Genetically engineered hydrogenases promote biophotocatalysis-mediated H2 production in the green alga Chlorella sp. DT

Abstract

Green algal hydrogenase (HydA) can produce hydrogen (H2) biophotocatalytically. Nevertheless, HydA is easily inhibited via its gas tunnel by O2, which is a byproduct of photosynthesis. To promote H2 production, the green alga Chlorella sp. HydA from the DT strain (CsHydAc.DT, encoded by CshydAc.DT) was genetically engineered to modify amino acid residues A105I, V265W, G113I, or V273I around the gas tunnel to prevent O2 accessing the enzyme active site. The mutated-CshydAc.DT genes were generated and homologously transformed into DT. Then the mutated-CsHydAc.DT transgenics with single or double mutations were selected. Using an in vitro assay, it was found that the DT transgenics produced up to 7-fold more H2 than the wild type (WT) in the presence of 5% O2 during biophotocatalysis. Furthermore, using an in vivo assay for several days, one transgenic with double mutations was capable of producing H2 up to 30-fold more than the WT in the presence of arbitrary O2 during biophotocatalysis.