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Biosynthesis of Veratrum californicum specialty chemicals in Camelina sativa seed

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Abstract

Economically feasible systems for heterologous production of complex secondary metabolites originating from difficult to cultivate species are in demand since Escherichia coli and Saccharomyces cerevisiae are not always suitable for expression of plant and animal genes. An emerging oilseed crop, Camelina sativa, has recently been engineered to produce novel oil profiles, jet fuel precursors, and small molecules of industrial interest. To establish C. sativa as a system for the production of medicinally relevant compounds, we introduced four genes from Veratrum californicum involved in steroid alkaloid biosynthesis. Together, these four genes produce verazine, the hypothesized precursor to cyclopamine, a medicinally relevant steroid alkaloid whose analogs are currently being tested for cancer therapy in clinical trials. The future supply of this potential cancer treatment is uncertain as V. californicum is slow-growing and not amendable to cultivation. Moreover, the complex stereochemistry of cyclopamine results in low-yield syntheses. Herein, we successfully engineered C. sativa to synthesize verazine, as well as other V. californicum secondary metabolites, in seed. In addition, we have clarified the stereochemistry of verazine and related V. californicum metabolites.

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Abbreviations

GAD2:

Glutamate decarboxylase 2 from Arabidopsis thaliana

GABA:

γ-Aminobutyric acid

GABAT1:

γ-Aminobutyrate transaminase 1 from Veratrum californicum

CYP90B27:

Cholesterol 22-hydroxylase from Veratrum californicum

CYP94N1:

22-Hydroxycholesterol 26-hydroxylase/oxidase from Veratrum californicum

CYP90G1:

22-Hydroxy-26-aminocholesterol 22-oxidase from Veratrum californicum

CO:

Binary vector containing CYP90B27 and CYP94N1

CTOG:

Binary vector containing CYP90B27, CYP94N1, GABAT1, and GAD2

CXTOG:

Binary vector containing CYP90B27, CYP94N1, GABAT1, GAD2, and CYP90G1

References

  • Abdel-Kader MS, Bahler BD, Malone S, Werkhoven MC, van Troon F, David, Wisse JH, Bursuker I, Neddermann KM, Mamber SW, Kingston DG (1998) DNA-damaging steroidal alkaloids from Eclipta alba from the suriname rainforest. J Nat Prod 61:1202–1208

    Article  CAS  PubMed  Google Scholar 

  • Asomaning J, Mussone P, Bressler DC (2014) Two-stage thermal conversion of inedible lipid feedstocks to renewable chemicals and fuels. Bioresour Technol 158:55–62

    Article  CAS  PubMed  Google Scholar 

  • Atanasov AG, Waltenberger B, Pferschy-Wenzig EM, Linder T, Wawrosch C, Uhrin P, Temml V, Wang L, Schwaiger S, Heiss EH, Rollinger JM, Schuster D, Breuss JM, Bochkov V, Mihovilovic MD, Kopp B, Bauer R, Dirsch VM, Stuppner H (2015) Discovery and resupply of pharmacologically active plant-derived natural products: A review. Biotechnol Adv 33:1582–1614

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Augustin JM, Higashi Y, Feng X, Kutchan TM (2015a) Production of mono- and sesquiterpenes in Camelina sativa oilseed. Planta 242:693–708

    Article  CAS  PubMed  Google Scholar 

  • Augustin MM, Ruzicka DR, Shukla AK, Augustin JM, Starks CM, O’Neil-Johnson M, McKain MR, Evans BS, Barrett MD, Smithson A, Wong GK, Deyholos MK, Edger PP, Pires JC, Leebens-Mack JH, Mann DA, Kutchan TM (2015b) Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells. Plant J 82:991–1003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bahra M, Kamphues C, Boas-Knoop S, Lippert S, Esendik U, Schuller U, Hartmann W, Waha A, Neuhaus P, Heppner F, Pietsch T, Koch A (2012) Combination of hedgehog signaling blockage and chemotherapy leads to tumor reduction in pancreatic adenocarcinomas. Pancreas 41:222–229

    Article  CAS  PubMed  Google Scholar 

  • Bansal S, Durrett TP (2016) Camelina sativa: an ideal platform for the metabolic engineering and field production of industrial lipids. Biochimie 120:9–16

    Article  CAS  PubMed  Google Scholar 

  • Batsaikhan BE, Yoshikawa K, Kurita N, Iwata T, Takasu C, Kashihara H, Shimada M (2014) Cyclopamine decreased the expression of Sonic Hedgehog and its downstream genes in colon cancer stem cells. Anticancer Res 34:6339–6344

