Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5-fluorocytosine toxicity in HT1080 cells

Tiana D. Warren, Krishna Patel, Jordan L. Rivera, James R. Eshleman, Marc Ostermeier

Research output: Contribution to journalArticlepeer-review

Abstract

Improved prodrug-activating enzymes have the potential to increase the therapeutic efficacy of gene-directed enzyme prodrug therapy (GDEPT). Yeast cytosine deaminase (yCD) is commonly used to convert the prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic 5-fluorouracil for GDEPT. Mutagenesis studies on yCD aimed at improving its application in GDEPT have been limited to subsets of residues or have sought to improve a single property of the enzyme. We performed comprehensive site-saturation mutagenesis (CSM) on yCD designed to create all 2,983 possible unique protein mutants with a single amino acid substitution. We identified active variants through Escherichia coli genetic complementation and screened these mutants, and combinations thereof, for increased ability to sensitize E. coli and HT1080 fibrosarcoma cells to 5-FC. Several mutants identified in this study showed increased sensitization ability for both E. coli and HT1080 cells indicating that CSM is an effective directed evolution tool for identifying unexpectedly beneficial mutations.

Original languageEnglish (US)
Article numbere16688
JournalAIChE Journal
Volume66
Issue number3
DOIs
StatePublished - Mar 1 2020

Keywords

  • 5-fluorocytosine
  • directed evolution
  • enzyme prodrug therapy
  • protein engineering
  • yeast cytosine deaminase

ASJC Scopus subject areas

  • Biotechnology
  • Environmental Engineering
  • Chemical Engineering(all)

Fingerprint Dive into the research topics of 'Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5-fluorocytosine toxicity in HT1080 cells'. Together they form a unique fingerprint.

Cite this