[4S04e] 19. Marine Biotechology - Part 3

5:30 PM - 5:50 PM JST(8:30 AM - 8:50 AM UTC)

[4Marine-13-KL] Challenges in artificial chromosome development for microalgae aiming genetic engineering

○Yoshiaki Maeda¹, Hyun-Sik Yun¹, Momoka Tokin¹, Takehito Sugasawa², Iwane Suzuki¹ (1. Institute of Life and Environmental Sciences, University of Tsukuba (Japan), 2. Institute of Medicine, University of Tsukuba (Japan))

Keywords:

diatom,autonomous replication sequence,origin recognition complexe,chromatin immunoprecipitation-sequencing,Phaeodactylum tricornutum

[Purpose]Microalgae have been recognized as promising hosts for the production of valuable compounds from carbon dioxide due to their photosynthetic activity. Genetic engineering is a promising approach to enhance the productivity and/or the variation of target molecules of interest. However, the development of genetic engineering tools for microalgae is still limited. We have aimed to develop artificial chromosome vectors that enable large-scale genetic engineering of diatoms, a group of microalgae, by introducing various functional genes simultaneously. Towards this goal, native autonomous replication sequences (ARSs) were recently mapped on the genome sequence of the marine diatom Phaeodactylum tricornutum by chromatin immunoprecipitation-sequencing (ChIP-Seq), because ARSs are essential elements for DNA replication in the cell. Subsequently, we developed the vector libraries, in which each vector contains different ARS candidates, to screen the ARSs potentially showing high activity of vector replication. In addition, we assessed whether any modifications occurred in the vectors introduced in the diatom. [Method]For the ChIP-seq study, transformant strains overexpressing the recombinant subunits of origin recognition complexes (ORCs), which bind to ARSs, were constructed with electroporation. ORC2 and ORC4 subunits fused with a FLAG-tag were pulled-down along with the associated DNA molecules using anti-FLAG antibody. After library preparation, the MiSeq platform was used to obtain the sequence information. Then, ARS candidates were mapped on the genome sequence of P. tricornutum. The candidate sequences were individually inserted into the backbone vector containing a bleomycin-resistance gene, oriT, and a native centromere of P. tricornutum. Some of the vectors were introduced to the diatom by bacterial conjugation. Subsequently, the introduced sequences were assessed using Sanger and Nanopore sequencing. [Results & Consideration]The ChIP-seq study revealed 69 promising candidates of ARSs recognized in the genome. The ARSs with approximately 50% (44.39–52.92%) GC contents did not have particularly conserved motifs. By contrast, Saccharomyces cerevisiae possesses AT-rich ARSs containing highly conserved motifs, suggesting that the diatom and yeast would have different mechanisms for ORC to recognize ARSs. Artificial chromosome vectors were constructed with the candidates. After transferring some of the vectors to the diatom, we found some insertions and deletions in the vectors. At this moment, the molecular mechanism(s) causing these mutation remains unclear. [Conclusion]Our study provides insight into DNA replication mechanisms in diatoms, and also specifies the issue to be addressed for the development of artificial chromosomes for the engineering of diatoms. In the future study, we will perform the screening assay to elucidate the highly active ARSs for the construction of artificial chromosome vectors for diatoms.