant protection. Pest Manag Sci. 75(9):2368-2377. doi: 10.1002/ps.5410.

Leveau A., Reed J., Qiao X., Stephenson M. J., Mugford S. T., Melton R. E., Rant J. C., Vickerstaff R., Langdon T., Osbourn A. (2019) Towards take-all control: a C-21ß oxidase required for acylation of triterpene defence compounds in oat. New Phytologist. 221(3):1544-1555. doi: 10.1111/nph.15456.

Liu Z, Suarez Duran HG, Harnvanichvech Y, Stephenson MJ, Schranz ME, Nelson D, Medema MH, Osbourn A. (2019) Drivers of metabolic diversification: how dynamic genomic neighborhoods generate new biosynthetic pathways in the Brassicaceae. New Phytol. Nov 26. doi: 10.1111/nph.16338.

Orme A., Louveau T., Stephenson M.J., Appelhagen I., Melton R.E., Cheema J., Li Y.,Zhao Q., Zhang L., Fan D., Tian Q., Vickerstaff R.J., Langdon T., Han B., Osbourn A. (2019) A non-canonical vacuolar sugar transferase required for biosynthesis of antimicrobial defense compounds in oat. PNAS 116 (52) 27105-27114 https://doi.org/10.1073/pnas.1914652116

Stephenson M. J., Field R. A., Osbourn A. E. (2019) The protosteryl and dammarenyl cation dichotomy in polycyclic triterpene biosynthesis revisited: has this ‘rule’ finally been broken? Nat Prod Rep. doi: 10.1039/c8np00096d. [Epub ahead of print]

Appelhagen I, Wulff-Vester AK, Wendell M, Hvoslef-Eide AK, Russell J, Oertel A, Martens S, Mock HP, Martin C, Matros A (2018). Colour bio-factories: Towards scale-up production of anthocyanins in plant cell cultures. Metab Eng. 48:218-232. doi: 10.1016/j.ymben.2018.06.004.

Boachon B, Buell CR, Crisovan E, Dudareva N, Garcia N, Godden G, Henry L, Kamileen MO, Kates HR, Kilgore MB, Lichman BR, Mavrodiev EV, Newton L, Rodriguez-Lopez C, O'Connor SE, Soltis D, Soltis P, Vaillancourt B, Wiegert-Rininger K, Zhao D (2018). Phylogenomic Mining of the Mints Reveals Multiple Mechanisms Contributing to the Evolution of Chemical Diversity in Lamiaceae. Mol Plant. 11(8):1084-1096. doi: 10.1016/j.molp.2018.06.002.

Dangl J, Osbourn A, Orzaez D et al. (2018).  What is the next frontier in plant engineering?  Cell 174(3): 499.

Fu R, Martin C, and Zhang Y (2018). Next Generation Plant Metabolic Engineering, Inspired by an Ancient Chinese Irrigation System. Mol Plant. 11(1):47-57. doi: 10.1016/j.molp.2017.09.002.

Galdon-Armero J, Fullana-Pericas M, Mulet PA, Conesa MA, Martin C, Galmes J. (2018). The ratio of trichomes to stomata is associated with water use efficiency in Solanum lycopersicum (tomato). Plant J. 96(3):607-619. doi: 10.1111/tpj.14055.

Hems ES, Wagstaff BA, Saalbach G, Field RA. (2018). CuAAC click chemistry for the enhanced detection of novel alkyne-based natural product toxins. Chem Commun (Camb). 54(86):12234-12237. doi: 10.1039/c8cc05113e. Erratum in: Chem Commun (Camb). 54(93):13161.

Li Y, Wang H, Zhang Y, Martin C. (2018). Can the world's favorite fruit, tomato, provide an effective biosynthetic chassis for high-value metabolites? Plant Cell Rep. 37(10):1443-1450. doi: 10.1007/s00299-018-2283-8.

