Robust expression of heterologous genes by selection marker fusion system in improved Chlamydomonas strains

Chlamydomonas is a very attractive candidate plant cell factory. However, its main drawback is the difficulty to find the transformants that robustly express heterologous genes randomly inserted in the nuclear genome. We previously showed that domestic squalene synthase (SQS) gene of Chlamydomonas was much more efficiently overexpressed in a mutant strain [UV-mediated mutant (UVM) 4] than in wild type. In this study, we evaluated the possibility of a new mutant strain, met1, which contains a tag in the maintenance type methyltransferase gene that is expected to play a key role in the maintenance of transcriptional gene silencing. The versatile usefulness of the UVM4 strain to express heterologous genes was also analyzed. We failed to overexpress CrSSL3 cDNA, which is the codon-adjusted squalene synthase-like gene originated from Botryococcus braunii, using the common expression cassette in the wild-type CC-1690 and UVM4 strains. However, we succeeded in isolating western blot-positive transformants through the combinational use of the UVM4 strain and ble2A expression system of which expression cassette bears a fused ORF of the target gene and the antibiotic resistance gene ble via the foot-and-mouth disease virus (FMDV) self-cleaving 2A sequence. It is noteworthy that even with this system, huge deviations in the accumulated protein levels were still observed among the UVM4 transformants.