Claudia Catalanotti

Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment containing the plasmid-free marker versus entire linearized plasmid with the marker) and in the strategies used to maintain and store transformants.

Fermentation metabolism and its evolution in algae

Fermentation or anoxic metabolism allows unicellular organisms to colonize environments that become anoxic. Free-living unicellular algae capable of a photoautotrophic lifestyle can also use a range of metabolic circuitry associated with different branches of fermentation metabolism. While algae that perform mixed-acid fermentation are widespread, the use of anaerobic respiration is more typical of eukaryotic heterotrophs. The occurrence of a core set of fermentation pathways among the algae provides insights into the evolutionary origins of these pathways, which were likely derived from a common ancestral eukaryote. Based on genomic, transcriptomic, and biochemical studies, anaerobic energy metabolism has been examined in more detail in Chlamydomonas reinhardtii (Chlamydomonas) than in any other photosynthetic protist. This green alga is metabolically flexible and can sustain energy generation and maintain cellular redox balance under a variety of different environmental conditions. Fermentation metabolism in Chlamydomonas appears to be highly controlled, and the flexible use of the different branches of fermentation metabolism has been demonstrated in studies of various metabolic mutants. Additionally, when Chlamydomonas ferments polysaccharides, it has the ability to eliminate part of the reductant (to sustain glycolysis) through the production of H2, a molecule that can be developed as a source of renewable energy. To date, little is known about the specific role(s) of the different branches of fermentation metabolism, how photosynthetic eukaryotes sense changes in environmental O2 levels, and the mechanisms involved in controlling these responses, at both the transcriptional and post-transcriptional levels. In this review, we focus on fermentation metabolism in Chlamydomonas and other protists, with only a brief discussion of plant fermentation when relevant, since it is thoroughly discussed in other articles in this volume.

Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

This article describes novel ways that algae may adjust metabolite trafficking when specific branches of fermentation metabolism are blocked. This rerouting of metabolites allows for continued glycolytic energy production under anoxic conditions, which is critical for the cell’s survival. Mechanisms associated with this reengineering of metabolism are almost completely unexplored. 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 H2 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 H2 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 the ADH1 Gene of Chlamydomonas reinhardtii Elicits Metabolic Restructuring during Anaerobiosis

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 CO2 was significantly reduced, but fermentative H2 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.

Mutagenesis and phenotypic selection as a strategy toward domestication of Chlamydomonas reinhardtii strains for improved performance in photobioreactors.

Microalgae have a valuable potential for biofuels production. As a matter of fact, algae can produce different molecules with high energy content, including molecular hydrogen (H2) by the activity of a chloroplastic hydrogenase fueled by reducing power derived from water and light energy. The efficiency of this reaction, however, is limited and depends from an intricate relationships between oxygenic photosynthesis and mitochondrial respiration. The way toward obtaining algal strains with high productivity in photobioreactors requires engineering of their metabolism at multiple levels in a process comparable to domestication of crops that were derived from their wild ancestors through accumulation of genetic traits providing improved productivity under conditions of intensive cultivation as well as improved nutritional/industrial properties. This holds true for the production of any biofuels from algae: there is the need to isolate multiple traits to be combined and produce organisms with increased performances. Among the different limitations in H2 productivity, we identified three with a major relevance, namely: (i) the light distribution through the mass culture; (ii) the strong sensitivity of the hydrogenase to even very low oxygen concentrations; and (iii) the presence of alternative pathways, such as the cyclic electron transport, competing for reducing equivalents with hydrogenase and H2 production. In order to identify potentially favorable mutations, we generated a collection of random mutants in Chlamydomonas reinhardtii which were selected through phenotype analysis for: (i) a reduced photosynthetic antenna size, and thus a lower culture optical density; (ii) an altered photosystem II activity as a tool to manipulate the oxygen concentration within the culture; and (iii) State 1–State 2 transition mutants, for a reduced cyclic electron flow and maximized electrons flow toward the hydrogenase. Such a broad approach has been possible thanks to the high throughput application of absorption/fluorescence optical spectroscopy methods. Strong and weak points of this approach are discussed.

