title: Altered fermentative metabolism in Chlamydomonas reinhardtii mutants lacking pyruvate formate lyase and both pyruvate formate lyase and alcohol dehydrogenase.
abstract: Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H(2) production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H(2) production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305, USA. ccatal1@stanford.edu
authors.forename: Claudia
authors.initials: C
authors.lastname: Catalanotti
authors.forename: Alexandra
authors.initials: A
authors.lastname: Dubini
authors.forename: Venkataramanan
authors.initials: V
authors.lastname: Subramanian
authors.forename: Wenqiang
authors.initials: W
authors.lastname: Yang
authors.forename: Leonardo
authors.initials: L
authors.lastname: Magneschi
authors.forename: Florence
authors.initials: F
authors.lastname: Mus
authors.forename: Michael
authors.initials: M
authors.lastname: Seibert
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Posewitz
authors.forename: Arthur R
authors.initials: AR
authors.lastname: Grossman
mesh_terms: D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D005561:Formates / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006859:Hydrogen / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010084:Oxidation-Reduction; D019289:Pyruvic Acid / Q000378:metabolism
chemical_list: D005561:Formates; C030544:formic acid; D019344:Lactic Acid; D000431:Ethanol; D006859:Hydrogen; D019289:Pyruvic Acid; D000426:Alcohol Dehydrogenase; D000123:Acetyltransferases; C022560:formate C-acetyltransferase