In the present study, DNA binding with one finger (DOF)-type transcription factors were screened from the Chlorella vulgaris genome database. The candidate DOF transcription factor was endogenously overexpressed in C. vulgaris to improve neutral lipid production. The protein expression vector contains the heat shock protein 70 and ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit promoters and self-cleaving 2A peptide to reduce the transgene-silencing effect of C. vulgaris. A total of 74 phleomycin-resistant transformants were obtained. Under nitrogen-deficient conditions, the transformant CvDOF#3 showed approximately 1.5-fold higher neutral lipid content per cell compared to the original strain and also showed a His-tagged DOF candidate protein expression of 0.6%. Microscopic observations revealed that CvDOF#3 cells were larger in size. However, the observed differences in average cell diameter between CvDOF#3 and control cells were not statistically significant. These results indicated that the protein expression vector harboring the dual promoters and the 2A peptide, when used in combination with enzymatic cell wall degradation and glass bead transformation, could be useful for transgene and protein expression in C. vulgaris. Further experiment is necessary to confirm the expression efficiency of the HSP70 and RBCS dual promoter and 2A peptide strategy after construction of homologous recombination system in C. vulgaris. Our findings suggested that the overexpression of the endogenous DOF-type transcription factor can be used for improving the lipid content in C. vulgaris.
Keywords: Chlorella vulgaris; DOF transcriptional factor; Lipid production; Transgene.