Alternative Acetate Production Pathways in Chlamydomonas reinhardtii during Dark Anoxia and the Dominant Role of Chloroplasts in Fermentative Acetate Production

Acetate is a primary Chlamydomonas fermentative product and is linked to dark, anoxic ATP biosynthesis. Chlamydomonas ack/pat mutants were isolated to further characterize fermentation networks, revealing that chloroplast pathways are dominant in this alga, and that despite blocking the primary ATP-generating routes to acetate, Chlamydomonas retains the metabolic flexibility to produce acetate. Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism

Significance Our results suggest that particular ferredoxins in photosynthetic organisms are tailored to serve as electron carriers that sustain day-time and night-time metabolism and that the chloroplast-localized ferredoxin-5 (FDX5) appears to function in the desaturation of fatty acids required for maintaining the correct ratio of the dominant lipids in the thylakoid membranes and the integration of chloroplast and mitochondrial metabolism, which is absolutely required for growth in the dark. The most important messages from this work are that redox components associated with critical activities in photosynthetic organisms must be tuned to the redox conditions of the cells and the overall carbon budget of photosynthetic cells requires an understanding of metabolic features that accompany the movement of cells between light and dark conditions. Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions.

Novel Thylakoid Membrane GreenCut Protein CPLD38 Impacts Accumulation of the Cytochrome b6f Complex and Associated Regulatory Processes

Background: The GreenCut is a group of ∼600 green-lineage-specific proteins hypothetically involved in photosynthesis. Results: A Chlamydomonas reinhardtii mutant disrupted for GreenCut gene CPLD38 has a marked reduction in cytochrome b6f and an increase in chlororespiration. Conclusion: CPLD38 is essential for accumulating cytochrome b6f and balancing chlororespiration and photosynthesis. Significance: This analysis demonstrates the importance of a GreenCut protein in photosynthesis. Based on previous comparative genomic analyses, a set of nearly 600 polypeptides was identified that is present in green algae and flowering and nonflowering plants but is not present (or is highly diverged) in nonphotosynthetic organisms. The gene encoding one of these “GreenCut” proteins, CPLD38, is in the same operon as ndhL in most cyanobacteria; the NdhL protein is part of a complex essential for cyanobacterial respiration. A cpld38 mutant of Chlamydomonas reinhardtii does not grow on minimal medium, is high light-sensitive under photoheterotrophic conditions, has lower accumulation of photosynthetic complexes, reduced photosynthetic electron flow to P700+, and reduced photochemical efficiency of photosystem II (ΦPSII); these phenotypes are rescued by a wild-type copy of CPLD38. Single turnover flash experiments and biochemical analyses demonstrated that cytochrome b6f function was severely compromised, and the levels of transcripts and polypeptide subunits of the cytochrome b6f complex were also significantly lower in the cpld38 mutant. Furthermore, subunits of the cytochrome b6f complex in mutant cells turned over much more rapidly than in wild-type cells. Interestingly, PTOX2 and NDA2, two major proteins involved in chlororespiration, were more than 5-fold higher in mutants relative to wild-type cells, suggesting a shift in the cpld38 mutant from photosynthesis toward chlororespiratory metabolism, which is supported by experiments that quantify the reduction state of the plastoquinone pool. Together, these findings support the hypothesis that CPLD38 impacts the stability of the cytochrome b6f complex and possibly plays a role in balancing redox inputs to the quinone pool from photosynthesis and chlororespiration.

Algae after dark: mechanisms to cope with anoxic/hypoxic conditions

Chlamydomonas reinhardtii is a unicellular, soil-dwelling (and aquatic) green alga that has significant metabolic flexibility for balancing redox equivalents and generating ATP when it experiences hypoxic/anoxic conditions. The diversity of pathways available to ferment sugars is often revealed in mutants in which the activities of specific branches of fermentative metabolism have been eliminated; compensatory pathways that have little activity in parental strains under standard laboratory fermentative conditions are often activated. The ways in which these pathways are regulated and integrated have not been extensively explored. In this review, we primarily discuss the intricacies of dark anoxic metabolism in Chlamydomonas, but also discuss aspects of dark oxic metabolism, the utilization of acetate, and the relatively uncharacterized but critical interactions that link chloroplastic and mitochondrial metabolic networks.