No abstract

The production of optimized strains of a specific phenotype requires the construction and testing of a large number of genome modifications and combinations thereof. Most bacterial iterative genome-editing methods include essential steps to eliminate selection markers, or to cure plasmids. Additionally, the presence of escapers leads to time-consuming separate single clone picking and subsequent cultivation steps. Herein, we report a genome-editing method based on a Rock-Paper-Scissors (RPS) strategy. Each of three constructed sgRNA plasmids can cure, or be cured by, the other two plasmids in the system; plasmids from a previous round of editing can be cured while the current round of editing takes place. Due to the enhanced curing efficiency and embedded double check mechanism, separate steps for plasmid curing or confirmation are not necessary, and only two times of cultivation are needed per genome-editing round. This method was successfully demonstrated in Escherichia coli and Klebsiella pneumoniae with both gene deletions and replacements. To the best of our knowledge, this is the fastest and most robust iterative genome-editing method, with the least times of cultivation decreasing the possibilities of spontaneous genome mutations.

Nucleosides and purine analogues have multiple functions in cell physiology, food additives, and pharmaceuticals, and some are produced on a large scale using different microorganisms. However, biosynthesis of purines is still lacking. In the present study, we engineered the de novo purine biosynthesis pathway, branched pathways, and a global regulator to ensure highly efficient hypoxanthine production by Escherichia coli. The engineered strain Q2973 produced 1243 mg/L hypoxanthine in fed-batch fermentation, accompanied by an extremely low accumulation of byproducts such as acetate and xanthine. We also performed global gene expression analysis to illustrate the mechanism for improving hypoxanthine production. This study demonstrated the feasibility of large-scale hypoxanthine production byan engineered E. coli strain, and provides a reference for subsequent studies on purine analogues and nucleosides.

Phenolic compounds are the most ubiquitously distributed pollutants, and are highly toxic to living organisms, however the detailed mechanism how phenols exert toxic effects remains elusive. Here, Escherichia coli and phloroglucinol are adapted as proxy to elucidate the molecular mechanism of phenols' toxicity. We demonstrated that phloroglucinol complexed with iron and promoted the generation of hydroxyl radicals in Fenton reaction, leading to reducing power depletion and lipid peroxidation, and further leading to ferroptosis-like cell death of E. coli. This ferroptotic death can be triggered by various phenols in diverse organisms, from bacteria to mammalian cells. Furthermore, we demonstrated that phloroglucinol-induced ferroptosis suppressed tumor growth in mice effectively, indicating phloroglucinol as promising drug for therapy-resistant cancers. It's also discovered that repression of this ferroptosis-like cell death benefited microbial degradation or production of desired phenolic compounds, showing great application potential in biotechnology field.