Efficient Agrobacterium tumefaciens-mediated stable genetic transformation of green microalgae, Chlorella sorokiniana

The green oleaginous microalgae, Chlorella sorokiniana, is a highly productive Chlorella species and a potential host for the production of biofuel, nutraceuticals, and recombinant therapeutic proteins. The lack of a stable and efficient genetic transformation system is the major bottleneck in improving this species. We report an efficient and stable Agrobacterium tumefaciens-mediated transformation system for the first time in C. sorokiniana. Cocultivation of C. sorokiniana cells (optical density at λ680 = 1.0) with Agrobacterium at a cell density of OD600 = 0.6, on BG11 agar medium (pH 5.6) supplemented with 100 μM of acetosyringone, for three days at 25 ± 2 °C in the dark, resulted in significantly higher transformation efficiency (220 ± 5 hygromycin-resistant colonies per 106 cells). Transformed cells primarily selected on BG11 liquid medium with 30 mg/L hygromycin followed by selecting homogenous transformants on BG11 agar medium with 75 mg/L hygromycin. PCR analysis confirmed the presence of hptII, and the absence of virG amplification ruled out the Agrobacterium contamination in transformed microalgal cells. Southern hybridization confirmed the integration of the hptII gene into the genome of C. sorokiniana. The qRT-PCR and Western blot analyses confirmed hptII and GUS gene expression in the transgenic cell lines. The specific growth rate, biomass doubling time, PSII activity, and fatty-acid profile of transformed cells were found similar to wild-type untransformed cells, clearly indicating the growth and basic metabolic processes not compromised by transgene expression. This protocol can facilitate opportunities for future production of biofuel, carotenoids, nutraceuticals, and therapeutic proteins.The online version contains supplementary material available at 10.1007/s13205-021-02750-7.