TY - JOUR

T1 - Novel transformation strategies improve efficiency up to 10-fold in stramenopile algae

AU - Poveda Huertes, Daniel

AU - Patwari, Payal

AU - Günther, Jan

AU - Fabris, Michele

AU - Andersen-Ranberg, Johan

N1 - Publisher Copyright: © 2023 The Authors

PY - 2023

Y1 - 2023

N2 - Stramenopile algae have the potential to become the light-driven photosynthetic biofactories of the future, but the transformation technologies required to reach this goal remain sub-optimal. Nannochloropsis oceanica and Phaeodactylum tricornutum were used as experimental systems for electroporation-mediated transformation. Two transformation approaches were developed; 1) timed transformation of synchronized cells and 2) addition of saponins as transformation adjuvants. Transformation efficiency was increased ~8 times using synchronized N. oceanica cultures transformed in the G2/M phase, in comparison to state-of-the-art methods based on transformation of non-synchronized cells. For P. tricornutum the transformation was up to 5 times more efficient in non-synchronized conditions. N. oceanica and P. tricornutum responded differently upon exposure to different saponin plant extracts. Saponin treatments enhanced P. tricornutum and N. oceanica transformation efficiencies ~2 and ~2.5 times, respectively. Combining cell synchronization and saponin transformation adjuvant treatment, improves transformation efficiency in N. oceanica, results in a >10-fold improvement of the transformation efficiency for N. oceanica. In addition, a protocol for directed ribonucleoprotein (RNP)-mediated genome engineering of DNA constructs with short flanking arms (50 bp) in N. oceanica was established, enabling improved RNP targeted non-homologous end-joining (NHEJ) gene editing. In conclusion, this study expands the toolbox for stramenopile genome engineering, promoting their use as model organisms and sustainable biofactories.

AB - Stramenopile algae have the potential to become the light-driven photosynthetic biofactories of the future, but the transformation technologies required to reach this goal remain sub-optimal. Nannochloropsis oceanica and Phaeodactylum tricornutum were used as experimental systems for electroporation-mediated transformation. Two transformation approaches were developed; 1) timed transformation of synchronized cells and 2) addition of saponins as transformation adjuvants. Transformation efficiency was increased ~8 times using synchronized N. oceanica cultures transformed in the G2/M phase, in comparison to state-of-the-art methods based on transformation of non-synchronized cells. For P. tricornutum the transformation was up to 5 times more efficient in non-synchronized conditions. N. oceanica and P. tricornutum responded differently upon exposure to different saponin plant extracts. Saponin treatments enhanced P. tricornutum and N. oceanica transformation efficiencies ~2 and ~2.5 times, respectively. Combining cell synchronization and saponin transformation adjuvant treatment, improves transformation efficiency in N. oceanica, results in a >10-fold improvement of the transformation efficiency for N. oceanica. In addition, a protocol for directed ribonucleoprotein (RNP)-mediated genome engineering of DNA constructs with short flanking arms (50 bp) in N. oceanica was established, enabling improved RNP targeted non-homologous end-joining (NHEJ) gene editing. In conclusion, this study expands the toolbox for stramenopile genome engineering, promoting their use as model organisms and sustainable biofactories.

KW - Cell cycle synchronization

KW - DNA transformation

KW - Genome editing

KW - Nannochloropsis oceanica

KW - Phaeodactylum tricornutum

KW - Saponins

U2 - 10.1016/j.algal.2023.103165

DO - 10.1016/j.algal.2023.103165

M3 - Journal article

AN - SCOPUS:85164358386

VL - 74

JO - Algal Research

JF - Algal Research

SN - 2211-9264

M1 - 103165

ER -