CRISPR/Cas9-based Genome Editing in Pseudomonas aeruginosa and Cytidine Deaminase-Mediated Base Editing in Pseudomonas SpeciesPseudomonas species are a large class of gram-negative bacteria that exhibit significant biomedical, ecological, and industrial importance. Despite the extensive research and wide applications, genetic manipulation in Pseudomonas species, in particular in the major human pathogen Pseudomonas aeruginosa, remains a laborious endeavor. Here we report the development of a genome editing method pCasPA/pACRISPR by harnessing the CRISPR/Cas9 and the phage λ-Red recombination systems. The method allows for efficient and scarless genetic manipulation in P. aeruginosa. By engineering the fusion of the cytidine deaminase APOBEC1 and the Cas9 nickase, we further develop a base editing system pnCasPA-BEC, which enables highly efficient gene inactivation and point mutations in a variety of Pseudomonas species, such as P. aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas syringae. Application of the two genome editing methods will dramatically accelerate a wide variety of investigations, such as bacterial physiology study, drug target exploration, and metabolic engineering.
Deep eutectic solvents as novel extraction media for phenolic compounds from model oilTongnian Gu, Mingliang Zhang, Ting Tan et al.|Chemical Communications|2014 Deep eutectic solvents (DES) as a new kind of green solvent were used for the first time to excellently extract phenolic compounds from model oil. It was also proved that DES could be used to extract other polar compounds from non-polar or weakly-polar solvents by liquid-phase microextraction.
Highly efficient base editing in <i>Staphylococcus aureus</i> using an engineered CRISPR RNA-guided cytidine deaminaseTongnian Gu, Siqi Zhao, Yishuang Pi et al.|Chemical Science|2018 and provides critical insights into the development of base-editing tools in other microbes.
Transition-Metal-Free Stereospecific Cross-Coupling with Alkenylboronic Acids as NucleophilesChengxi Li, Yuanyuan Zhang, Qi Sun et al.|Journal of the American Chemical Society|2016 We herein report a transition-metal-free cross-coupling between secondary alkyl halides/mesylates and aryl/alkenylboronic acid, providing expedited access to a series of nonchiral/chiral coupling products in moderate to good yields. Stereospecific SN2-type coupling is developed for the first time with alkenylboronic acids as pure nucleophiles, offering an attractive alternative to the stereospecific transition-metal-catalyzed C(sp(2))-C(sp(3)) cross-coupling.
Mechanistic insights into staphylopine-mediated metal acquisitionLiqiang Song, Yifei Zhang, Weizhong Chen et al.|Proceedings of the National Academy of Sciences|2018 , plays a central role in transition-metal acquisition and bacterial virulence. The StP-like biosynthesis loci are present in various pathogens, and the proteins responsible for StP/metal transportation have been determined. However, the molecular mechanisms of how StP/metal complexes are recognized and transported remain unknown. We report multiple structures of the extracytoplasmic solute-binding protein CntA from the StP/metal transportation system in apo form and in complex with StP and three different metals. We elucidated a sophisticated metal-bound StP recognition mechanism and determined that StP/metal binding triggers a notable interdomain conformational change in CntA. Furthermore, CRISPR/Cas9-mediated single-base substitution mutations and biochemical analysis highlight the importance of StP/metal recognition for StP/metal acquisition. These discoveries provide critical insights into the study of novel metal-acquisition mechanisms in microbes.