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.

A Mutant in theADH1Gene ofChlamydomonas reinhardtiiElicits Metabolic Restructuring during Anaerobiosis

“…A similar coupling between PFL and AdhE in E. coli has been reported (for review, see Clark, 1989). Although other pathways in Chlamydomonas may generate acetaldehyde or acetyl-CoA (substrates for ADH1 and ethanol production), elevated CO 2 evolution in sodium hypophosphite-treated cultures (in which PFL activity is irreversibly blocked; Knappe et al, 1984;Plaga et al, 1988) implicates PDC3/ADH coupled reactions in the production of ethanol (Hemschemeier et al, 2008;Catalanotti et al, 2012). ADH1 and/or one of the other two putative ADH proteins in Chlamydomonas could potentially work in conjunction with PDC3 (previously referred to as PDC1 and PDC by Mus et al [2007] and Terashima et al [2010], respectively) to help manage intracellular redox conditions during anaerobiosis, especially when PFL1 activity is reduced or impaired.…”

“…ADH1 and/or one of the other two putative ADH proteins in Chlamydomonas could potentially work in conjunction with PDC3 (previously referred to as PDC1 and PDC by Mus et al [2007] and Terashima et al [2010], respectively) to help manage intracellular redox conditions during anaerobiosis, especially when PFL1 activity is reduced or impaired. However, the work presented here suggests that it is predominantly ADH1 that catalyzes the reduction of the acetaldehyde generated by PDC3 (Catalanotti et al, 2012;this paper), at least under the conditions used in our experiments. This coupling raises some issues concerning the way in which ADH1 accesses acetaldehyde, since the ADH1 protein was found exclusively in chloroplasts of Chlamydomonas, while PDC3 (which has no apparent transit peptide) was putatively cytoplasmic; acetaldehyde would have to traffic into plastids to be reduced to ethanol.…”

“…The adh1 strain did show some extracellular lactate accumulation, which was not observed in wild-type cells (Fig. 6C); however, a significantly larger increase in lactate accumulation was observed in strains lacking PFL1 (Philipps et al, 2011;Catalanotti et al, 2012). Furthermore, transcript levels for LDH were identical in adh1 and wild-type cells over the entire anoxic time course (Fig.…”

“…The metabolic pathway that leads to the fermentative production of succinate is unveiled in the hydEF-1 mutant (Dubini et al, 2009) and is depicted in the figure in green. An increase in the production of lactate, which is almost undetectable in fermenting wild-type cells, has been observed in the pfl1 mutants (Philipps et al, 2011;Catalanotti et al, 2012) and is highlighted in orange. ACK1, Acetate kinase isoform 1; ACK2, acetate kinase isoform 2; ADH, alcohol dehydrogenase (ADH1, ADH2, or ADH3 could perform this reaction; see text); ADH1, acetaldehyde/alcohol dehydrogenase; FDX, ferredoxin; FMR, fumarate reductase; FUM, fumarase; HYDA1 and HYDA2, two putative hydrogenases; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; MME4, malic enzyme; PAT1, phosphate acetyltransferase isoform 1; PAT2, phosphate acetyltransferase isoform 2; PDC3, pyruvate decarboxylase; PEPC, phosphenolpyruvate carboxylase; PFL1, pyruvate formate lyase; PFR1, pyruvate:ferredoxin oxidoreductase; PYC, pyruvate carboxylase; PYK, pyruvate kinase.…”

“…This figure was modified from Grossman et al (2011). fermentation metabolism (Dubini et al, 2009;Grossman et al, 2011;Philipps et al, 2011;Catalanotti et al, 2012). For example, in the Chlamydomonas hydEF-1 mutant (Dubini et al, 2009), pyruvate metabolism is redirected to the reverse tricarboxylic acid reactions, while in the pfl1 mutant (Philipps et al, 2011;Catalanotti et al, 2012), there is a marked increase in lactate production and a smaller increase in ethanol synthesis; both of these metabolites are linked to NADH reoxidation (Fig.…”