Development of efficient genetic-transformation- and genome-editing systems, and the isolation of a CRISPR/Cas9-mediated high-oil mutant in the unicellular green alga Parachlorella kessleri strain NIES-2152

Abstract

Abstract Background Parachlorella kessleri strain NIES-2152, a unicellular green alga, has the characteristics of rapid growth and high lipid productivity; thus this strain has been considered to be a candidate for use in biofuel production. However, the commercialization of biofuels requires significant cost reduction, and strain improvements may be needed to achieve this objective. Results In the first part of this study, we developed a genetic transformation method applicable to P. kessleri as follows. During the cell cycle of P. kessleri, daughter cells develop within the mother cell wall. Therefore, P. kessleri cells are encompassed by two cell-wall layers: a daughter cell wall and a mother cell wall, during most of its cell cycle except for a brief period after the cleavage of the mother cell wall. We found that cells just hatched out of the mother cell wall exhibited high efficiency in taking up DNA through electroporation. After optimizing the electroporation conditions, we achieved a transformation efficiency of 3.2 to 4.6 × 10−5 transformants per input cell. Previously, we had isolated a mutant of P. kessleri named strain PK4 that accumulated higher concentrations of lipids than the wild-type strain. Resequencing of the PK4 genome had identified three genes with potential defects associated with the high lipid phenotype. In the second part of this study, we delivered Cas9-gRNA ribonucleoproteins (RNPs) targeting each of the three genes into P. kessleri cells using the same protocol as the genetic transformation, and successfully disrupted these three genes separately. The disruptants of one gene encoding a plastidic ATP/ADP translocase exhibited >30% higher lipid productivity than that of the wild-type strain under diurnal conditions. Conclusions We established an effective gene-editing method applicable to P. kessleri. Using this method, we succeeded in creating strains showing significantly higher lipid productivity than the wild-type strain, especially under diurnal rhythms.