Establishment of an optimized electroporation method for Halomonas sp. YK44 and its application in the coproduction of PHB and isobutanol

Abstract

Many Halomonas spp. thrive in high-salinity environments, and their resistance to high salt levels allows for their cultivation in non-sterile conditions. Despite their robustness and potential poly (3-hydroxybutyrate) (PHB) production capability, there are relatively few reports on the engineering of various Halomonas species, and there are still some difficulties in genetically engineering novel Halomonas strains. In particular, conjugation as a transformation method has been employed more frequently than electroporation in Halomonas; however, electroporation is necessary for the accelerated engineering of Halomonas and increased time efficiency. To touch this issue, we collected Halomonas strains and evaluated their PHB production and electroporation efficiencies resulting that the Halomonas sp. YK44 showed the highest electroporation efficiency with high PHB production among the various Halomonas strains. A series of electroporation protocol optimization experiments were conducted to identify optimal conditions for Halomonas sp. YK44 such as main culturing for 10 h, utilizing a DNA concentration of 150–200 μg/mL, and performing electroporation at 2.1 kV, followed by a washing step using 10% glycerol and a recovery period of 36 h with pBBR1MCS2. By introducing isobutanol biosynthetic genes using an optimized electroporation protocol, the highest isobutanol production was obtained at 196 mg/L with 63% PHB content simultaneously and the higher PHB production was obtained at 6.6 g/L with 152 mg/L isobutanol. Our approach showed the overall process to identify a suitable Halomonas host by applying general electroporation methods, optimizing electroporation protocols, and demonstrated the first coproduction of PHB and isobutanol in Halomonas.