Bioretrosynthetic construction of a didanosine biosynthetic pathway.

Birmingham WR, Starbird CA, Panosian TD, Nannemann DP, Iverson TM, Bachmann BO
Nat Chem Biol. 2014 10 (5): 392-9

PMID: 24657930 · PMCID: PMC4017637 · DOI:10.1038/nchembio.1494

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.

MeSH Terms (5)

Biocatalysis Didanosine Directed Molecular Evolution Enzymes Models, Molecular

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