A method for the preparation of electrocompetent cells to transform unicellular green algae, Coccomyxa (Trebouxiophyceae, Chlorophyta) strains Obi and KJ
2020
Tatara, Hiroshi | Sato, Yusuke | Kasai, Yuki | Hagiwara, Daisuke | Makino, Arisa | Imamura, Sousuke | Abe, Jun | Yoshimitsu, Yuya | Harayama, Shigeaki
Unicellular green algae, Coccomyxa spp. strains Obi and KJ are capable of growing at broad pH ranges, and accumulate lipids at levels higher than 50% of the dry cell weight under nitrogen-depleted conditions. An efficient genetic transformation method applicable to the two strains is required for their genetic improvement to facilitate their application in commercial production of biofuels. After an extended trial and error period, we fortuitously determined conditions required for the transformation of the strains through electroporation. First, the growth of the strains in isotonic medium e.g., minimal salt medium containing 0.6 M sorbitol or mannitol was required. Second, the growth under a light/dark cycle was critical. In the dark period in the cycle, cells contained two or four endospores surrounded by a mother-cell wall. Within half an hour after the beginning of the light period, the mother-cell wall was cleaved, and unicellular autospores that were electrocompetent were released. The application of a single electroporation pulse at an electric-field strength of 10 kV cm⁻¹ to the autospores yielded transformants at a frequency of >10⁻⁶ per input cell for strain KJ and >10⁻⁷ per input cell for strain Obi. The transformation method is relatively simple, and provided several to several dozen higher transformation frequencies than those applying the biolistic bombardment method that we previously developed. The present study also provides insights that could facilitate the development of genetic transformation systems for microalgae, which reproduce by endospore formation.
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