Enhanced Hydrogen Photo-Production in Microalgae by Computational based Genetic Engineering

Hydrogen photo-production in green algae, catalyzed by the enzyme [FeFe]-hydrogenase (hydA), is considered a promising source of renewable clean energy. Yet, a significant increase in hydrogen production efficiency is necessary for industrial scale-up. The main obstacles hindering the process are the enzyme’s oxygen sensitivity and its poor competing abilities for photosynthetic electrons. In previous work we fused hydA to the electron carrier ferredoxin, thus forming a fd-hydA synthetic enzyme which significantly outcompeted the native hydA in competition for electrons in vitro.

Recently, we expressed our fd-hydA fusion in live C. reinhardtii cells. From comparison of the hydrogen production features in our engineered clones to those of the wild-type strain, we reached 2 important conclusions: (a) the H₂ production rate, normalized by the amount of enzymes, is 4.5-fold higher in fd-hydA clones, (b) the fd-hydA enzyme showed elevated O₂ tolerance. These findings emphasized the important role fd-hydA might play in moving towards commercialization. However, the levels of heterologous expression received were lower than the wild-type hydA protein levels, therefore higher protein levels should be achieved for the full potential of the synthetic enzyme to be unlocked.

To this end, we analyzed C. reinhardtii genomic data and implemented our findings into synthetic fd-hydA sequences, using an array of sequence optimization algorithms. In vivo gene expression of clones transformed with these sequences showed that we were able to significantly increase heterologous expression. This was achieved mainly by eliminating splicing signals (Fig. 2), selecting optimal codons and optimizing the folding energy of mRNA secondary structures in the translation initiation vicinity. Overall, we were able to isolate new clones with H₂ production rates up to 4.4-fold higher than the previous highest expressing clone.