Louveau T, Orme A, Pfalzgraf H, Stephenson MJ, Melton R, Saalbach G, Hemmings AM, Leveau A, Rejzek M, Vickerstaff RJ, Langdon T, Field RA, Osbourn A. (2018) Analysis of Two New Arabinosyltransferases Belonging to the Carbohydrate Active Enzyme (CAZY) Glycosyl Transferase Family1 Provides Insights into Disease Resistance and Sugar Donor Specificity. Plant Cell. 30(12):3038-3057. doi: 10.1105/tpc.18.00641.

Martin C (2018) A role for plant science in underpinning the objective of global nutritional security? Ann Bot. 122(4):541-553. doi: 10.1093/aob/mcy118.

Nützmann HW, Scazzocchio C, Osbourn A (2018). Metabolic gene clusters in eukaryotes. Annu Rev Genet. 52:159-183 doi: 10.1146/annurev-genet-120417-031237.

Reed J, Osbourn A (2018). Engineering terpenoid production through transient expression in Nicotiana benthamiana. Plant Cell Rep. 37(10):1431-1441. doi: 10.1007/s00299-018-2296-3.

Soubeyrand E, Johnson T, Latimer S, Block A, Kim J, Colquhoun TA,  Butelli E, Martin C, Wilson MA, Basset G (2018). The Peroxidative Cleavage of Kaempferol Contributes to the Biosynthesis of the Benzenoid Moiety of Ubiquinone in Plants. Plant Cell. 30(12):2910-2921. doi: 10.1105/tpc.18.00688.

Stewart C, Patron N, Hanson A, Jez J, (2018). Plant metabolic engineering in the synthetic biology era: plant chassis selection. Plant Cell Rep. 37(10):1357-1358. doi: 10.1007/s00299-018-2342-1.

Wulff-Vester A, Wendell M, Matros A, Appelhagen I, Martin C, Hvoslef-Eide AK (2018) ANTHOcyanin production Platform Using Suspension Cultures in custom-made bioreactors. IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT. 2018: 54, S77-S77

Xu JJ, Fang X, Li CY, Zhao Q, Martin C, Chen XY, Yang L. (2018) Characterization of Arabidopsis thaliana Hydroxyphenylpyruvate Reductases in the Tyrosine Conversion Pathway. Front Plant Sci. 9:1305. doi: 10.3389/fpls.2018.01305.

Xue Z, Tan Z, Huang A, Zhou Y, Sun J, Wang X, Thimmappa RB, Stephenson MJ, Osbourn A, Qi X (2018). Identification of key amino acid residues determining product specificity of 2,3-oxidosqualene cyclase in Oryza species. New Phytol. 218(3):1076-1088. doi: 10.1111/nph.15080.

Stephenson MJ, Reed J, Brouwer B, Osbourn A (2018). Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale. J Vis Exp. (138). doi: 10.3791/58169.

Boobier S, Osbourn A, Mitchell JBO (2017). Can human experts predict solubility better than computers? J Cheminform. 9(1):63. doi: 10.1186/s13321-017-0250-y.

Kautsar SA, Suarez Duran HG, Blin K, Osbourn A, Medema MH (2017). plantiSMASH: automated identification, annotation and expression analysis of plant biosynthetic gene clusters. Nucleic Acids Res. 45(W1):W55-W63. doi: 10.1093/nar/gkx305.

Kries H, Kellner F, Kamileen MO, O'Connor SE. (2017). Inverted stereocontrol of iridoid synthase in snapdragon. J Biol Chem. 292(35):14659-14667. doi: 10.1074/jbc.M117.800979.

Osbourn A. (2017) Painting with betalains. Nat Plants. 3(11):852-853. doi: 10.1038/s41477-017-0049-x.

Owen C, Patron NJ, Huang A, Osbourn A (2017). Harnessing plant metabolic diversity. Curr Opin Chem Biol. 40:24-30. doi: 10.1016/j.cbpa.2017.04.015.

