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Bioretrosynthetic construction of a didanosine biosynthetic pathway

Nature Chemical Biology volume 10, pages 392–399 (2014)Cite this article

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Abstract

Concatenation of engineered biocatalysts into multistep pathways markedly increases their utility, but the development of generalizable assembly methods remains a major challenge. Herein we evaluate 'bioretrosynthesis', which is an application of the retrograde evolution hypothesis, for biosynthetic pathway construction. To test bioretrosynthesis, we engineered a pathway for synthesis of the antiretroviral nucleoside analog didanosine (2′,3′-dideoxyinosine). Applying both directed evolution– and structure-based approaches, we began pathway construction with a retro-extension from an engineered purine nucleoside phosphorylase and evolved 1,5-phosphopentomutase to accept the substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate selectivity and threefold increased turnover in cell lysate. A subsequent retrograde pathway extension, via ribokinase engineering, resulted in a didanosine pathway with a 9,500-fold change in nucleoside production selectivity and 50-fold increase in didanosine production. Unexpectedly, the result of this bioretrosynthetic step was not a retro-extension from phosphopentomutase but rather the discovery of a fortuitous pathway-shortening bypass via the engineered ribokinase.

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Figure 1: Model inosine biosynthetic pathway and proposed bioretrosynthesis of didanosine.
Figure 2: First shell residues targeted for PPM saturation mutagenesis.
Figure 3: Lineage and characterization of PPM variants through generations of evolution.
Figure 4: Optimization of RK via didanosine production assay.
Figure 5: Selectivity and activity changes in selected variant enzymes as assessed using coupled assays.
Figure 6: Progression of pathway evolution throughout enzyme engineering stages.

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Acknowledgements

We thank V. Phelan, K. McCulloch and C. Goodwin for assistance with data acquisition. We also thank J. Zang (Chinese Academy of Sciences), A. Joachimiak (Argonne National Laboratory and University of Chicago), J. Deutscher (Centre national de la recherche scientifique) and S. Ealick (Cornell University) for expression plasmids used in this study. This work was supported by the US National Institutes of Health (NIH) grant T32 GM065086 and the D. Stanley and Ann T. Tarbell Endowment fund (W.R.B.), NIH training grant 5T32GM008320, a US National Science Foundation individual graduate fellowship DGE:0909667 (C.A.S.), NIH training grants T32NS07491 and 5T32GM008320 (T.D.P.), NIH training grant T90 DA022873 (D.P.N.), NIH grants GM079419 (to T.M.I.) and GM077189 (B.O.B.) and the Vanderbilt Institute of Chemical Biology. The use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357. The use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). The use of the Vanderbilt robotic crystallization facility was supported by NIH grant S10 RR026915.

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Author notes

  1. William R Birmingham, Timothy D Panosian & David P Nannemann

    Present address: Present address: Department of Chemistry, University of Manchester, Manchester, UK (W.R.B.); Monsanto Co., Chesterfield, Missouri, USA (T.D.P.); EMD Serono, Inc., Rockland, Massachusetts, USA (D.P.N.).,

Authors and Affiliations

  1. Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee, USA

    William R Birmingham, T M Iverson & Brian O Bachmann

  2. Chemical and Physical Biology Program, Vanderbilt University Medical Center, Nashville, Tennessee, USA

    Chrystal A Starbird

  3. Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, USA

    Timothy D Panosian & T M Iverson

  4. Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA

    David P Nannemann & Brian O Bachmann

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Contributions

B.O.B. supervised bioretrosynthetic studies, and T.M.I. supervised the X-ray crystallographic work. W.R.B. and D.P.N. designed assays. W.R.B. performed assays, screened mutagenesis libraries, performed kinetic characterization and tested enzymes in the biosynthetic pathway studies. C.A.S. determined the crystal structures of V158L and 4H11 PPM variants. T.D.P. determined crystal structures of wild-type PPM and the S154A and S154G variants. D.P.N. established initial synthesis routes of dideoxyribose and dideoxyribose 5-phosphate. W.R.B., C.A.S., T.M.I. and B.O.B. wrote the paper.

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Correspondence to T M Iverson or Brian O Bachmann.

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Supplementary Note, Supplementary Results, Supplementary Figures 1–20 and Supplementary Tables 1–7. (PDF 4014 kb)

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Birmingham, W., Starbird, C., Panosian, T. et al. Bioretrosynthetic construction of a didanosine biosynthetic pathway. Nat Chem Biol 10, 392–399 (2014). https://doi.org/10.1038/nchembio.1494

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