Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system ofZymomonas mobilisfor genome engineering

Establishment of production platform organisms through prokaryotic engineering represents an efficient means to generate alternatives for yielding renewable biochemicals and biofuels from sustainable resources.Zymomonas mobilis, a natural facultative anaerobic ethanologen, possesses many attractive physiological attributes, making it an important industrial microorganism. To facilitate the broad applications of this strain for biorefinery, an efficient genome engineering toolkit forZ. mobiliswas established in this study by repurposing the endogenous Type I-F CRISPR-Cas system upon its functional characterization, and further updated. This toolkit includes a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Using the updated toolkit, genome engineering purposes were achieved with efficiencies of up to 100%, including knockout ofcas3gene, replacement ofcas3with the mCherry-encodingrfpgene, nucleotide substitutions incas3, and deletion of two large genomic fragments up to 10 kb. This study established thus far the most efficient, straightforward and convenient genome engineering toolkit forZ. mobilis, and laid a foundation for further native CRISPRi studies inZ.mobilis, which extended the application scope of CRISPR-based technologies, and could also be applied to other industrial microorganisms with unexploited endogenous CRISPR-Cas systems.