Development of an endogenous type I-E CRISPR-Cas-based gene editing tool for Halomonas nigrificans X339: Applications in enhancing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production

Halomonas nigrificans X339 is a versatile halophilic bacterium capable of synthesizing polyhydroxyalkanoates (PHAs) using a broad range of carbon sources. In this study, we genetically engineered the strain to enhance propionate assimilation and boost the production of poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (PHBV). We first developed an endogenous CRISPR-Cas-based gene editing tool tailored for this strain, enabling precise genetic modifications. Using this tool, we successfully knocked out the prpC gene, encoding 2-methylcitrate synthase, involved in propionate catabolism. The resulting mutant ΔprpC exhibited a significant increase in propionate-to-3-hydroxyvalerate (3HV) conversion rate, from 1.8 % to 70.5 %, and an improvement in 3HV molar fraction of PHBV, from 0.28 % to 9.76 %, in cultures supplemented with 1 g/L propionate. However, this modification also impaired propionate tolerance of ΔprpC, reducing cell dry weight (CDW) from 15.05 g/L to 8.26 g/L and PHBV content from 51.60 % to 29.67 %. To address this, we further deleted a 23.6-kb exopolysaccharide (EPS) synthesis gene cluster (PS1) in ΔprpC to limit carbon flux toward EPS synthesis. Surprisingly, this deletion partially restored propionate tolerance, allowing the double mutant (ΔPS1ΔprpC) to reach a CDW of 12.91 g/L, PHBV content of 62.5 %, and 3HV molar fraction of 7.57 %, with a propionate-to-3HV conversion rate up to 100 %. These findings highlight the utility of the endogenous CRISPR-Cas-based editing tool in optimizing metabolic pathways, thereby establishing H. nigrificans X339 as a promising chassis for efficient PHBV production. • An endogenous CRISPR-Cas system is first developed as a gene editing tool in Halomonas . • The prpC gene (1.1 kb) and EPS gene cluster (23.6 kb) are simultaneously deleted in H. nigrificans . • EPS knockout in ΔprpC remarkably improved the propionate tolerance of cells. • EPS knockout in ΔprpC enhanced PHBV yield by 3.34-fold. • EPS knockout in ΔprpC improved propionate-to-3HV conversion to 100 %.