Isolation of Chlamydomonas Mutants with Improved Oxygen-Tolerance

Timothy Flynn; Maria L. Ghirardi; Michael Seibert

Isolation of Chlamydomonas Mutants with Improved Oxygen-Tolerance

Timothy Flynn, Maria L. Ghirardi, Michael Seibert

Bioenergy Science and Technology

National Renewable Energy Laboratory

Research output: Contribution to conference › Paper › peer-review

3 Scopus Citations

Abstract

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 language	American English
Pages	846-850
Number of pages	5
State	Published - 22 Aug 1999
Event	218th ACS National Meeting - New Orleans, Louisiana Duration: 22 Aug 1999 → 26 Aug 1999

Conference

Conference	218th ACS National Meeting
City	New Orleans, Louisiana
Period	22/08/99 → 26/08/99

NREL Publication Number

NREL/CP-29039

Cite this

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title = "Isolation of Chlamydomonas Mutants with Improved Oxygen-Tolerance",

abstract = "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.",

author = "Timothy Flynn and Ghirardi, {Maria L.} and Michael Seibert",

year = "1999",

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language = "American English",

pages = "846--850",

note = "218th ACS National Meeting ; Conference date: 22-08-1999 Through 26-08-1999",

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TY - CONF

T1 - Isolation of Chlamydomonas Mutants with Improved Oxygen-Tolerance

AU - Flynn, Timothy

AU - Ghirardi, Maria L.

AU - Seibert, Michael

PY - 1999/8/22

Y1 - 1999/8/22

N2 - 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.

AB - 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.

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M3 - Paper

AN - SCOPUS:0039186012

SP - 846

EP - 850

T2 - 218th ACS National Meeting

Y2 - 22 August 1999 through 26 August 1999

ER -

Isolation of Chlamydomonas Mutants with Improved Oxygen-Tolerance

Abstract

Conference

NREL Publication Number

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