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Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid

Abstract

Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving.

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Figure 1: Expression of PUFA synthase in Arabidopsis.
Figure 2: Expression of PUFA synthase in canola.
Figure 3: Characterization of DHA-producing canola events.

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References

  1. Abedi, E. & Sahari, M.A. Long-chain polyunsaturated fatty acid sources and evaluation of their nutritional and functional properties. Food Sci. Nutr. 2, 443–463 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Metz, J.G. et al. Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293, 290–293 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Shulse, C.N. & Allen, E.E. Widespread occurrence of secondary lipid biosynthesis potential in microbial lineages. PLoS One 6, e20146 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Barclay, W., Weaver, C. & Metz, J.G. in Single Cell Oils (eds. Z. Cohen & C. Ratledge) (AOCS Press, Urbana, IL, 2005).

  5. Petrie, J.R. et al. Metabolic engineering plant seeds with fish oil-like levels of DHA. PLoS One 7, e49165 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wu, G. et al. Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants. Nat. Biotechnol. 23, 1013–1017 (2005).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  8. Petrie, J.R. et al. Metabolic engineering Camelina sativa with fish oil-like levels of DHA. PLoS One 9, e85061 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Frankel, E.N. Lipid Oxidation 2nd edn. (The Oily Press, Bridgwater, UK, 2005).

  10. Metz, J.G. et al. Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochytrium: release of the products as free fatty acids. Plant Physiol. Biochem. 47, 472–478 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Staunton, J. & Weissman, K.J. Polyketide biosynthesis: a millennium review. Nat. Prod. Rep. 18, 380–416 (2001).

    Article  CAS  PubMed  Google Scholar 

  12. Leibundgut, M., Maier, T., Jenni, S. & Ban, N. The multienzyme architecture of eukaryotic fatty acid synthases. Curr. Opin. Struct. Biol. 18, 714–725 (2008).

    Article  CAS  PubMed  Google Scholar 

  13. Apt, K.E., Richter, L., Simpson, D. & Zirkle, R. Polyunsaturated fatty acid synthase nucleic acid molecules and polypeptides, compositions, and methods of making and uses thereof. US patent application 20100266564A1 (2010).

  14. Copp, J.N. & Neilan, B.A. The phosphopantetheinyl transferase superfamily: phylogenetic analysis and functional implications in Cyanobacteria. Appl. Environ. Microbiol. 72, 2298–2305 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hauvermale, A. et al. Fatty acid production in Schizochytrium sp.: Involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase. Lipids 41, 739–747 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Ullrich, K.K., Hiss, M. & Rensing, S.A. Means to optimize protein expression in transgenic plants. Curr. Opin. Biotechnol. 32, 61–67 (2015).

    Article  CAS  PubMed  Google Scholar 

  17. Peremarti, A. et al. Promoter diversity in multigene transformation. Plant Mol. Biol. 73, 363–378 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Naqvi, S. et al. When more is better: multigene engineering in plants. Trends Plant Sci. 15, 48–56 (2010).

    Article  CAS  PubMed  Google Scholar 

  19. Dietz-Pfeilstetter, A. Stability of transgene expression as a challenge for genetic engineering. Plant Sci. 179, 164–167 (2010).

    Article  CAS  Google Scholar 

  20. Weaver, C.A., Zirkle, R., Doherty, D.H. & G, M.J. Chimeric PUFA polyketide synthase systems and uses thereof. US patent application 8309796B2 (2012).

  21. Hoffman, L.M. & Donaldson, D.D. Characterization of two Phaseolus vulgaris phytohemagglutinin genes closely linked on the chromosome. EMBO J. 4, 883–889 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gudynaite-Savitch, L., Johnson, D.A. & Miki, B.L.A. Strategies to mitigate transgene-promoter interactions. Plant Biotechnol. J. 7, 472–485 (2009).

    Article  CAS  PubMed  Google Scholar 

  23. Fourmann, M. et al. The two genes homologous to Arabidopsis FAE1 co-segregate with the two loci governing erucic acid content in Brassica napus. Theor. Appl. Genet. 96, 852–858 (1998).

