title: A mutant in the ADH1 gene of Chlamydomonas reinhardtii elicits metabolic restructuring during anaerobiosis.
abstract: The green alga Chlamydomonas reinhardtii has numerous genes encoding enzymes that function in fermentative pathways. Among these, the bifunctional alcohol/acetaldehyde dehydrogenase (ADH1), highly homologous to the Escherichia coli AdhE enzyme, is proposed to be a key component of fermentative metabolism. To investigate the physiological role of ADH1 in dark anoxic metabolism, a Chlamydomonas adh1 mutant was generated. We detected no ethanol synthesis in this mutant when it was placed under anoxia; the two other ADH homologs encoded on the Chlamydomonas genome do not appear to participate in ethanol production under our experimental conditions. Pyruvate formate lyase, acetate kinase, and hydrogenase protein levels were similar in wild-type cells and the adh1 mutant, while the mutant had significantly more pyruvate:ferredoxin oxidoreductase. Furthermore, a marked change in metabolite levels (in addition to ethanol) synthesized by the mutant under anoxic conditions was observed; formate levels were reduced, acetate levels were elevated, and the production of CO(2) was significantly reduced, but fermentative H(2) production was unchanged relative to wild-type cells. Of particular interest is the finding that the mutant accumulates high levels of extracellular glycerol, which requires NADH as a substrate for its synthesis. Lactate production is also increased slightly in the mutant relative to the control strain. These findings demonstrate a restructuring of fermentative metabolism in the adh1 mutant in a way that sustains the recycling (oxidation) of NADH and the survival of the mutant (similar to wild-type cell survival) during dark anoxic growth.
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305, USA. magneschi@sssup.it
authors.forename: Leonardo
authors.initials: L
authors.lastname: Magneschi
authors.forename: Claudia
authors.initials: C
authors.lastname: Catalanotti
authors.forename: Venkataramanan
authors.initials: V
authors.lastname: Subramanian
authors.forename: Alexandra
authors.initials: A
authors.lastname: Dubini
authors.forename: Wenqiang
authors.initials: W
authors.lastname: Yang
authors.forename: Florence
authors.initials: F
authors.lastname: Mus
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Posewitz
authors.forename: Michael
authors.initials: M
authors.lastname: Seibert
authors.forename: Pierdomenico
authors.initials: P
authors.lastname: Perata
authors.forename: Arthur R
authors.initials: AR
authors.lastname: Grossman
mesh_terms: D000084:Acetate Kinase / Q000235:genetics / Q000378:metabolism; D000085:Acetates / Q000378:metabolism; D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000693:Anaerobiosis; D015153:Blotting, Western; D002245:Carbon Dioxide / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D005561:Formates / Q000378:metabolism; D017343:Genes, Plant; D005990:Glycerol / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D055442:Metabolome; D009243:NAD / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D050743:Pyruvate Synthase / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D000085:Acetates; D005561:Formates; D010940:Plant Proteins; D009243:NAD; C030544:formic acid; D002245:Carbon Dioxide; D019344:Lactic Acid; D000431:Ethanol; D006859:Hydrogen; D000426:Alcohol Dehydrogenase; D050743:Pyruvate Synthase; D000123:Acetyltransferases; C022560:formate C-acetyltransferase; D000084:Acetate Kinase; D005990:Glycerol