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Title: Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to the Antimalarial Drug Fosmidomycin in E. coli

Abstract

Sequence to activity mapping technologies are rapidly developing, enabling the generation and isolation of mutations conferring novel phenotypes. Here we used the CRISPR enabled trackable genome engineering (CREATE) technology to investigate the inhibition of the essential ispC gene in its native genomic context in Escherichia coli. We created a full saturation library of 33 sites proximal to the ligand binding pocket and challenged this library with the antimalarial drug fosmidomycin, which targets the ispC gene product, DXR. This selection is especially challenging since it is relatively weak in E. coli, with multiple naturally occurring pathways for resistance. We identified several previously unreported mutations that confer fosmidomycin resistance, in highly conserved sites that also exist in pathogens including the malaria-inducing Plasmodium falciparum. This approach may have implications for the isolation of resistance-conferring mutations and may affect the design of future generations of fosmidomycin-based drugs.

Authors:
ORCiD logo [1]; ORCiD logo [1];  [2];  [3];  [4]; ORCiD logo [4]; ORCiD logo [5]; ORCiD logo [1]
  1. Renewable and Sustainable Energy Institute, University of Colorado Boulder, 027 UCB, Boulder, Colorado 80309, United States; Department of Chemical and Biological Engineering, University of Colorado Boulder, 596 UCB, Boulder, Colorado 80309, United States
  2. Renewable and Sustainable Energy Institute, University of Colorado Boulder, 027 UCB, Boulder, Colorado 80309, United States; Department of Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, 347 UCB, Boulder, Colorado 80309, United States
  3. Department of Chemical and Biological Engineering, University of Colorado Boulder, 596 UCB, Boulder, Colorado 80309, United States
  4. Renewable and Sustainable Energy Institute, University of Colorado Boulder, 027 UCB, Boulder, Colorado 80309, United States
  5. Renewable and Sustainable Energy Institute, University of Colorado Boulder, 027 UCB, Boulder, Colorado 80309, United States; Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, Colorado 80401, United States
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1491138
Report Number(s):
NREL/JA-2700-73080
Journal ID: ISSN 2161-5063
DOE Contract Number:  
AC36-08GO28308
Resource Type:
Journal Article
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 7; Journal Issue: 12; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; acquired resistance; CRISPR/Cas9; deoxyxylulose phosphate reductoisomerase; fosmidomycin; malaria; sequence to activity mapping

Citation Formats

Pines, Gur, Oh, Eun Joong, Bassalo, Marcelo C., Choudhury, Alaksh, Garst, Andrew D., Fankhauser, Reilly G., Eckert, Carrie A., and Gill, Ryan T. Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to the Antimalarial Drug Fosmidomycin in E. coli. United States: N. p., 2018. Web. doi:10.1021/acssynbio.8b00219.
Pines, Gur, Oh, Eun Joong, Bassalo, Marcelo C., Choudhury, Alaksh, Garst, Andrew D., Fankhauser, Reilly G., Eckert, Carrie A., & Gill, Ryan T. Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to the Antimalarial Drug Fosmidomycin in E. coli. United States. doi:10.1021/acssynbio.8b00219.
Pines, Gur, Oh, Eun Joong, Bassalo, Marcelo C., Choudhury, Alaksh, Garst, Andrew D., Fankhauser, Reilly G., Eckert, Carrie A., and Gill, Ryan T. Wed . "Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to the Antimalarial Drug Fosmidomycin in E. coli". United States. doi:10.1021/acssynbio.8b00219.
@article{osti_1491138,
title = {Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to the Antimalarial Drug Fosmidomycin in E. coli},
author = {Pines, Gur and Oh, Eun Joong and Bassalo, Marcelo C. and Choudhury, Alaksh and Garst, Andrew D. and Fankhauser, Reilly G. and Eckert, Carrie A. and Gill, Ryan T.},
abstractNote = {Sequence to activity mapping technologies are rapidly developing, enabling the generation and isolation of mutations conferring novel phenotypes. Here we used the CRISPR enabled trackable genome engineering (CREATE) technology to investigate the inhibition of the essential ispC gene in its native genomic context in Escherichia coli. We created a full saturation library of 33 sites proximal to the ligand binding pocket and challenged this library with the antimalarial drug fosmidomycin, which targets the ispC gene product, DXR. This selection is especially challenging since it is relatively weak in E. coli, with multiple naturally occurring pathways for resistance. We identified several previously unreported mutations that confer fosmidomycin resistance, in highly conserved sites that also exist in pathogens including the malaria-inducing Plasmodium falciparum. This approach may have implications for the isolation of resistance-conferring mutations and may affect the design of future generations of fosmidomycin-based drugs.},
doi = {10.1021/acssynbio.8b00219},
journal = {ACS Synthetic Biology},
issn = {2161-5063},
number = 12,
volume = 7,
place = {United States},
year = {2018},
month = {11}
}