The next logical step in the development of unnatural biocatalytic transformations is their concatenation into multi-step biosynthetic pathways. We have adapted the design paradigm of retrosynthesis, historically used as a planning tool in chemical synthesis, as an applied experimental evolutionary method for the generation of a new biosynthetic pathway for the nucleoside analog didanosine. Specifically, we use selective pressure from the last step in a biochemical sequence to drive the evolution of biocatalytic genes in a retro-consecutive fashion. As a biosynthetic retro-extension from our previously engineered human purine nucleoside phosphorylase, we have used structure-guided and whole gene mutagenesis to evolve B. cereus phosphopentomutase to accept the non-natural substrate 2,3-dideoxyribose 5‑phosphate with ~700-fold change in substrate specificity and 3-fold increased activity in cell lysate. Further pathway extension using an engineered E. coli ribokinase mutant provided ~50-fold total increase in dideoxyinosine production and a 9,500-fold change in nucleoside production specificity in a five step-biosynthetic pathway beginning from the sugar analog 2,3‑dideoxyribose (Didanosine) and including an ATP regeneration cycle. A surprising benefit of applying bioretrosynthesis in this system was the discovery of a pathway-shortening biochemical bypass via the engineered ribokinase.

Adapting the retrograde evolution hypothesis to build new biosynthetic pathways for unatural molecules

249th National Meeting (2015)

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