Gehrke Sebastian S, Kumar Garima, Yokubynas Nicole A, Côté Jean-Philippe, Wang Wenliang, French Shawn, MacNair Craig R, Wright Gerard D, Brown Eric D
Michael G. DeGroote Institute of Infectious Disease Research, Department of Biochemistry and Biomedical Science, McMaster University , 1200 Main Street West, Hamilton Ontario L8N 3ZS, Canada.
ACS Infect Dis. 2017 Dec 8;3(12):955-965. doi: 10.1021/acsinfecdis.7b00149. Epub 2017 Nov 7.
Actinomycete secondary metabolites are a renowned source of antibacterial chemical scaffolds. Herein, we present a target-specific approach that increases the detection of antimetabolites from natural sources by screening actinomycete-derived extracts against nutrient transporter deletion strains. On the basis of the growth rescue patterns of a collection of 22 Escherichia coli (E. coli) auxotrophic deletion strains representative of the major nutrient biosynthetic pathways, we demonstrate that antimetabolite detection from actinomycete-derived extracts prepared using traditional extraction platforms is masked by nutrient supplementation. In particular, we find poor sensitivity for the detection of antimetabolites targeting vitamin biosynthesis. To circumvent this and as a proof of principle, we exploit the differential activity of actinomycete extracts against E. coli ΔyigM, a biotin transporter deletion strain versus wildtype E. coli. We achieve more than a 100-fold increase in antimetabolite sensitivity using this method and demonstrate a successful bioassay-guided purification of the known biotin antimetabolite, amiclenomycin. Our findings provide a unique solution to uncover the full potential of naturally derived antibiotics.
放线菌次级代谢产物是著名的抗菌化学支架来源。在此，我们提出一种靶向特异性方法，通过针对营养转运体缺失菌株筛选放线菌衍生提取物，增加从天然来源中抗代谢物的检测。基于代表主要营养生物合成途径的22株大肠杆菌营养缺陷型缺失菌株的生长拯救模式，我们证明使用传统提取平台制备的放线菌衍生提取物中的抗代谢物检测被营养补充所掩盖。特别是，我们发现针对维生素生物合成的抗代谢物检测灵敏度较低。为了规避这一问题并作为原理证明，我们利用放线菌提取物对大肠杆菌ΔyigM（一种生物素转运体缺失菌株）与野生型大肠杆菌的差异活性。使用这种方法，我们将抗代谢物灵敏度提高了100倍以上，并成功地通过生物测定指导纯化了已知的生物素抗代谢物氨甲环霉素。我们的发现为揭示天然抗生素的全部潜力提供了独特的解决方案。
