A pyruvate formate lyase‐deficient Chlamydomonas reinhardtii strain provides evidence for a link between fermentation and hydrogen production in green algae

Abstract

SUMMARYThe green alga Chlamydomonas reinhardtii has a complex anaerobic metabolism characterized by a plastidic hydrogenase (HYD1) coupled to photosynthesis and a bacterial-type fermentation system in which pyruvate formate lyase (PFL1) is the central fermentative enzyme. To identify mutant strains with altered hydrogen metabolism, a C. reinhardtii nuclear transformant library was screened. Mutant strain 48F5 showed lower light-dependent hydrogen (H 2 ) evolution rates and reduced in vitro hydrogenase activity, and fermentative H 2 production in the dark was enhanced. The transformant has a single integration of the paromomycin resistance cassette within the PFL1 gene, and is unable to synthesize PFL1 protein. 48F5 secretes no formate, but produces more ethanol, D-lactate and CO 2 than the wild type. Moreover, HYD1 transcript and HYD1 protein levels were lower in the pfl1 mutant strain. Complementation of strain 48F5 with an intact copy of the PFL1 gene restored formate excretion and hydrogenase activity to the wild type level. This analysis shows that the PFL1 pathway has a significant impact on hydrogen metabolism in C. reinhardtii.

A Mutant in theADH1Gene ofChlamydomonas reinhardtiiElicits Metabolic Restructuring during Anaerobiosis

“…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.…”

“…Analogous to the consequences of metabolic blocks that have been reported in previous studies (Dubini et al, 2009;Philipps et al, 2011;Catalanotti et al, 2012), a block in the conversion of acetaldehyde and acetylCoA to ethanol leads to the rerouting of metabolites to other pathways in the fermentation network; some of these pathways are marginally active in wild-type cells. For the adh1 mutant placed under anoxic conditions, the level of extracellular formate is decreased while the level of acetate is increased relative to wildAnaerobic Metabolism in Chlamydomonas adh1 type cells.…”

“…Previous work has shown that blocking various reactions associated with fermentation metabolism can cause changes in the flow of metabolites through other branches of the fermentative network. For example, Chlamydomonas strains lacking PFL1 (Philipps et al, 2011;Catalanotti et al, 2012) compensate for the loss of this activity by increasing the synthesis of lactate through the activity of LDH, an enzyme that in wild-type cells would compete with PFL1 for the substrate pyruvate. Hence, a proportion of the pyruvate that accumulates in the pfl1 mutant would be metabolized by LDH, resulting in the accumulation of lactate and the reoxidation of 1 mol of NADH, which would help sustain the catabolism of polysaccharides and the synthesis of ATP in the mutant (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.…”