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

doi:10.1002/aic.16688

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

School of Medicine

Research output: Contribution to journal › Article › peer-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 language	English (US)
Article number	e16688
Journal	AIChE Journal
Volume	66
Issue number	3
DOIs	https://doi.org/10.1002/aic.16688
State	Published - 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)

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title = "Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5-fluorocytosine toxicity in HT1080 cells",

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.",

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note = "Funding Information: This work was supported by the National Institute of Health (R01GM066972 to M.O.), the National Science Foundation (CBET-1803805 to M.O.) and the National Science Foundation Graduate Research Fellowship (DGE-1232825 to T.D.W.). Funding Information: National Institutes of Health, Grant/Award Number: R01GM066972 ; National Science Foundation Graduate Research Fellowship; National Science Foundation, Grant/Award Numbers: CBET-1803805, DGE-1232825 Publisher Copyright: {\textcopyright} 2019 American Institute of Chemical Engineers",

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

Abstract

Keywords

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