    Article  CAS  Google Scholar 

  24. Schwender, J. et al. Quantitative multilevel analysis of central metabolism in developing oilseeds of oilseed rape during in vitro culture. Plant Physiol. 168, 828–848 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Napier, J.A., Haslam, R.P., Beaudoin, F. & Cahoon, E.B. Understanding and manipulating plant lipid composition: Metabolic engineering leads the way. Curr. Opin. Plant Biol. 19, 68–75 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lau, W. & Sattely, E.S. Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone. Science 349, 1224–1228 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Yalpani, N., Altier, D.J., Barbour, E., Cigan, A.L. & Scelonge, C.J. Production of 6-methylsalicylic acid by expression of a fungal polyketide synthase activates disease resistance in tobacco. Plant Cell 13, 1401–1409 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hertweck, C. The biosynthetic logic of polyketide diversity. Angew. Chem. Int. Ed. Engl. 48, 4688–4716 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Weissman, K.J. & Leadlay, P.F. Combinatorial biosynthesis of reduced polyketides. Nat. Rev. Microbiol. 3, 925–936 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Salem, N. Jr. & Eggersdorfer, M. Is the world supply of omega-3 fatty acids adequate for optimal human nutrition? Curr. Opin. Clin. Nutr. Metab. Care 18, 147–154 (2015).

    Article  CAS  PubMed  Google Scholar 

  31. Xue, Z. et al. Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat. Biotechnol. 31, 734–740 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Petrie, J.R. & Singh, S.P. Lipid comprising docosapentaenoic acid. US patent application 20150374654A1 (2015).

  33. Jones, P.J. et al. DHA-enriched high-oleic acid canola oil improves lipid profile and lowers predicted cardiovascular disease risk in the canola oil multicenter randomized controlled trial. Am. J. Clin. Nutr. 100, 88–97 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kridl, J.C. et al. Isolation and characterization of an expressed napin gene from Brassica rapa. Seed Sci. Res. 1, 209–219 (1991).

    Article  CAS  Google Scholar 

  35. Ayele, M. et al. Whole genome shotgun sequencing of Brassica oleracea and its application to gene discovery and annotation in Arabidopsis. Genome Res. 15, 487–495 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Verdaguer, B., de Kochko, A., Fux, C.I., Beachy, R.N. & Fauquet, C. Functional organization of the cassava vein mosaic virus (CsVMV) promoter. Plant Mol. Biol. 37, 1055–1067 (1998).

    Article  CAS  PubMed  Google Scholar 

  37. Bustos, M.M., Begum, D., Kalkan, F.A., Battraw, M.J. & Hall, T.C. Positive and negative cis-acting DNA domains are required for spatial and temporal regulation of gene expression by a seed storage protein promoter. EMBO J. 10, 1469–1479 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Krebbers, E. et al. Determination of the processing sites of an Arabidopsis 2S-albumin and characterization of the complete gene family. Plant Physiol. 87, 859–866 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Barker, R.F., Idler, K.B., Thompson, D.V. & Kemp, J.D. Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol. Biol. 2, 335–350 (1983).

    Article  CAS  PubMed  Google Scholar 

  40. de Silva, J. et al. The isolation and sequence analysis of two seed-expressed acyl carrier protein genes from Brassica napus. Plant Mol. Biol. 14, 537–548 (1990).

    Article  CAS  PubMed  Google Scholar 

  41. Clough, S.J. & Bent, A.F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Article  CAS  PubMed  Google Scholar 

  42. Smith, M.A., Moon, H., Chowrira, G. & Kunst, L. Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana. Planta 217, 507–516 (2003).

    Article  CAS  PubMed  Google Scholar 

  43. Hepburn, A.G. et al. The use of pNJ5000 as an intermediate vector for the genetic manipulation of Agrobacterium Ti-plasmids. J. Gen. Microbiol. 131, 2961–2969 (1985).

    CAS  PubMed  Google Scholar 

  44. Mayerhofer, R. et al. Complexities of chromosome landing in a highly duplicated genome: toward map-based cloning of a gene controlling blackleg resistance in Brassica napus. Genetics 171, 1977–1988 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. De Block, M., De Brouwer, D. & Tenning, P. Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants. Plant Physiol. 91, 694–701 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Weng, H. et al. Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with HMG I/Y as an endogenous reference gene. Plant Mol. Biol. Rep. 22, 289–300 (2004).

    Article  CAS  Google Scholar 

  47. Murray, M.G. & Thompson, W.F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8, 4321–4325 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Quinlan, A.R. & Hall, I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Krzywinski, M. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Official Methods and Recommended Practices of the AOCS 6th edn. (AOCS Press, Champaign, IL, USA, 2013).

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Acknowledgements

We thank P. Roessler for early inspiration for this project. At Dow AgroSciences, we thank B. Martindale and L. Juberg for greenhouse support, M. Landes, A. Walker, B. Case, C. Ransom, R. Preuss, K. Ubayasena, S. Chennareddy, G. Booher and P. Nelson for technical assistance, M. Foster and A. Beach for providing recombinant protein standards, P. Graupner for triacylgycerol positional analysis and A. Wang for statistical analyses. The fae1/fad3 Arabidopsis mutant was obtained from M. Smith (NRC Canada, Saskatoon, Canada).