    CAS  PubMed  Google Scholar 

  • Berman DM, Karhadkar SS, Hallahan AR, Pritchard JI, Eberhart CG, Watkins DN, Chen JK, Cooper MK, Taipale J, Olson JM, Beachy PA (2002) Medulloblastoma growth inhibition by hedgehog pathway blockade. Science 297:1559–1561

    Article  CAS  PubMed  Google Scholar 

  • Betancor MB, Sprague M, Sayanova O, Usher S, Campbell PJ, Napier JA, Caballero MJ, Tocher DR (2015a) Evaluation of a high-EPA oil from transgenic in feeds for Atlantic salmon (L.): effects on tissue fatty acid composition, histology and gene expression. Aquaculture 444:1–12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Betancor MB, Sprague M, Usher S, Sayanova O, Campbell PJ, Napier JA, Tocher DR (2015b) A nutritionally-enhanced oil from transgenic Camelina sativa effectively replaces fish oil as a source of eicosapentaenoic acid for fish. Sci Rep 5:8104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen JK, Taipale J, Cooper MK, Beachy PA (2002) Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev 16:2743–2748

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Finamore E, Minale L, Riccio R, Rinaldo G, Zollo F (1991) Novel Marine Polyhydroxylated Steroids from the Starfish Myxoderma-Platyacanthum. J Org Chem 56:1146–1153

    Article  CAS  Google Scholar 

  • Heretsch P, Tzagkaroulaki L, Giannis A (2010) Cyclopamine and hedgehog signaling: chemistry, biology, medical perspectives. Angew Chem Int Ed Engl 49:3418–3427

    Article  CAS  PubMed  Google Scholar 

  • Iven T, Hornung E, Heilmann M, Feussner I (2015) Synthesis of oleyl oleate wax esters in Arabidopsis thaliana and Camelina sativa seed oil. Plant Biotechnol J 14:252–259

    Article  PubMed  Google Scholar 

  • Jimeno A, Weiss GJ, Miller WH Jr, Gettinger S, Eigl BJ, Chang AL, Dunbar J, Devens S, Faia K, Skliris G, Kutok J, Lewis KD, Tibes R, Sharfman WH, Ross RW, Rudin CM (2013) Phase I study of the Hedgehog pathway inhibitor IPI-926 in adult patients with solid tumors. Clin Cancer Res 19:2766–2774

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kagale S, Koh C, Nixon J, Bollina V, Clarke WE, Tuteja R, Spillane C, Robinson SJ, Links MG, Clarke C, Higgins EE, Huebert T, Sharpe AG, Parkin IA (2014) The emerging biofuel crop Camelina sativa retains a highly undifferentiated hexaploid genome structure. Nat Commun 5:3706

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Keeler RF, Binns W (1968) Teratogenic compounds in Veratrum californicum (Durand) V. Comparison of cyclopian effects of steroidal alkaloids from the plant and structurally related compounds from other sources. Teratology 1:5–10

    Article  CAS  PubMed  Google Scholar 

  • Kim HJ, Silva JE, Vu HS, Mockaitis K, Nam JW, Cahoon EB (2015) Toward production of jet fuel functionality in oilseeds: identification of FatB acyl-acyl carrier protein thioesterases and evaluation of combinatorial expression strategies in Camelina seeds. J Exp Bot 66:4251–4265

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kristensen C, Morant M, Olsen CE, Ekstrom CT, Galbraith DW, Moller BL, Bak S (2005) Metabolic engineering of dhurrin in transgenic Arabidopsis plants with marginal inadvertent effects on the metabolome and transcriptome. Proc Natl Acad Sci USA 102:1779–1784

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li-Beisson Y, Shorrosh B, Beisson F, Andersson MX, Arondel V, Bates PD, Baud S, Bird D, Debono A, Durrett TP, Franke RB, Graham IA, Katayama K, Kelly AA, Larson T, Markham JE, Miquel M, Molina I, Nishida I, Rowland O, Samuels L, Schmid KM, Wada H, Welti R, Xu C, Zallot R and Ohlrogge J (2013) Acyl-lipid metabolism. Arabidopsis Book, vol 11, p e0161

  • Lin TL, Wang QH, Brown P, Peacock C, Merchant AA, Brennan S, Jones E, McGovern K, Watkins DN, Sakamoto KM, Matsui W (2010) Self-renewal of acute lymphocytic leukemia cells is limited by the Hedgehog pathway inhibitors cyclopamine and IPI-926. PLoS One 5:e15262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu J, Rice A, McGlew K, Shaw V, Park H, Clemente T, Pollard M, Ohlrogge J, Durrett TP (2015) Metabolic engineering of oilseed crops to produce high levels of novel acetyl glyceride oils with reduced viscosity, freezing point and calorific value. Plant Biotechnol J 13:858–865

