Development of a CRISPR/Cas9 genome editing toolbox for <i>Corynebacterium stationis</i> and its application in hypoxanthine biosynthesis.

ABSTRACT: Corynebacterium stationis, a high-GC Gram-positive bacterium with significant industrial potential, has faced limitations due to the lack of efficient genetic tools. In this study, we developed a CRISPR/Cas9-based genome editing platform specifically tailored for C. stationis. First, electroporation efficiency was optimized to 1.81 ± 0.16 × 10⁵ CFU (colony forming units)/μg plasmid DNA through medium selection, pulse parameter adjustments (2.5 kV, 2 pulses), and concentration optimization of cell wall-weakening agents (3.0 % glycine, 0.25 % isoniazid). Three functional shuttle vectors (p99E-pCG1, p19-Kan, p19-Spe) were constructed, enabling stable heterologous gene expression. By engineering a tightly regulated Cas9 expression system (Plac promoter with dual

SUBMITTER: Ouyang Z 

PROVIDER: S-EPMC12271617 | biostudies-literature | 2025 Dec

REPOSITORIES: biostudies-literature