The emergence of pathogens for which all current treatments are ineffective has placed the antibiotic resistance crisis front and center for many communities. To address the trend of ever-increasing incidences of antibiotic resistance, anti-virulence strategies have been proposed as a promising solution. We aim to explore inhibition of bacterial nitric oxide (NO) defenses as a broad spectrum anti-virulence strategy, due to the importance of NO to innate immunity. To identify druggable genetic mediators of NO defense in bacteria, I used transposon insertion sequencing (Tn-seq) to screen Escherichia coli genome. Previously, it was found that low-NO-tolerance mutant (Δhmp) would cheat to obtain better fitness during NO treatment. While the extracellular NO donor fails to distinguish NO tolerance based on strain growth, we found that an intracellular NO donor generates a 5-fold difference in growth between wildtype and Δhmp during the assay. Using the intracellular NO donor in Tn-seq, we discovered several genes in branch-chain amino acid synthesis and Entner-Doudoroff shunt, are important for NO detoxification and recovery of Escherichia coli.