Isolation of Chlamydomonas Mutants with Improved Oxygen-Tolerance

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The photoproduction of H2 from water by anaerobically-induced algae is catalyzed by a bidirectional hydrogenase, an enzyme that is rapidly deactivated by exposure to low levels of O2. We have developed two selective pressures, with which a mutant's survival depends on hydrogenase activity in the presence of O2, and a chemochromic screening method to quickly identify and isolate desirable O2-tolerant hydrogenase mutants. The clones that still produced H2 after exposure to O2 were further characterized by gas chromatography for maximal H2-production rate and tolerance to O2. The best mutant obtained by a single round of mutagenesis/selection/screening maintained up to 35% of its maximal H2-production rate (measured with no exposure to O2) following deactivation of the enzyme by 2% O2 for 2 minutes, a condition that almost totally inactivates wild-type (WT) H2 production. A mutant that underwent a second round of mutagenesis and selection exhibited 49% of its maximal activity following the deactivation treatment. This represents a 10-fold improvement over the WT strain and suggests that even better mutants will be forthcoming.

Original languageAmerican English
Number of pages5
StatePublished - 22 Aug 1999
Event218th ACS National Meeting - New Orleans, Louisiana
Duration: 22 Aug 199926 Aug 1999


Conference218th ACS National Meeting
CityNew Orleans, Louisiana

NREL Publication Number

  • NREL/CP-29039


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