CRISPR/Cas9 editing genome of extremophile Halomonas spp

Extremophiles are suitable chassis for developing the next generation industrial biotechnology (NGIB) due to their resistance to microbial contamination. However, engineering extremophiles are not an easy task. Halomonas, an industrially interesting halophile able to grow under unsterile and continuous conditions in large-scale processes, can only be engineered using suicide plasmid-mediated two-step homologous recombination which is very laborious and time-consuming (up to half a year). A convenient approach for the engineering of halophiles that can possibly be extended to other extremophiles is therefore urgently required. To meet this requirement, a rapid, efficient and scarless method via CRISPR/Cas9 system was developed in this study for genome editing in Halomonas. The method achieved the highest efficiency of 100%. When eight different mutants were constructed via this special CRISPR/Cas9 method to study the combinatorial influences of four different genes on the glucose catabolism in H. bluephagenesis TD01, it took only three weeks to complete the deletion and insertion of up to 4.5 kb DNA. H. bluephagenesis was designed to produce a microbial copolymer P(3HB-co-3HV) consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). The CRISPR/Cas9 was employed to delete the prpC gene in H. bluephagenesis TD01. Shake flask studies showed that the 3HV fraction in the copolymers increased approximately 16-folds, demonstrating enhanced effectiveness of the ΔprpC mutant to synthesize PHBV. This genome engineering strategy significantly speeds up the studies on Halomonas engineering, opening up a wide area for developing NGIB.