Inducible high level expression of a variant ΔD19A,D58A-ferredoxin-hydrogenase fusion increases photohydrogen production efficiency in the green alga Chlamydomonas reinhardtii

Oxygenic photosynthetic organisms use solar energy to split water into protons, electrons, and oxygen. Unicellular green algae like Chlamydomonas reinhardtii have evolved the additional ability to reduce protons to produce hydrogen (H2) via chloroplast hydrogenases (HYDA1 and HYDA2) under light-driven anaerobic conditions. In wild type cells, H2 production is limited by proton and electron supply to HYDA; most electrons are diverted to ferredoxin-NADP+-reductase. To enhance electron supply to HYDA and improve H2 production, variant ferredoxins with reduced ferredoxin-NADP+-reductase affinity have been demonstrated in vitro. Ferredoxin-hydrogenase fusions have also been tested, but achieving robust expression in vivo has been challenging. To improve efficiency, we here combine (1) a ferredoxin-HYDA fusion containing (2) a mutant FDX1 ferredoxin (D19A, D58A) with lowered affinity for ferredoxin-NADP+-reductase, utilising (3) a strongly inducible lhcbm9 promoter for controllable expression, and (4) a transgene construct containing introns for higher nuclear expression levels. Fusion constructs with 15 (pDK6-15) or 25 (pDK6-25) amino acid linkers were expressed in the nucleus of a HYDA1/HYDA2 knockout strain. Under illuminated, sulfur-deprived conditions, the variant ferredoxin-HYDA fusion strains yielded 3.0 and 4.6-fold more H2 than the wild type background strain over a 6-day period (typical purity range attained; 92–97%), confirming that the variant ferredoxin fused to HYDA is functional in vivo, strongly inducible, expresses at a similar level to the native HYDA gene, and significantly increases light-driven H2 production over wild type levels, likely by reducing losses to other electron acceptors such as ferredoxin-NADP+-reductase.