Our results indicated that G418 was a potent selection agent able to
completely block the cell growth of C. vulgaris CBS 15-2075 at a concentration
of 30 μg/mL. The inhibitory effect of G418 on cell growth has also
been demonstrated in other Chlorella strains including Chlorella
ellipsoidea [42,43], C. sp. [44], and C. sorokiniana [37]. It is worth noting
that the selection concentration of G418 in these studies varies greatly
from as low as 30 μg/mL to as high as 1 mg/mL, indicating the considerable
difference in antibiotic sensitivity across Chlorella strains. Intriguingly,
we found that G418 at a low concentration of 2.5 μg/mL in
liquid culture promoted the growth of C. vulgaris unexpectedly
(Fig. 2a). This growth-promoting effect might be attributed to the positive
reallocation of the metabolic flux in C. vulgaris cells in response to
the reduced protein synthesis caused by G418. The E. coli derived nptII
gene encodes a neomycin phosphotransferase that confers resistance
to G418 [45] and has been used as a dominant selectable marker for
yeast [46], plant [47], and some microalgae [48]. This selectable marker
gene also worked well for C. vulgaris in our study