Genome engineering a polyploid archaeon chassis Haloferax mediterranei using the endogenous type I-B CRISPR-Cas system for efficient biopolymer production

Haloferax mediterranei is a promising archaeon for producing the high-value-added biopolymer poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (PHBV). However, its metabolic engineering has been largely hampered by the scarcity of efficient genome-editing tools. Here, we developed a versatile genome-editing platform by harnessing the endogenous type I-B CRISPR-Cas system. This toolkit enables highly efficient and precise gene deletion (>90% efficiency), multiplex editing, gene insertion, and large-scale chromosomal deletions (up to 24.8 kb). Additionally, we implemented a Cas3-mediated bidirectional deletion strategy facilitated by recombination inhibition. Using these tools, we constructed genome-reduced chassis with deletions of up to 43.2 kb, which exhibited superior PHBV synthesis without compromising cell growth. One optimal strain, ES1Δ123Δ24.9, achieved a PHBV production of 27.44 g/L from glucose in a 7-L bioreactor, with a conversion rate of 0.38 g PHBV/g glucose—the highest reported carbon source conversion rate among archaeal PHBV producers to date. These genome-reduced H. mediterranei strains represent an advanced chassis for the production of PHBV and other valuable biochemicals.