Functional analysis of the putative centromere sequences of marine oleaginous diatom Fistulifera solaris

Abstract

Trans-kingdom conjugation-based transformation with oriT-containing episomal plasmids is an emerging technique to achieve genetic engineering in diatoms. The native centromeres lacking any consensus motifs and budding yeast-derived centromere/autonomous replication sequence accompanied by a histidine synthase gene (CEN6-ARSH4-HIS3) supported the plasmid maintenance in model diatoms. However, it remained elusive whether these host-vector systems are universally applicable in industrially important non-model diatom species. In this study, we found the consensus motifs in the putative centromeres of Fistulifera solaris; some exhibited characteristic dispersion patterns along the chromosomes. Furthermore, we investigated whether (1) oriT-containing plasmids could be transferred from the Escherichia coli strain S17-1 expressing tra genes to the oleaginous diatom F. solaris, and (2) the putative centromeres of F. solaris and yeast CEN6-ARSH4-HIS3 could support the episomal maintenance in F. solaris. As a result, transformant clones were successfully obtained through a drug-registrant selection process. PCR testing further confirmed the transfer of the plasmid sequences in the diatom. Some of the centromeres containing the consensus motifs marginally contributed to the transformation; however, the differences in terms of the transformation efficiency in the presence or absence of F. solaris-derived putative centromeres and yeast-derived CEN6-ARSH4-HIS3 were not statistically significant. The episome rescue experiment did not provide evidence that the transferred plasmids were maintained as episomes. These results suggest that these centromeric sequences might not be solely responsible for episomal maintenance in F. solaris.