Establishment of genetic tools for genomic DNA engineering of Halomonas sp. KM-1, a bacterium with potential for biochemical production

<jats:title>Abstract</jats:title><jats:p><jats:italic>Halomonas</jats:italic> 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 <jats:italic>Halomonas</jats:italic> for suitable fermentation performance are limited. In this study, we developed two independent basic vectors for <jats:italic>Halomonas</jats:italic>, named pUCpHAw and pHA1AT_32, consisting of <jats:italic>ori</jats:italic> regions from two plasmids isolated from <jats:italic>Halomonas</jats:italic> sp. A020, and chloramphenicol- and tetracycline-resistant genes as cloning markers, respectively. These vectors can independently transform and co-transform the <jats:italic>Halomonas</jats:italic> 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 <jats:italic>hcp</jats:italic> promoter, various genes, such as <jats:italic>phaA</jats:italic> and EGFP, were highly expressed. To establish a gene disruption system, the <jats:italic>Streptococcus pyogenes cas9</jats:italic> gene and guide RNA for the <jats:italic>pyrF</jats:italic> gene, a yeast <jats:italic>URA3</jats:italic> 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.</jats:p>