Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas

An inducible chloroplast gene expression system was developed in Chlamydomonas reinhardtii by taking advantage of the properties of the copper-sensitive cytochrome c6 promoter and of the nucleus-encoded Nac2 chloroplast protein. This protein is specifically required for the stable accumulation of the chloroplast psbD RNA and acts on its 5′ UTR. A construct containing the Nac2 coding sequence fused to the cytochrome c6 promoter was introduced into the nac2-26 mutant strain deficient in Nac2. In this transformant, psbD is expressed in copper-depleted but not in copper-replete medium. Because psbD encodes the D2 reaction center polypeptide of photosystem II (PSII), the repression of psbD leads to the loss of PSII. We have tested this system for hydrogen production. Upon addition of copper to cells pregrown in copper-deficient medium, PSII levels declined to a level at which oxygen consumption by respiration exceeded oxygen evolution by PSII. The resulting anaerobic conditions led to the induction of hydrogenase activity. Because the Cyc6 promoter is also induced under anaerobic conditions, this system opens possibilities for sustained cycling hydrogen production. Moreover, this inducible gene expression system is applicable to any chloroplast gene by replacing its 5′ UTR with the psbD 5′ UTR in the same genetic background. To make these strains phototrophic, the 5′ UTR of the psbD gene was replaced by the petA 5′ UTR. As an example, we show that the reporter gene aadA driven by the psbD 5′ UTR confers resistance to spectinomycin in the absence of copper and sensitivity in its presence in the culture medium.