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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

BioProject

References

  1. 1.

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

  2. 2.

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

  3. 3.

    & Widespread occurrence of secondary lipid biosynthesis potential in microbial lineages. PLoS One 6, e20146 (2011).

  4. 4.

    , & in Single Cell Oils (eds. Z. Cohen & C. Ratledge) (AOCS Press, Urbana, IL, 2005).

  5. 5.

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

  6. 6.

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

  7. 7.

    , , & 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).

  8. 8.

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

  9. 9.

    Lipid Oxidation 2nd edn. (The Oily Press, Bridgwater, UK, 2005).

  10. 10.

    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).

  11. 11.

    & Polyketide biosynthesis: a millennium review. Nat. Prod. Rep. 18, 380–416 (2001).

  12. 12.

    , , & The multienzyme architecture of eukaryotic fatty acid synthases. Curr. Opin. Struct. Biol. 18, 714–725 (2008).

  13. 13.

    , , & Polyunsaturated fatty acid synthase nucleic acid molecules and polypeptides, compositions, and methods of making and uses thereof. US patent application 20100266564A1 (2010).

  14. 14.

    & The phosphopantetheinyl transferase superfamily: phylogenetic analysis and functional implications in Cyanobacteria. Appl. Environ. Microbiol. 72, 2298–2305 (2006).

  15. 15.

    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).

  16. 16.

    , & Means to optimize protein expression in transgenic plants. Curr. Opin. Biotechnol. 32, 61–67 (2015).

  17. 17.

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

  18. 18.

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

  19. 19.

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

  20. 20.

    , , & Chimeric PUFA polyketide synthase systems and uses thereof. US patent application 8309796B2 (2012).

  21. 21.

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

  22. 22.

    , & Strategies to mitigate transgene-promoter interactions. Plant Biotechnol. J. 7, 472–485 (2009).

  23. 23.

    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).

  24. 24.

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

  25. 25.

    , , & Understanding and manipulating plant lipid composition: Metabolic engineering leads the way. Curr. Opin. Plant Biol. 19, 68–75 (2014).

  26. 26.

    & Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone. Science 349, 1224–1228 (2015).

  27. 27.

    , , , & Production of 6-methylsalicylic acid by expression of a fungal polyketide synthase activates disease resistance in tobacco. Plant Cell 13, 1401–1409 (2001).

  28. 28.

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

  29. 29.

    & Combinatorial biosynthesis of reduced polyketides. Nat. Rev. Microbiol. 3, 925–936 (2005).

  30. 30.

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

  31. 31.

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

  32. 32.

    & Lipid comprising docosapentaenoic acid. US patent application 20150374654A1 (2015).

  33. 33.

    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).

  34. 34.

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

  35. 35.

    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).

  36. 36.

    , , , & Functional organization of the cassava vein mosaic virus (CsVMV) promoter. Plant Mol. Biol. 37, 1055–1067 (1998).

  37. 37.

    , , , & 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).

  38. 38.

    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).

  39. 39.

    , , & Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol. Biol. 2, 335–350 (1983).

  40. 40.

    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).

  41. 41.

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

  42. 42.

    , , & Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana. Planta 217, 507–516 (2003).

  43. 43.

    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).

  44. 44.

    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).

  45. 45.

    , & 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).

  46. 46.

    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).

  47. 47.

    & Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8, 4321–4325 (1980).

  48. 48.

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

  49. 49.

    & BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

  50. 50.

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

  51. 51.

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

Download references

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).

Author information

Author notes

    • James G Metz

    Present address: Metz Consultancy, Longmont, Colorado, USA.

Affiliations

  1. Dow AgroSciences LLC, Research and Development, Indianapolis, Indiana, USA.

    • Terence A Walsh
    • , Scott A Bevan
    • , Daniel J Gachotte
    • , Cory M Larsen
    • , William A Moskal
    • , P A Owens Merlo
    • , Lyudmila V Sidorenko
    • , Ronnie E Hampton
    • , Virginia Stoltz
    • , Dayakar Pareddy
    • , Geny I Anthony
    • , Pudota B Bhaskar
    • , Pradeep R Marri
    • , Lauren M Clark
    • , Wei Chen
    • , Patrick S Adu-Peasah
    •  & Steven T Wensing
  2. DSM Nutritional Products LLC, Columbia, Maryland, USA.

    • Ross Zirkle
  3. DSM Nutritional Products LLC, Boulder, Colorado, USA.

    • James G Metz

Authors

  1. Search for Terence A Walsh in:

  2. Search for Scott A Bevan in:

  3. Search for Daniel J Gachotte in:

  4. Search for Cory M Larsen in:

  5. Search for William A Moskal in:

  6. Search for P A Owens Merlo in:

  7. Search for Lyudmila V Sidorenko in:

  8. Search for Ronnie E Hampton in:

  9. Search for Virginia Stoltz in:

  10. Search for Dayakar Pareddy in:

  11. Search for Geny I Anthony in:

  12. Search for Pudota B Bhaskar in:

  13. Search for Pradeep R Marri in:

  14. Search for Lauren M Clark in:

  15. Search for Wei Chen in:

  16. Search for Patrick S Adu-Peasah in:

  17. Search for Steven T Wensing in:

  18. Search for Ross Zirkle in:

  19. Search for James G Metz in:

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.

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.

Corresponding author

Correspondence to Terence A Walsh.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–4 and Supplementary Tables 1–10

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nbt.3585

Further reading