Bioretrosynthetic construction of a didanosine biosynthetic pathway

Nat Chem Biol. 2014 May;10(5):392-9. doi: 10.1038/nchembio.1494. Epub 2014 Mar 23.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Biocatalysis
  • Didanosine / metabolism*
  • Directed Molecular Evolution
  • Enzymes / metabolism
  • Models, Molecular


  • Enzymes
  • Didanosine

Associated data

  • PDB/4LR7
  • PDB/4LR8
  • PDB/4LR9
  • PDB/4LRA
  • PDB/4LRB
  • PDB/4LRC
  • PDB/4LRD
  • PDB/4LRE
  • PDB/4LRF