title: Bioretrosynthetic construction of a didanosine biosynthetic pathway.
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.
authors.affiliation: 1] Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee, USA. [2].
authors.forename: William R
authors.initials: WR
authors.lastname: Birmingham
authors.affiliation: Chemical and Physical Biology Program, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: Chrystal A
authors.initials: CA
authors.lastname: Starbird
authors.affiliation: 1] Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, USA. [2].
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Panosian
authors.affiliation: 1] Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA. [2].
authors.forename: David P
authors.initials: DP
authors.lastname: Nannemann
authors.affiliation: 1] Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee, USA. [2] Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: T M
authors.initials: TM
authors.lastname: Iverson
authors.affiliation: 1] Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee, USA. [2] Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA.
authors.forename: Brian O
authors.initials: BO
authors.lastname: Bachmann
mesh_terms: D055162:Biocatalysis; D016049:Didanosine / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004798:Enzymes / Q000378:metabolism; D008958:Models, Molecular
chemical_list: D004798:Enzymes; D016049:Didanosine