Accumulation of O 2-tolerant phenotypes in H 2-producing strains of Chlamydomonas reinhardtii by sequential applications of chemical mutagenesis and selection

Abstract

The photoproduction of hydrogen by anaerobically induced algae is catalyzed by a bidirectional hydrogenase that is rapidly inactivated by oxygen. We isolated two generations of Chlamydomonas reinhardtii strains with H 2-evolving activities of up to 10 times the O 2-tolerance seen in the wild-type (WT). These isolates were generated by two sequential selections, consisting of random chemical mutagenesis, enrichment for H 2-metabolism clones following exposure to increasing amounts of O 2, and screening using a chemochromic sensor. The selected strains were characterized by two types of assays and classified as those that (a) can evolve H 2 following exposure to O 2 concentrations that inactive the WT strain and (b) in addition, are able to quickly reactivate H 2-production activity once O 2 is removed. These results suggest that O 2-tolerance can be increased by successive rounds of mutagenesis, selection, and screening, demonstrating that the WT phenotype can be improved by genetic means. Other results show that the hydrogenase is less sensitive to O 2 when it is actively catalyzing H 2 evolution.