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Title page for ETD etd-05112010-084149

Type of Document Dissertation
Author Scism, Robert Allen
Author's Email Address rscism@gmail.com
URN etd-05112010-084149
Title Directed evolution and pathway engineering for nucleotide analogue biosynthesis
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Brian Bachmann Committee Chair
David Wright Committee Member
Lawrence Marnett Committee Member
Michael Stone Committee Member
Richard Armstrong Committee Member
  • atp regeneration
  • atp recycling
  • CLEA
  • immobilized
  • glutaraldehyde
  • biocatalysis
Date of Defense 2010-05-05
Availability unrestricted
Nucleotide analogues are essential pharmaceuticals with challenges in preparation due to need for control over regio and stereochemistry, and protecting group manipulations. Enzymatic biosynthesis of these analogues provides solutions to these synthetic issues, but suffers from stringent substrate requirements of the enzymes. Directed evolution presents a possible solution to amend these enzymes toward alternative substrate acceptance. We propose a superior method for nucleotide analogue production, achieved through directed evolution of nucleotide synthesizing enzymes, using an in vivo screening method. Combination of the evolved biocatalyst into an engineered biosynthetic pathway will provide an efficient system for nucleotide analogue production from ribose.

This dissertation describes using directed evolution as a means to alter whole cell turnover rates and active site specificity of hypoxanthine phosphoribosyl transferase (HPRT) for increased production of the nucleotide analogue ribavirin monophosphate. A novel in vivo screening method for negative selection of variant HPRT enzymes is introduced, and the subsequent validation of the screening method and characterization of the mutant encoded enzymes is described.

The utilization of the evolved HPRT as a general nucleotide analogue biocatalyst is detailed. A method for assaying general nucleobase turnover, and end product purification and characterization is also described. Moreover, the addition of a series of enzymes which allow a multistep synthesis of nucleotides from ribose is illustrated. Inclusion of additional enzymes for cofactor recycling adds utility and relieves byproduct inhibition. Finally, stability and reusability of the pathway is increased by self-immobilization in a cross-linked enzyme aggregate.

The sum of this work presents a significant improvement over current methods in nucleotide synthesis by alleviating current impediments due to lack of control over regio and stereochemistry, and by eliminating need for isolation of unstable intermediates, thus providing a green synthetic alternative in the synthesis of nucleotide analogues.

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