Reed J, Stephenson MJ, Miettinen K, Brouwer B, Leveau A, Brett P, Goss RJM, Goossens A, O'Connell MA, Osbourn A. (2017). A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like molecules. Metab Eng. 42:185-193. doi: 10.1016/j.ymben.2017.06.012.

Scarano A, Butelli E, De Santis S, Cavalcanti E, Hill L, De Angelis M, Giovinazzo G, Chieppa M, Martin C, Santino A. (2017). Combined Dietary Anthocyanins, Flavonols, and Stilbenoids Alleviate Inflammatory Bowel Disease Symptoms in Mice. Front Nutr. 4:75. doi: 10.3389/fnut.2017.00075.

Sherden NH, Lichman B, Caputi L, Zhao D, Kamileen MO, Buell CR, O'Connor SE (2017). Identification of iridoid synthases from Nepeta species: Iridoid cyclization does not determine nepetalactone stereochemistry. Phytochemistry. 145:48-56. doi: 10.1016/j.phytochem.2017.10.004.

Alagna F, Geu-Flores F, Kries H, Panara F, Baldoni L, O'Connor SE, Osbourn A. (2016) Identification and characterization of the iridoid synthase involved in oleuropein biosynthesis in olive (Olea europaea) fruits. J Biol Chem. 291(11):5542-54. doi: 10.1074/jbc.M115.701276.

Medema, MH & Osbourn, A (2016) Computational genomic identification and functional reconstitution of plant natural product biosynthetic pathways. Nat Prod Rep. 33(8):951-62. doi: 10.1039/c6np00035e.

Nguyen TD, Faraldos JA, Vardakou M, Salmon M, O'Maille PE, Ro DK (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):622-627. doi: 10.1016/j.bbrc.2016.09.165.

Nützmann HW, Huang A, Osbourn A (2016). Plant metabolic clusters – from genetics to genomics. New Phytol. 211(3):771-89. doi: 10.1111/nph.13981.

Osbourn A., Morgan J. (2016) Editorial overview: Plant biotechnology. Curr Opin Biotechnol. 37:153-154. doi: 10.1016/j.copbio.2015.12.006.

Salmon M, Thimmappa R, Minto R, Melton R, Hughes R, O'Maille P, Hemmings AM, Osbourn A (2016) A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases. Proc Natl Acad Sci U S A. 113(30):E4407-14. doi: 10.1073/pnas.1605509113.

Yu N, Nützmann HW, MacDonald JT, Moore B, Field B, Berriri S, Trick M, Rosser SJ, Kumar SV, Freemont PS, Osbourn A (2016). Delineation of metabolic gene clusters in plant genomes by chromatin signatures. Nucleic Acids Res. 44(5):2255-65. doi: 10.1093/nar/gkw100.

Zhao Q, Zhang Y, Wang G, Hill L, Weng JK, Chen XY, Xue H, Martin C (2016). A specialized flavone biosynthetic pathway has evolved in the medicinal plant, Scutellaria baicalensis. Sci Adv. 2(4):e1501780. doi: 10.1126/sciadv.1501780.

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 DK, Shang Y, Huang S. (2016) Convergence and divergence of bitterness biosynthesis and regulation in Cucurbitaceae. Nat Plants. 2:16183. doi: 10.1038/nplants.2016.183.

Boutanaev AM, Moses T, Zi J, Nelson DR, Mugford ST, Peters RJ, Osbourn A (2015). Investigation of terpene diversification across multiple sequenced plant genomes. Proc Natl Acad Sci U S A. 112(1):E81-8. doi: 10.1073/pnas.1419547112.

