Ectoine (1,4,5,6-tetrahydro–2-methyl-4-pyrimidinecarboxylic acid) and its derivatives are compatible solutes produced intracellularly by halophilic or halotolerant bacteria to combat salt stress. These compounds are critical for maintaining the stability of cellular macromolecules and membranes, and they are widely used in biomedicine, food processing, and the fine chemical industry (Liu et al., 2021). Ectoine biosynthesis originates from L-aspartate-4-semialdehyde (ASA) and involves a three-step enzymatic cascade catalyzed by diaminobutyrate transaminase (EctB), diaminobutyrate acetyltransferase (EctA), and ectoine synthase (EctC) (Mais et al., 2020). Chassis cell modification through metabolic engineering, gene editing, and gene knockouts has emerged as a prominent approach to enhancing ectoine production (Ye and Chen, 2021). Among these strategies, CRISPR/Cas9-based gene editing has gained significant attention for its high efficiency, low cost, and limited potential for off-target effects (Arshad et al., 2025). For instance, Wilding-Steele (Wilding-Steele et al., 2021) applied CRISPR/Cas9 to sequentially knock out ldhA and the ptb-buk operon in Streptococcus pyogenes, enhancing its butanol output. Qin (Qin et al., 2018) used CRISPR/Cas9 to disrupt the prpC gene in Halomonas bluephagenesis TD01, resulting in a 16-fold increase in 3-hydroxyvalerate (3HV) content. Similarly, Chen (Chen et al., (2019) knocked out sdhE and icl in H. bluephagenesis TD08AB using CRISPR/Cas9, leading to a two-fold increase in 3HV accumulation compared to the parental strain.

Optimizing or simplifying secondary metabolite biosynthetic pathways is a central goal in systems metabolic engineering. Targeted gene editing can enhance metabolite yield by eliminating competing or degradative pathways. However, gene knockout can also disrupt metabolic flux, potentially causing the accumulation or depletion of intermediate metabolites. Targeted metabolomics strategies afford a high degree of accuracy and reproducibility conducive to the quantitative analysis of pathway-specific metabolites (Wang et al., 2022). In our previous study, we isolated the wild-type H. campaniensis strain XH26 from Xiaochaidan Salt Lake in China, and found that it produced intracellular ectoine at a concentration of 0.36 g/L (Wang et al., 2023). Lysine is a byproduct of ectoine synthesis, and to improve ectoine production, deletion of the key lysine biosynthesis gene dapA in strain XH26 resulted in loss of viability. Betaine is also a byproduct of ectoine synthesis, in the present study, we employed CRISPR/Cas9 to knockout hom (involved in betaine biosynthesis) and doeA (involved in ectoine degradation) in the XH26 strain in an effort to block competing pathways and enhance ectoine biosynthesis (Fig. 1). Targeted metabolomics and RT-qPCR were further employed to compare metabolite yields and pathway responses between wild-type and mutant strains. These findings provide a theoretical framework for engineering strains with a superior capacity for ectoine production.