Pines Gur, Oh Eun Joong, Bassalo Marcelo C, Choudhury Alaksh, Garst Andrew D, Fankhauser Reilly G, Eckert Carrie A, Gill Ryan T
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.
ACS Synth Biol. 2018 Dec 21;7(12):2824-2832. doi: 10.1021/acssynbio.8b00219. Epub 2018 Dec 7.
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.
序列到活性映射技术正在迅速发展，能够产生和分离赋予新表型的突变。在这里，我们使用了基于CRISPR的可追踪基因组工程（CREATE）技术，在大肠杆菌的天然基因组环境中研究必需基因ispC的抑制作用。我们在配体结合口袋附近创建了一个包含33个位点的完全饱和文库，并用靶向ispC基因产物DXR的抗疟药物磷霉素对该文库进行筛选。这种筛选尤其具有挑战性，因为它在大肠杆菌中的作用相对较弱，且存在多种天然的抗性途径。我们在高度保守的位点上鉴定出了几个以前未报道的赋予磷霉素抗性的突变，这些位点在包括引发疟疾的恶性疟原虫在内的病原体中也存在。这种方法可能对分离赋予抗性的突变有影响，并且可能会影响下一代基于磷霉素的药物设计。
