Green algal hydrogenase (HydA) can produce hydrogen (H-2) biophotocatalytically. Nevertheless, HydA is easily inhibited via its gas tunnel by O-2, which is a byproduct of photosynthesis. To promote H-2 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 O-2 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 H-2 than the wild type (WT) in the presence of 5% O-2 during biophotocatalysis. Furthermore, using an in vivo assay for several days, one transgenic with double mutations was capable of producing H-2 up to 30-fold more than the WT in the presence of arbitrary O-2 during biophotocatalysis. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.