Medema MH, Kottmann R, Yilmaz P, Cummings M, Biggins JB, Blin K, de Bruijn I, Chooi YH, Claesen J, Coates RC, Cruz-Morales P, Duddela S, Düsterhus S, Edwards DJ, Fewer DP, Garg N, Geiger C, Gomez-Escribano JP, Greule A, Hadjithomas M, Haines AS, Helfrich EJ, Hillwig ML, Ishida K, Jones AC, Jones CS, Jungmann K, Kegler C, Kim HU, Kötter P, Krug D, Masschelein J, Melnik AV, Mantovani SM, Monroe EA, Moore M, Moss N, Nützmann HW, Pan G, Pati A, Petras D, Reen FJ, Rosconi F, Rui Z, Tian Z, Tobias NJ, Tsunematsu Y, Wiemann P, Wyckoff E, Yan X, Yim G, Yu F, Xie Y, Aigle B, Apel AK, Balibar CJ, Balskus EP, Barona-Gómez F, Bechthold A, Bode HB, Borriss R, Brady SF, Brakhage AA, Caffrey P, Cheng YQ, Clardy J, Cox RJ, De Mot R, Donadio S, Donia MS, van der Donk WA, Dorrestein PC, Doyle S, Driessen AJ, Ehling-Schulz M, Entian KD, Fischbach MA, Gerwick L, Gerwick WH, Gross H, Gust B, Hertweck C, Höfte M, Jensen SE, Ju J, Katz L, Kaysser L, Klassen JL, Keller NP, Kormanec J, Kuipers OP, Kuzuyama T, Kyrpides NC, Kwon HJ, Lautru S, Lavigne R, Lee CY, Linquan B, Liu X, Liu W, Luzhetskyy A, Mahmud T, Mast Y, Méndez C, Metsä-Ketelä M, Micklefield J, Mitchell DA, Moore BS, Moreira LM, Müller R, Neilan BA, Nett M, Nielsen J, O'Gara F, Oikawa H, Osbourn A, Osburne MS, Ostash B, Payne SM, Pernodet JL, Petricek M, Piel J, Ploux O, Raaijmakers JM, Salas JA, Schmitt EK, Scott B, Seipke RF, Shen B, Sherman DH, Sivonen K, Smanski MJ, Sosio M, Stegmann E, Süssmuth RD, Tahlan K, Thomas CM, Tang Y, Truman AW, Viaud M, Walton JD, Walsh CT, Weber T, van Wezel GP, Wilkinson B, Willey JM, Wohlleben W, Wright GD, Ziemert N, Zhang C, Zotchev SB, Breitling R, Takano E, Glöckner FO (2015). Minimum Information about a Biosynthetic Gene cluster. Nat Chem Biol. 11(9):625-31. doi: 10.1038/nchembio.1890.

Nützmann HW, Osbourn A (2015) Regulation of metabolic gene clusters in Arabidopsis thaliana. New Phytol. 205(2):503-10. doi: 10.1111/nph.13189.

Tohge T, Zhang Y, Peterek S, Matros A, Rallapalli G, Tandrón YA, Butelli E, Kallam K, Hertkorn N, Mock HP, Martin C, Fernie AR.(2015). Ectopic expression of snapdragon transcription factors facilitates the identification of genes encoding enzymes of anthocyanin decoration in tomato. Plant J. 83(4):686-704. doi: 10.1111/tpj.12920.

Zhang Y, Butelli E, Alseekh S, Tohge T, Rallapalli G, Luo J, Kawar PG, Hill L, Santino A, Fernie AR, Martin C (2015). Multilevel engineering facilitates the production of phenylpropanoid compounds in tomato. Nat Commun. 6:8635. doi: 10.1038/ncomms9635.

Zhang Y, De Stefano R, Robine M, Butelli E, Bulling K, Hill L, Rejzek M, Martin C, Schoonbeek HJ (2015). Different Reactive Oxygen Species Scavenging Properties of Flavonoids Determine Their Abilities to Extend the Shelf Life of Tomato. Plant Physiol. 169(3):1568-83. doi: 10.1104/pp.15.00346.