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Authors and Affiliations

Authors

Contributions

J.G.M. and R.Z. selected PUFA synthase genes, T.A.W., S.A.B., D.J.G., C.M.L., P.A.O.M. and L.V.S. were responsible for construct design strategy, plant transformation experiments and analyzed results. S.A.B. made the constructs. D.J.G., W.A.M., R.E.H. and V.S. performed technical analyses. D.P. and G.I.A. were responsible for canola transformation experiments. P.B.B. designed and analyzed field trials. P.R.M., L.M.C. and W.C. designed and conducted the canola whole genome sequencing experiments. P.S.A.-P. and S.T.W. designed and conducted oil processing and analyses. T.A.W. wrote the manuscript. All authors discussed results and reviewed the manuscript.

Corresponding author

Correspondence to Terence A Walsh.

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Competing interests

This work was part of the research and development programs of Dow AgroSciences LLC, a for-profit agricultural technology company. Part of the work was performed within a research collaboration with DSM Nutritional Products LLC. T.A.W., S.A.B., D.J.G., C.M.L., W.A.M., P.A.O.M., L.V.S., R.E.H., V.S., D.P., G.I.A., P.B.B., P.R.M., L.M.C., W.C., P.S.A.-P. and S.T.W. were employees of Dow AgroSciences LLC and R.Z. and J.G.M. were employees of DSM Nutritional Products LLC when contributing to this work. Some of the data were used in patent applications US2013 0150599 A1, US2014 0359900 and WO2015 081270 A1.

Integrated supplementary information

Supplementary Figure 1 LC-PUFA content of seed from individual Arabidopsis T1 events

Each bar represents the LC-PUFA content of the T2 seed from an individual T1 plant (one event). The events are sorted by DHA content and the blank space on the left of each box therefore represents the proportion of events that produced no LC-PUFAs. The microalgal source of the PUFA synthase genes used in each construct is noted. Note that the events produced using the Schizo9695 PUFA synthase genes produce more EPA (pale green) than those using the Schizo20888 PUFA synthase.

Supplementary Figure 2 Single seed analysis of the total PUFA synthase-derived LC-PUFA content of seed from T0 canola plants

Each bar represents the LC-PUFA content of the T2 seed from an individual T1 plant (one event). The events are sorted by DHA content and the blank space on the left of each box therefore represents the proportion of events that produced no LC-PUFAs. The microalgal source of the PUFA synthase genes used in each construct is noted. Note that the events produced using the Schizo9695 PUFA synthase genes produce more EPA (pale green) than those using the Schizo20888 PUFA synthase.

Supplementary Figure 3 Western blots of protein extracts from homozygous transgenic T2 canola seed expressing algal PUFA synthase

(a) PUFA synthase western blots. A single SDS-PAGE gel was loaded, run then cut as indicated by the dotted lines. The left-hand section was processed with anti-PFA1 antibody, the center panel with anti-PFA2 antibody and the right-hand panel with anti-PFA3 antibody. An appropriate purified protein standard was run adjacent to each seed extract. Little evidence for proteolytic degradation was noted for PFA2 and PFA3. The major PFA1 band accounts for 75% of the signal intensity in the transgenic seed extracts. (b) NoHetI western blot. The slightly larger size of the PFA2, PFA3 and NoHetI standards and the proteins expressed in plants is due to the 6xHis-tag on the standards.

Supplementary Figure 4 DHA, EPA and total LC-PUFA content of T4 seed from transgenic T3 canola plants grown in the field

PUFA synthase-derived fatty acids from transgenic events grown in the field at two different locations in 2014. (a) DHA, (b) EPA. (c) Total PUFA synthase-derived fatty acids (DHA+EPA+DPA(n-6)+ARA+GLA). The box plots show 25-50 and 50-75% quartiles (n=7-8 plots per event) and the whiskers extend to 1.5x the interquartile region. Outliers are shown as circles, means are shown by the widest bar. A very small amount of DHA (<0.15%) was detected in some DH12075 untransformed control plots and was attributed to occasional cross pollination from adjacent transgenic plots within the same field in the randomized plot design. No LC-PUFAs were detected in any greenhouse-grown material where pollen movement was strictly controlled by bagging flowering plants. Similarly no LC-PUFAs were detected in seed from other field studies in which plants were individually bagged prior to pollination.

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Walsh, T., Bevan, S., Gachotte, D. et al. Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid. Nat Biotechnol 34, 881–887 (2016). https://doi.org/10.1038/nbt.3585

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