    Article  CAS  PubMed  Google Scholar 

  • Lu C, Kang J (2008) Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation. Plant Cell Rep 27:273–278

    Article  CAS  PubMed  Google Scholar 

  • Ma R, Ritala A, Oksman-Caldentey KM, Rischer H (2006) Development of in vitro techniques for the important medicinal plant Veratrum californicum. Planta Med 72:1142–1148

    Article  CAS  PubMed  Google Scholar 

  • Malik MR, Yang W, Patterson N, Tang J, Wellinghoff RL, Preuss ML, Burkitt C, Sharma N, Ji Y, Jez JM, Peoples OP, Jaworski JG, Cahoon EB, Snell KD (2015) Production of high levels of poly-3-hydroxybutyrate in plastids of Camelina sativa seeds. Plant Biotechnol J 13:675–688

    Article  CAS  PubMed  Google Scholar 

  • Mansour MP, Shrestha P, Belide S, Petrie JR, Nichols PD, Singh SP (2014) Characterization of oilseed lipids from “DHA-producing Camelina sativa”: a new transformed land plant containing long-chain omega-3 oils. Nutrients 6:776–789

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ohyama K, Suzuki M, Kikuchi J, Saito K, Muranaka T (2009) Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis. Proc Natl Acad Sci USA 106:725–730

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A, McIntyre D, Honess D, Madhu B, Goldgraben MA, Caldwell ME, Allard D, Frese KK, Denicola G, Feig C, Combs C, Winter SP, Ireland-Zecchini H, Reichelt S, Howat WJ, Chang A, Dhara M, Wang L, Ruckert F, Grutzmann R, Pilarsky C, Izeradjene K, Hingorani SR, Huang P, Davies SE, Plunkett W, Egorin M, Hruban RH, Whitebread N, McGovern K, Adams J, Iacobuzio-Donahue C, Griffiths J, Tuveson DA (2009) Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 324:1457–1461

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Petrie JR, Shrestha P, Belide S, Kennedy Y, Lester G, Liu Q, Divi UK, Mulder RJ, Mansour MP, Nichols PD, Singh SP (2014) Metabolic engineering Camelina sativa with fish oil-like levels of DHA. PLoS One 9:e85061

    Article  PubMed  PubMed Central  Google Scholar 

  • Putnam DH, Budin JT, Field LA and M. BW (1993) Camelina: a promising low-input oilseed. Wiley New York

    Google Scholar 

  • Ruiz-Lopez N, Haslam RP, Napier JA, Sayanova O (2014) Successful high-level accumulation of fish oil omega-3 long-chain polyunsaturated fatty acids in a transgenic oilseed crop. Plant J 77:198–208

    Article  CAS  PubMed  Google Scholar 

  • Sawai S, Ohyama K, Yasumoto S, Seki H, Sakuma T, Yamamoto T, Takebayashi Y, Kojima M, Sakakibara H, Aoki T, Muranaka T, Saito K, Umemoto N (2014) Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato. Plant Cell 26:3763–3774

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shukla VKS, Dutta PC, Artz WE (2002) Camelina oil and its unusual cholesterol content. J Am Oil Chem Soc 79:965–969

    Article  CAS  Google Scholar 

  • Song J, Naylor-Adelberg J, White S, Mann D, Adelberg J (2014) Establishing clones of Veratrum californicum, a native medicinal species, for micropropagation. In Vitro Cell Dev Biol Plant 50:337–344

    Article  CAS  Google Scholar 

  • Taipale J, Chen JK, Cooper MK, Wang B, Mann RK, Milenkovic L, Scott MP, Beachy PA (2000) Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406:1005–1009

    Article  CAS  PubMed  Google Scholar 

  • Tremblay MR, Lescarbeau A, Grogan MJ, Tan E, Lin G, Austad BC, Yu LC, Behnke ML, Nair SJ, Hagel M, White K, Conley J, Manna JD, Alvarez-Diez TM, Hoyt J, Woodward CN, Sydor JR, Pink M, MacDougall J, Campbell MJ, Cushing J, Ferguson J, Curtis MS, McGovern K, Read MA, Palombella VJ, Adams J, Castro AC (2009) Discovery of a potent and orally active hedgehog pathway antagonist (IPI-926). J Med Chem 52:4400–4418

    Article  CAS  PubMed  Google Scholar 

  • Turano FJ, Fang TK (1998) Characterization of two glutamate decarboxylase cDNA clones from Arabidopsis. Plant Physiol 117:1411–1421

