Accumulation of O2-tolerant phenotypes in H2-producing strains of Chlamydomonas reinhardtii by sequential applications of chemical mutagenesis and selection

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 H2-evolving activities of up to 10 times the O2-tolerance seen in the wild-type (WT). These isolates were generated by two sequential selections, consisting of random chemical mutagenesis, enrichment for H2-metabolism clones following exposure to increasing amounts of O2, and screening using a chemochromic sensor. The selected strains were characterized by two types of assays and classified as those that (a) can evolve H2 following exposure to O2 concentrations that inactive the WT strain and (b) in addition, are able to quickly reactivate H2-production activity once O2 is removed. These results suggest that O2-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 O2 when it is actively catalyzing H2 evolution.