Overcoming the expression barrier of the ferredoxin‑hydrogenase chimera in Chlamydomonas reinhardtii supports a linear increment in photosynthetic hydrogen output

While the prospect of producing hydrogen from photosynthetic microalgae has long been described as one of the promising directions towards achieving a renewable fuel source, current endeavors towards this goal face serious limitations including the inefficient electron supply to hydrogenase and the enzyme's sensitivity to molecular oxygen. In this work we express our ferredoxin‑hydrogenase (fd-hyd) fusion enzyme in a hydrogenase knockout Chlamydomonas reinhardtii mutant and compare its hydrogen production traits to those of wild-type strains. We found that the active enzyme abundance in both systems is in linear correlation with photosynthetic hydrogen production, thus establishing that protein abundance is an additional important bottleneck in the process of hydrogen photo-production. We report here the isolation of two clones with high expression of fd-hyd; OP68 and R2D2, created by either nuclear or chloroplast transformation, respectively. The study of these clones shows that fd-hyd's roughly 4.5-fold higher H₂ production efficiency, compared to the native hydrogenase, is maintained in high expression regimes. By comparing a strain's active enzyme pool to its total protein amount, we observed that the engineered clones harbor a large non-mature enzyme pool, indicating that natural maturation of the fd-hyd saturates at a lower level than the native hydrogenase. Subsequently, we measured mRNA levels by quantitative PCR and observed that while the two clones express roughly the same amount of protein, R2D2 has considerably more mRNA. Thus improving transcription rates in nuclear transformants or translation efficiency in chloroplast transformants are both potential routes towards further increasing protein abundance. Finally, we show that these clones are able to continuously produce H₂ in sealed bioreactors for five days, significantly outcompeting their parental wild-type strain.