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Watkins DN, Berman DM, Burkholder SG, Wang B, Beachy PA, Baylin SB (2003) Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer. Nature 422:313–317

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Dr. David Kingston, Virginia Tech, for his kind gift of verazine. This work was supported by funds from Infinity Pharmaceuticals to T.M.K., an Indo-US (IUSSTF) Research Fellowship to A.K.S., International Cooperation Program of Outstanding Young Backbone Teachers for L.H., NSF funded Research Experiences for Undergraduates DBI-1156581 to C.K.H. and NIH 1R01DA025197-02 to T.M.K. This material is based upon work supported by the National Science Foundation under Grant No. DBI-1427621 for acquisition of the QTRAP LC–MS/MS. No funding source had any involvement in the design or execution of the experiments within, nor did they have any involvement in the preparation of this manuscript.

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Authors and Affiliations

  1. Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO, 63132, USA

    Megan M. Augustin, Ashutosh K. Shukla, Linna Han, Cynthia K. Holland & Toni M. Kutchan

  2. CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, Uttar Pradesh, 226015, India

    Ashutosh K. Shukla

  3. Sequoia Sciences, 1912 Innerbelt Business Center Dr., St. Louis, MO, 63114, USA

    Courtney M. Starks & Mark O’Neil-Johnson

  4. College of Pharmacy, Shandong University of Traditional Chinese Medicine, 4655 Daxue Road, Jinan, 250355, People’s Republic of China

    Linna Han

  5. Washington University, 1 Brookings Dr., St. Louis, MO, 63130, USA

    Cynthia K. Holland

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  1. Megan M. Augustin
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Corresponding author

Correspondence to Toni M. Kutchan.

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Author contributions

MMA, AKS, LH, and CKH constructed vectors for transformation. MMA and LH performed plant transformations and seed collection. MMA and LH extracted seeds for metabolite analysis. MMA ran all samples on the GC–MS and LC–MS/MS and analyzed the results. CMS and MOJ performed the Mosher ester analysis, NMR, and ELSD. MMA analyzed data and generated figures. MMA and TMK wrote the manuscript. TMK oversaw all work.

Additional information

Significance statement Biosynthesis of verazine in Camelina sativa seeds by heterologous expression of Veratrum californicum genes CYP90B27, CYP94N1, CYP90G1, GABAT1 and the Arabidopsis thaliana gene GAD2 was achieved. This is the first report of engineering C. sativa for the production of medicinally relevant compounds. In addition, the conformation of the synthesized verazine at C-20 was confirmed to be (S) and the stereochemistry of 22-keto-26-hydoxycholesterol at C-25 was determined to be most likely (R).

Electronic supplementary material

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11816_2017_427_MOESM1_ESM.pdf

Figure S1. PCR analysis of genomic DNA extracted from transgenic C. sativa leaves transformed with the Veratrum californicum genes CYP90B27, CYP94N1, GABAT1, and CYP90G1 and Arabidopsis thaliana GAD2. Transgenic C. sativa containing CYP90B27, CYP94N1, CYP90G1, GABAT1, and GAD2 (CXTOG) lines C, E, and K were propagated to the T3 generation prior to DNA extraction. DNA was extracted from leaves of 5 individual plants per line. Wild type DNA was used for a negative control while the purified pRSe3 plasmid containing all genes was used for a positive control (PDF 136 KB)

11816_2017_427_MOESM2_ESM.pdf

Figure S2. Mass spectra of naturally occurring and heterologously produced verazine. Samples were extracted and filtered prior to analysis by LC-MS/MS with a QTRAP 6500 using EPI scan for 398.3 m/z. Mass spectra of verazine from (a) Camelina sativa seeds expressing CYP90B27, CYP94N1, and GABAT1 from Veratrum californicum and GAD2 from Arabidopsis thaliana (CTOG), (b) C. sativa seeds expressing CYP90B27, CYP94N1, CYP90G1 and GABAT1 from V. californicum and GAD2 from A. thaliana (CXTOG), (c) V. californicum root extract, (d) authentic verazine standard, and (e) verazine produced by heterologous expression of CYP90B27, CYP94N1, CYP90G1, CPR, and GABAT1 in S. frugiperda Sf9 insect cells. Only select ions are shown for clarity. CPR refers to cytochrome P450 reductase from E. californica and GAD2 refers to glutamate decarboxylase 2 (PDF 86 KB)

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Augustin, M.M., Shukla, A.K., Starks, C.M. et al. Biosynthesis of Veratrum californicum specialty chemicals in Camelina sativa seed. Plant Biotechnol Rep 11, 29–41 (2017). https://doi.org/10.1007/s11816-017-0427-x

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