A Mutant in theADH1Gene ofChlamydomonas reinhardtiiElicits 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.

A novel screening method for hydrogenase-deficient mutants in Chlamydomonas reinhardtii based on in vivo chlorophyll fluorescence and photosystem II quantum yield

“…In contrast, adh1 mutant strain defective in acetalhedyde/alcohol dehydrogenase, although also exhibiting important metabolic changes in anaerobiosis is not affected in hydrogenase expression [47] or activity and thus displays a wild-type fluorescence signature.…”

“…pfl1 mutant is defective in pyruvate formate lyase and showed a 30-45 % residual hydrogenase activity and H 2 production compared to cc-124 wild-type strain [27,46]. It was here compared to a parental cc-125 wild-type strain and to another fermentation mutant strain (adh1) lacking bifunctional acetaldehyde/alcohol dehydrogenase for which no hydrogenase deficiency has been reported [47]. Mutants lacking mitochondrial respiratorychain complex III (dum11) or complexes I and III (dum22) have been also investigated.…”

COPPER RESPONSE REGULATOR1–Dependent and –Independent Responses of theChlamydomonas reinhardtiiTranscriptome to Dark Anoxia

“…In the light, these enzymes generate H 2 using photosynthetically provided electrons (Ghirardi et al, 2009;Hemschemeier and Happe, 2011). C. reinhardtii also employs a pyruvate:formate lyase (PFL1) and the enzymes involved in the PFL pathway, which form the backbone of the fermenting metabolism in (facultative) anaerobic bacteria like Escherichia coli (Atteia et al, 2006;Hemschemeier et al, 2008;Philipps et al, 2011;Magneschi et al, 2012). Additionally, the alga has a pyruvate:ferredoxin oxidoreductase (PFR1) (Mus et al, 2007;Hemschemeier et al, 2008;Terashima et al, 2010;van Lis et al, 2013;Noth et al, 2013).…”

Concerted Up-regulation of Aldehyde/Alcohol Dehydrogenase (ADHE) and Starch in Chlamydomonas reinhardtii Increases Survival under Dark Anoxia

“…PFO is expressed under anaerobic conditions and localizes to the chloroplast where it is coupled to H 2 production by iron-only hydrogenases via FDX1 (PetF) (18,19). ADHE is a mono-iron enzyme, which was found to be the major ethanol-producing enzyme in conditions of dark anoxia (22). Although it is now clear that the bacterial-type enzymes fully participate in the anaerobic life of C. reinhardtii, their physiological significance in the intricate anaerobic network (Fig.…”