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

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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.

OriginalsprogEngelsk
Artikelnummer103165
TidsskriftAlgal Research
Vol/bind74
Antal sider11
ISSN2211-9264
DOI
StatusUdgivet - 2023

Bibliografisk note

Funding Information:
This work was supported by funds provided by a Novo Nordisk Foundation grant NNF20OC0061048 to JAR and DPH; Novo Nordisk Foundation grant NNF21OC0070602 to DPH; JG is supported by a Novo Nordisk Foundation grant NNF20OC0060298 . PP and MF are supported by a Villum Fonden grant ( 37521 ) to MF.

Funding Information:
This work was supported by funds provided by a Novo Nordisk Foundation grant NNF20OC0061048 to JAR and DPH; Novo Nordisk Foundation grant NNF21OC0070602 to DPH; JG is supported by a Novo Nordisk Foundation grant NNF20OC0060298. PP and MF are supported by a Villum Fonden grant (37521) to MF. Quinoa seed powder was kindly provided by Associate Professor Bekzod Khakimov at the University of Copenhagen (UCPH). The tea seed extract was a gift from Prof. Nina Cedergreen (UCPH). The wild type C. reinhardtii C137 strain was kindly provided by Antonina Karakostova (UCPH). We thank Prof. Krishna K. Niyogi (University of California Berkeley) for providing the Nannochlropsis oceanica CMP1779 strain and Prof. Birger Lindberg Møller (UCPH) for discussion and feedback on the manuscript.

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© 2023 The Authors

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