According to the codon bias in the chloroplast genome of Chlamydomonas reinhardtii, the codon-optimized coding regions of both the ferrochelatase gene, hemH, from Bradyrhizobium japonicum and the leghemoglobin gene, lba, from Glycine max were synthesized de novo and transferred into the chloroplast of C reinhardtii. The expression level of hemH-lba protein was improved by 6.8 folds in the codon-optimized transgenic alga compared with the non-optimized one under both normal and anaerobic conditions. H-2 yield was 22% and the respiration rate was 44% higher in the codon-optimized transgenic algal cultures than those of the non-optimized ones, and was 450% and 134% higher than those of the control cultures, respectively. The transcript levels of hydA1 and hydA2 in the hemH-lba transgenic alga were also more stable and higher than those of the control alga. These results demonstrate that codon optimization increased the expression level of hemH-lba protein in the chloroplast of C. reinhardtii and improved algal H-2 yield by enhancing the respiration rate resulting in low 02 content in the medium and up regulation of the expression of hydA1 and hydA2 in cells, thereby confirming the potential of the utilization of leghemoglobins for H-2 production in green algae. (C) 2010 Elsevier Ltd. All rights reserved.