Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to the Antimalarial Drug Fosmidomycin in <i>E. coli</i> Gur Pines Eun Joong Oh Marcelo C. Bassalo Alaksh Choudhury Andrew D. Garst Reilly G. Fankhauser Carrie A. Eckert Ryan T. Gill 10.1021/acssynbio.8b00219.s001 https://acs.figshare.com/articles/journal_contribution/Genomic_Deoxyxylulose_Phosphate_Reductoisomerase_DXR_Mutations_Conferring_Resistance_to_the_Antimalarial_Drug_Fosmidomycin_in_i_E_coli_i_/7436303 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 <i>ispC</i> gene in its native genomic context in <i>Escherichia coli</i>. 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 <i>ispC</i> gene product, DXR. This selection is especially challenging since it is relatively weak in <i>E. coli</i>, 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 <i>Plasmodium falciparum</i>. This approach may have implications for the isolation of resistance-conferring mutations and may affect the design of future generations of fosmidomycin-based drugs. 2018-11-21 00:00:00 Antimalarial Drug Fosmidomycin E . coli Sequence E . coli site technology ispC gene product CREATE Mutations Conferring Resistance generation Genomic Deoxyxylulose Phosphate Reductoisomerase mutation resistance ligand binding pocket activity mapping technologies trackable genome engineering CRISPR . coli antimalarial drug fosmidomycin isolation DXR malaria-inducing Plasmodium falciparum