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Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform

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Abstract

The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways1,2,3 in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-l-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus4. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l−1) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.

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Figure 1: Functional validation of a DEHU transporter gene (Ac_DHT1) isolated via complementary strategies.
Figure 2: Evolutionary correlation among quinate (QutD), Ac_DHT1, and homologous transporters.
Figure 3: Engineering mannitol metabolism in S. cerevisiae.
Figure 4: Ethanol fermentation from mannitol and DEHU.

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Change history

  • 08 January 2014

    A present address was added for author Avinash Gill.

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Acknowledgements

We thank R. Schekman, J. H. D. Cate and P. A. Silver for critical discussion and suggestions. This work is supported by the DOE under an Advanced Research Projects Agency–Energy (ARPA-E) award (DE-AR0000006), by CORFO INNOVA CHILE (código 09CTEI-6866), and by Statoil ASA. This report was prepared as an account of work sponsored by an agency of the US government. Neither the US government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favouring by the US government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the US government or any agency thereof.

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Author notes

  1. Christine Nicole S. Santos, Drew D. Regitsky, Avinash Gill, Shital A. Tripathi & Vineet Rajgarhia

    Present address: Present addresses: Manus Biosynthesis Inc., 790 Memorial Drive, Suite 102, Cambridge, Massachusetts 02139 (C.N.S.S.); Calysta Energy, 1140 O'Brien Drive, Menlo Park, California 94025 (D.D.R.); Sutro Biopharma lnc., 310 Utah Avenue, Suite 150, South San Francisco, California 94080, USA (A.G.); Total New Energies USA, 5858 Horton Street, Emeryville, California 94560 (S.A.T.; V.R.).,

  2. Maria Enquist-Newman, Ann Marie E. Faust and Daniel D. Bravo: These authors contributed equally to this work.

Authors and Affiliations

  1. Bio Architecture Lab Inc., 604 Bancroft Way, Suite A, Berkeley, California 94710, USA,

    Maria Enquist-Newman, Ann Marie E. Faust, Daniel D. Bravo, Christine Nicole S. Santos, Ryan M. Raisner, Arthur Hanel, Preethi Sarvabhowman, Chi Le, Drew D. Regitsky, Susan R. Cooper, Alana Clark, Yessica Martinez, Joshua Goldsmith, Min Y. Cho, Paul D. Donohoue, Lily Luo, Brigit Lamberson, Pramila Tamrakar, Edward J. Kim, Jeffrey L. Villari, Avinash Gill, Shital A. Tripathi, Padma Karamchedu, Carlos J. Paredes, Vineet Rajgarhia, Richard B. Bailey, Nicholas L. Ohler, Candace Swimmer & Yasuo Yoshikuni

  2. Department of Chemical Engineering and Materials Science, Michigan State University, 2527 Engineering Building, East Lansing, Michigan 48824-1226, USA,

    Lars Peereboom & Dennis J. Miller

  3. Statoil ASA, Statoil Research Centre, Arkitekt Ebbells vei 10, Rotvoll, 7005 Trondheim, Norway,

    Hans Kristian Kotlar

  4. BALChile S.A., Badajoz 100, Oficina 1404, Las Condes, Santiago 7550000, Chile,

    Yasuo Yoshikuni

  5. BAL Biofuels S.A., Badajoz 100, Oficina 1404, Las Condes, Santiago 7550000, Chile,

    Yasuo Yoshikuni

Authors
  1. Maria Enquist-Newman
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  2. Ann Marie E. Faust
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  3. Daniel D. Bravo
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  4. Christine Nicole S. Santos
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  5. Ryan M. Raisner
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  6. Arthur Hanel
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  7. Preethi Sarvabhowman
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  8. Chi Le
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  32. Yasuo Yoshikuni
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Contributions

Y.Y. conceived the overall project. C.S., Y.Y., M.E.-N., N.L.O., R.B.B., H.K.K. and V.R. supervised the overall project. R.M.R., S.R.C., P.D.D., Y.M. and A.G. characterised the A. cruciatus alginate degradation profiles. A.M.E.F., M.E.-N., P.K., L.L., M.Y.C. and S.A.T. constructed all S. cerevisiae host strains for screening. R.M.R. and P.D.D. prepared A. cruciatus genomic DNA and total RNA for the high-throughput sequencing analysis. D.D.R., R.M.R. and Y.Y. assembled and analysed the RNA-seq data. C.N.S.S., P.T., M.E.-N., J.G. and C.J.P. constructed and screened the A. cruciatus cDNA library. M.E.-N. and J.L.V. identified yeast strains that can grow on mannitol and performed microarray analyses to isolate mannitol catabolism genes. A.M.E.F., M.E.-N., L.L., M.Y.C., P.D.D. and J.G. constructed all S. cerevisiae host strains for fermentation experiments. Y.Y., D.D.B., M.E.-N. and A.C. carried out all adaptation and ethanol fermentation experiments. A.H. and P.S. developed and carried out biochemical assays for enzyme characterisation. S.R.C., P.S., Y.Y., D.D.B., B.L. and A.H. performed all necessary analytical experiments and analysed all fermentation samples. L.P., C.L., E.J.K., N.L.O. and D.J.M. conducted sample preparation and the NMR analysis of the DEHU structure. C.L., P.S., D.D.B. and Y.Y. prepared media for fermentation, Y.Y., M.E.-N., A.M.E.F., C.S., D.D.B., C.N.S.S. and L.P. wrote and revised the manuscript.

Corresponding authors

Correspondence to Candace Swimmer or Yasuo Yoshikuni.

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Competing interests

Y.Y., J.L.V., S.R.C., V.R., S.A.T., M.E.-N., R.M.R., A.G., A.H., C.N.S.S., D.D.R, C.S., R.B.B. and A.M.E.F. are co-inventors of a patent application entitled “Methods and Compositions for Growth of Yeast on Alginate” (PCT/US2013/021328).

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Enquist-Newman, M., Faust, A., Bravo, D. et al. Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform. Nature 505, 239–243 (2014). https://doi.org/10.1038/nature12771

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