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Title page for ETD etd-11212013-085213


Type of Document Dissertation
Author Birmingham, William Ross
Author's Email Address william.r.birmingham@vanderbilt.edu
URN etd-11212013-085213
Title Bioretrosynthetic Construction of a Non-Natural Nucleoside Analog Biosynthetic Pathway
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Brian O. Bachmann Committee Chair
Carmelo J. Rizzo Committee Member
Lawrence J. Marnett Committee Member
Richard N. Armstrong Committee Member
Tina M. Iverson Committee Member
Keywords
  • directed evolution
  • retrograde evolution
  • bioretrosynthesis
  • dideoxyinosine
  • phosphopentomutase
  • ribokinase
Date of Defense 2013-10-15
Availability unrestricted
Abstract
Engineered biocatalysts have become increasingly sought after as replacements for individual chemical synthetic steps. However, their implementation in multistep sequences as biosynthetic pathways has proven to be a significant challenge, in part due to a lack of broadly applicable methods for their assembly. One strategy for construction of a de novo biosynthetic pathway can be inspired by the theory of retrograde evolution, where pathway enzymes are recruited and optimized in the reverse order of biosynthesis. Herein, this process, termed ‘bioretrosynthesis’, is demonstrated through the construction and evolution of a pathway for in vitro production of the antiretroviral nucleoside analog didanosine (2,3 dideoxyinosine). Using methods in structure guided mutagenesis and directed evolution, we have evolved phosphopentomutase to accept 2,3-dideoxyribose 5 phosphate with a 700-fold change in substrate selectivity and 3-fold improved turnover in cell lysate as a retrograde extension from an engineered purine nucleoside phosphorylase. A second pathway extension via evolution of ribokinase resulted in a didanosine biosynthetic pathway with 9,500-fold change in nucleoside production selectivity. Surprisingly, the product of bioretrosynthesis in this step was not a retrograde extension from phosphopentomutase, but instead resulted in the discovery of a pathway shortening bypass via a new phosphorylation regiochemistry in the engineered ribokinase.
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