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

Nature Biotechnology volume 34, pages 881–887 (2016)Cite this article

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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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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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  1. James G Metz

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

Authors and 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

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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.

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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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