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.

中文翻译：

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基因组中的脱氧木酮糖磷酸还原异构酶（DXR）突变赋予了抗疟疾药物磷霉素在大肠杆菌中的抗性。

序列到活性作图技术正在迅速发展，使得能够产生和分离赋予新表型的突变。在这里，我们使用了具有CRISPR功能的可追踪基因组工程（CREATE）技术来研究基本ispC基因在大肠杆菌中的天然基因组环境中的抑制作用。我们创建了一个完整的饱和文库，包含接近配体结合口袋的33个位点，并用抗疟药fosmidomycin攻击了该文库，该药物靶向ispC基因产物DXR。这种选择特别具有挑战性，因为它在大肠杆菌中相对较弱，具有多种自然发生的抗药性途径。我们确定了一些以前未报道的，赋予磷霉素抗性的突变，在高度保守的位置，也存在于包括疟疾的恶性疟原虫在内的病原体中。这种方法可能对分离抗药性突变具有影响，并可能影响基于磷霉素的下一代药物的设计。