DEVELOPMENT OF AN EFFICIENT ALGAL H2-PRODUCTION SYSTEM

The ultimate goal of our research is to generate Chlamydomonas reinhardtii mutants that are sufficiently tolerant to O2 to produce H2 under aerobic conditions. We have been addressing this goal by means of both classical genetic and molecular biology approaches. Two generations of algal mutants were produced by application of chemical mutagenesis, selection, and screening to a wild-type algal strain. The H2 production activity of the mutants showed up to 9 times higher tolerance to O2 when compared to the parental strain. However, since the algal hydrogenase gene sequence was not known, it was not possible to determine whether the chemical mutagenesis that gave rise to the O2 tolerant phenotypes was due to a single-point mutation affecting the hydrogenase gene or whether it was due to a secondary mutation in another gene. We have concomitantly pursued a molecular biology approach, in order to enhance the probability of ultimately obtaining a commercially-viable organism. Our purpose was to first clone the hydrogenase gene and then to use molecular genetic means to generate random mutations in the hydrogenase gene. The transformed genes would be used in the future to re-transform wild-type C. reinhardtii, and O2-tolerance of the resulting transformants would be determined by chemochromic screening, as was done for the classical mutants. Current results include the successful cloning and sequencing of not only the Fe-only reversible hydrogenase gene but also a second Fe-only hydrogenase gene in C. reinhardtii. Preliminary studies suggesting differences in the expression of the two genes are also reported. The cloning of the algal hydrogenase will also allow us to re-analyze our mutants obtained previously by the classical mutagenesis approach. This will determine the specific locus of each genotype and may help guide us in possible future site-directed mutagenesis approaches to improving O2 toleranc