Improvement of molecular tools for the model diatom Thalassiosira pseudonana

The centric diatom Thalassiosira pseudonana is a model organism for studies of diatom biology, especially the biomineralization of its frustule. Methods for genetic transformation of this species have primarily been limited to biolistic transformation and conjugation. Biolistic methods present significant disadvantages in terms of accessibility and handling, while conjugation requires a diatom-specific backbone and a helper plasmid. In this study an efficient and straightforward transformation method using electroporation was established. Two electroporation systems were investigated, and reliable protocols were implemented. Transformation efficiency, with an average of 88 colony-forming cells and reaching up to 1324 per reaction, is more than sufficient for genome editing or other approaches and presenting up to a 3-fold improvement compared to biolistic transformation. A major advantage of this method is the possibility of genomic integration of any plasmid, making the usage of diatom specific vectors redundant while ensuring stable expression over time. Furthermore, a new small, strong and reliable native promoter-terminator pair was explored using the reporter gene mNeonGreen . Fluorescence microscopy and flow cytometry showed stable mNeonGreen fluorescence over time. Expression of target genes was further enhanced by inserting the small native LHCF9 intron. Its unique positioning within the start codon of the corresponding gene makes it universally usable. Combining the LHCF9 intron with its native promoter strongly increased reporter gene expression; this was confirmed on protein level using a fluorescence reporter. In summary, these new tools can be helpful accessing T. pseudonana for genome editing as well as for improving production of valuable compounds. • A straightforward electroporation approach for T. pseudonana was established. • Expression was enhanced by extending the LHCF9 promoter with its first intron. • The new small promoter/terminator pair FTR1A also provided high expression. • The LHCF9 intron also enhanced expression driven by the FTR1A promoter.