Sustainable photosynthetic H2-production mediated by artificial miRNA silencing of OEE2 gene in green alga Chlamydomonas reinhardtii

• OEE2 expression in C. reinhardtii was tightly correlated with sulfur deprivation.
• A heat-inducible expression vector containing amiRNA-OEE2 was constructed.
• Expression of OEE2 was conditionally regulated by the amiRNA-OEE2.
• The knock-down effect of amiRNA-OEE2 improved photosynthetic-H2 yield.
• A new strategy on regulation of photo-H2 production was successfully demonstrated.

Hydrogen (H2) is considered as the most attractive alternative fuel and photosynthetic-H2 production by green microalgae is regarded as the most promising biofuel. However, incompatibility in the simultaneous O2 and H2 evolution from microalgae prevented the H2 production industry, and the inconvenience of culture medium change for sulfur deprivation treatment also makes it not cost-effective. In this study, a new strategy on regulation of H2 production by a model microalga Chlamydomonas reinhardtii was investigated by using artificial miRNA (amiRNA) technology. A heat-inducible expression vector containing the amiRNA targeting OEE2 gene (a photosystem II related protein, oxygen evolving enhancer (OEE2)) was constructed and transformed into C. reinhardtii. With a more rapidly O2 consumption, the transgenic alga showed that OEE2 gene knock down was correlated with the higher H2 production. The highest H2 yield was observed after the second heat shock made to the same transgenic algal culture, about 2-fold more than the control under the same condition. With no need to change medium, our results suggested a prospective way for continuous H2 production by green algae for industry applications.