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Biotechnology

Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

James Kirby, Minobu Nishimoto, Ruthie W. N. Chow, Edward E. K. Baidoo, George Wang, Joel Martin, Wendy Schackwitz, Rossana Chan, Jeffrey L. Fortman, Jay D. Keasling
R. E. Parales, Editor
James Kirby
California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USAJoint BioEnergy Institute, Emeryville, California, USA
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Minobu Nishimoto
California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USAJoint BioEnergy Institute, Emeryville, California, USA
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Ruthie W. N. Chow
California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA
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Edward E. K. Baidoo
Joint BioEnergy Institute, Emeryville, California, USA
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George Wang
Joint BioEnergy Institute, Emeryville, California, USA
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Joel Martin
Joint Genome Institute, Walnut Creek, California, USA
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Wendy Schackwitz
Joint Genome Institute, Walnut Creek, California, USA
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Rossana Chan
California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA
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Jeffrey L. Fortman
California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA
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Jay D. Keasling
California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USAJoint BioEnergy Institute, Emeryville, California, USADepartment of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, California, USAPhysical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA
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R. E. Parales
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DOI: 10.1128/AEM.02920-14
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ABSTRACT

Terpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5 sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of a dxs deletion in Escherichia coli grown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-type E. coli yajO gene, annotated as a putative xylose reductase, or via various mutations in the native ribB gene. In vitro analysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway in E. coli for production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

FOOTNOTES

    • Received 5 September 2014.
    • Accepted 9 October 2014.
    • Accepted manuscript posted online 17 October 2014.
  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02920-14.

  • Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate
James Kirby, Minobu Nishimoto, Ruthie W. N. Chow, Edward E. K. Baidoo, George Wang, Joel Martin, Wendy Schackwitz, Rossana Chan, Jeffrey L. Fortman, Jay D. Keasling
Applied and Environmental Microbiology Dec 2014, 81 (1) 130-138; DOI: 10.1128/AEM.02920-14

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Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate
James Kirby, Minobu Nishimoto, Ruthie W. N. Chow, Edward E. K. Baidoo, George Wang, Joel Martin, Wendy Schackwitz, Rossana Chan, Jeffrey L. Fortman, Jay D. Keasling
Applied and Environmental Microbiology Dec 2014, 81 (1) 130-138; DOI: 10.1128/AEM.02920-14
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