Optogenetic regulation of artificial microRNA improves H2 production in green alga Chlamydomonas reinhardti

Background: Chlamydomonas reinhardtii is an ideal model organism not only for the study of basic metabolic processes in both plants and animals but also the production of biofuels including hydrogen. Transgenic analysis of C. reinhardtii is now well established and very convenient, but inducible exogenous gene expression systems remain under-studied. The most commonly used heat shock-inducible system has serious effects on algal cell growth and is difficult and costly to control in large-scale culture. Previous studies of hydrogen photoproduction in Chlamydomonas also use this heat-inducible system to activate target gene transcription and hydrogen synthesis. Results: Here we describe a blue light-inducible system with which we achieved optogenetic regulation of target gene expression in C. reinhardtii. This light-inducible system was engineered in a photosynthetic organism for the first time. The photo- inducible heterodimerizing proteins CRY2 and CIB1 were fused to VP16 transcription activation domain and the GAL4 DNA-binding domain, respectively. This scheme allows for transcription activation of the target gene downstream of the activation sequence in response to blue light. Using this system, we successfully engineered blue light-inducible hydrogen-producing transgenic alga. The transgenic alga was cultured under red light and grew approximately normally until logarithmic phase. When illuminated with blue light, the transgenic alga expressed the artificial miRNA targeting photosynthetic system D1 protein, and altered hydrogen production was observed. Conclusions: The light-inducible system successfully activated the artificial miRNA and, consequently, regulation of its target gene under blue light. Moreover, hydrogen production was enhanced using this system, indicating a more convenient and efficient approach for gene expression regulation in large-scale microalgae cultivation. This optogenetic gene control system is a useful tool for gene regulation and also establishes a novel way to improve hydrogen production in green algae.