Functionality of XVE-inducible system and toxicity assessment of the bacterial PezT toxin in microalga, Messastrum gracile SE-MC4

This study investigated the effects of the heterologous expression of the pezT toxin gene from the bacterium Streptococcus pneumoniae in the green microalga, Messastrum gracile SE-MC4. The microalgae was co-transformed with the pMDC150_35S activator vector (containing the XVE expression cassette) and the pMDC221_pezTGFP responder vector (containing pezT-GFP fusion genes) using Agrobacterium-mediated transformation. The stable transgenic line, M. gracile SE-MC4 (pezT-GFP) was confirmed through antibiotic selection and PCR validation. Upon induction with 17-β-estradiol, GFP signals were observed in the transgenic M. gracile SE-MC4 (pezT-GFP) cells, indicating successful transformation and expression of the transgene cassettes. Furthermore, the functionality and action of the PezT toxin was demonstrated with a significant reduction of 52% in cell viability of transgenic cells, compared to wild-type cells. This finding suggests the potential use of bacterial toxins as a novel approach for harvesting microalgal cellular contents for various microalgae-based biotechnological applications. It also can be a containment system to prevent the accidental release of transgenic microalgae and used for transgenic microalgae selection, as an alternative to antibiotic selection currently widely used.