Synthetic Biology Toolbox, Including a Single-Plasmid CRISPR-Cas9 System to Biologically Engineer the Electrogenic, Metal-Resistant Bacterium Cupriavidus metallidurans CH34

Abstract

Cupriavidus metallidurans CH34 exhibits extraordinary metabolic versatility, including chemolithoautotrophic growth; degradation of BTEX (benzene, toluene, ethylbenzene, xylene); high resistance to numerous metals; biomineralization of gold, platinum, silver, and uranium; and accumulation of polyhydroxybutyrate (PHB). These qualities make it a valuable host for biotechnological applications such as bioremediation, bioprocessing, and the generation of bioelectricity in microbial fuel cells (MFCs). However, the lack of genetic tools for strain development and studying its fundamental physiology represents a bottleneck to boosting its commercial applications. In this study, inducible and constitutive promoter libraries were built and characterized, providing the first comprehensive list of biological parts that can be used to regulate protein expression and optimize the CRISPR-Cas9 genome editing tools for this host. A single-plasmid CRISPR-Cas9 system that can be delivered by both conjugation and electroporation was developed, and its efficiency was demonstrated by successfully targeting the pyrE locus. The CRISPR-Cas9 system was next used to target candidate genes encoding type IV pili, hypothesized by us to be involved in extracellular electron transfer (EET) in this organism. Single and double deletion strains (ΔpilA, ΔpilE, and ΔpilAE) were successfully generated. Additionally, the CRISPR-Cas9 tool was validated for constructing genomic insertions (ΔpilAE::gf p and ΔpilAE::λ Pr gfp). Finally, as type IV pili are believed to play an important role in extracellular electron transfer to solid surfaces, C. metallidurans CH34 ΔpilAE was further studied by means of cyclic voltammetry using disposable screen-printed carbon electrodes. Under these conditions, we demonstrated that C. metallidurans CH34 could generate extracellular currents; however, no difference in the intensity of the current peaks was found in the ΔpilAE double deletion strain when compared to the wild type. This finding suggests that the deleted type IV pili candidate genes are not involved in extracellular electron transfer under these conditions. Nevertheless, these experiments revealed the presence of different redox centers likely to be involved in both mediated electron transfer (MET) and direct electron transfer (DET), the first interpretation of extracellular electron transfer mechanisms in C. metallidurans CH34.

Tunable Nanomaterials of Intracellular Crystallization for In Situ Biolabeling and Biomedical Imaging

“…It was found that the induced resistance was mediated by copper oxidase CopA, which was reduced to Au(0) nanoparticles in the periplasm by the intermediate detoxification Au complex. , This is a biological way for bacteria to detoxify metal ions and an unnatural process of biomineralization. Second, there are studies on the complex structure of biosynthetic DNA–gold in Escherichia coli and Staphylococcus, which has fluorescence effect and is used for the bioresponsive fluorescence imaging of bacteria. , Different bacteria were used to explore the biosynthesis of gold nanoparticles, revealing that different bacterial biosynthetic pathways are inconsistent, leading to differences in efficiency and gold nanostructures. − …”

Genetic modification strategies for electroporation and CRISPR-Cas-based technologies in the non-competent Gram-negative bacteriumAcinetobactersp. Tol 5

“…Genetic manipulation is essential not only for elucidating the biological functions of bacterial genes, but also for effectively employing bacteria in synthetic biology applications encompassing industrial, environmental, and biomedical fields. CRISPR-Cas-based technologies have remarkably enhanced the efficiency of correlating genotype and phenotype [3][4][5] , and constructed advantageous bacteria for synthetic biology applications [6][7][8][9] . However, these techniques exhibit efficient performance in E. coli and some closely related enterobacteria, while functioning inefficiently in other bacterial species.…”