Halomonas species, which are aerobic, alkaliphilic, and moderately halophilic bacteria, produce diverse biochemicals. To identify food-related Halomonas strains for bioremediation and the industrial production of biochemicals, 20 strains were isolated from edible seashells, shrimp, and umeboshi (pickled Japanese plum) factory effluents. All isolates were phylogenetically classified into a large clade of Halomonas species. Most isolates, which grew in wide pH (6-13) and salt concentration (0-14%) ranges, exhibited the intracellular accumulation of poly(3-hydroxybutyrate) granules. The characteristics of these isolates varied. A020 isolated from umeboshi factory effluents exhibited enhanced stress tolerance and proliferation and comprised two plasmids. IMZ03 and A020 grew to more than 200 OD 600 , while IMZ03 produced 3.5% 3-hydroxybutyrate in inorganic medium supplemented with 10% sucrose. The mucus of TK1-1 cultured on agar medium comprised approximately 64 mM of ectoine. Whole-genome sequencing of A020 was performed to elucidate its origin and genomic characteristics. The genome analysis revealed a region exhibiting synteny with a large virus genome isolated from the ocean, but did not identify any predictable pathogenic genes. Therefore, saline foods and related materials may be suitable resources for isolating Halomonas strains exhibiting unique, useful, and innocuous features.

Halomonas species are halophilic and alkaliphilic bacteria, which exhibit potential for industrial production of a variety of chemicals, such as polyhydroxyalkanoates and ectoine, by fermentation because of their favorable characteristics, including high-density culturing capacity and low risk of contamination. However, genetic tools to modify the metabolism of Halomonas for suitable fermentation performance are limited. In this study, we developed two independent basic vectors for Halomonas, named pUCpHAw and pHA1AT_32, consisting of ori regions from two plasmids isolated from Halomonas sp. A020, and chloramphenicol- and tetracycline-resistant genes as cloning markers, respectively. These vectors can independently transform and co-transform the Halomonas sp. KM-1 (KM-1). A protein that was highly and constitutively accumulated was identified as a hemolysin coregulated protein (Hcp) based on proteome analysis of KM-1. Using the hcp promoter, various genes, such as phaA and EGFP, were highly expressed. To establish a gene disruption system, the Streptococcus pyogenes cas9 gene and guide RNA for the pyrF gene, a yeast URA3 homologue, were expressed in pUCpHAw and pHA1AT_32, respectively. As a result, gene disruption mutants were isolated based on phenotypes, 5-fluoroorotic acid resistance, and uracil auxotrophy. A combination of KM-1 and these vectors could be a suitable platform for industrial chemical and protein production.