Abstract
BACKGROUND: Sulfur-deprived cultivation of Chlamydomonas reinhardtii, referred as "two-stage culture" transferring the cells from regular algal medium to sulfur-deplete one, has been extensively studied to improve photobio-H(2) production in this green microalga. During sulfur-deprivation treatment, the synthesis of a key component of photosystem II complex, D1 protein, was inhibited and improved photobio-H(2) production could be established in C. reinhardtii. However, separation of algal cells from a regular liquid culture medium to a sulfur-deprived one is not only a discontinuous process, but also a cost- and time-consuming operation. More applicable and economic alternatives for sustained H(2) production by C. reinhardtii are still highly required. RESULTS: In the present study, a significant improvement in photobio-H(2) production was observed in the transgenic green microalga C. reinhardtii, which employed a newly designed strategy based on a heat-inducible artificial miRNA (amiRNA) expression system targeting D1-encoded gene, psbA. A transgenic algal strain referred as "amiRNA-D1" has been successfully obtained by transforming the expression vector containing a heat-inducible promoter. After heat shock conducted in the same algal cultures, the expression of amiRNA-D1 was detected increased 15-fold accompanied with a 73% decrease of target gene psbA. More interestingly, this transgenic alga accumulated about 60% more H(2) content than the wild-type strain CC-849 at the end of 7-day cultivation. CONCLUSIONS: The photobio-H(2) production in the engineered transgenic alga was significantly improved. Without imposing any nutrient-deprived stress, this novel strategy provided a convenient and efficient way for regulation of photobio-H(2) production in green microalga by simply "turn on" the expression of a designed amiRNA.