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Issue 1, July 2004

Biological & Biomedical Sciences

A Putative Pyruvate: Ferredoxin Oxidoreductase Gene in Chlamydomonas reinhardtii is Expressed under Hydrogen Production Conditions

Jonathan Meuser
University of California at Davis
Advisor: Maria Ghirardi, Ph.D.
National Renewably Energy Laboratory
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Abstract

In an anaerobic environment, the unicellular green algae Chlamydomonas reinhardtii produces hydrogen (H2) gas and is a potential source of this clean, renewable fuel. C. reinhardtii can produce H2 anaerobically in either light or dark through photoproductive and fermentative pathways, respectively. The goal of this work was to identify the link between fermentative carbon metabolism and dark, anaerobic hydrogen production in C. reinhardtii. Though H2 photoproduction pathways are well-studied, less is known about the role of fermentative pathways in dark H2 production. As fermentative pathways are better elucidated, H2 production levels may be improved by more efficient conversion of carbon substrates. Both of C. reinhardtii’s [Fe]-hydrogenases, HydA1 and HydA2, catalyze H2 production using electrons from ferredoxin. One product of fermentative carbon metabolism is pyruvate. In several species of anaerobic microbes, the decarboxylation of pyruvate to acetyl-coA by pyruvate:oxidoreductase (PFOR) is linked to hydrogen production via the reduction of ferredoxin. The completion of the C. reinhardtii genome has allowed us to use bioinformatics to identify a putative PFOR gene based on a blast search. A single gene was identified that encoded a putative protein with four orthologous functional subunits with high similarity to other known PFORs. Northern blot data of RNA from anaerobically induced cells demonstrated that the putative PFOR gene is expressed. Moreover, expression revealed that the putative PFOR is transcribed concomitantly with hydrogenase genes HydA1 and HydA2 under anaerobic conditions. The identification in C. reinhardtii of a putative PFOR gene and evidence of its coexpression with HydA1 and HydA2 suggests fermentative carbon metabolism and dark, anaerobic H2 production may be linked via ferredoxin. This new PFOR could explain the source of electrons in dark, fermentative H2 production, a discovery with the potential to improve dark H2 conversion efficiency.

 

Introduction

The study of Chlamydomonas reinhardtii fermentative biology began with Hans Gaffron’s discovery of H2 production in Scenedesmus obliquus and Chlamydomonas moewusii in 1939 (Gaffron 1939; Gaffron and Rubin 1942). C. reinhardtii has a dynamic metabolism, and is able to regulate biochemical pathways to obtain energy and recycle necessary cofactors in response to changes in growth conditions (Klein and Betz 1978; Ohta and Miura 1987; Geffler and Gibbs 1984). While an aerobic and illuminated environment activates photoautotrophic pathways, an anaerobic environment activates fermentative, H2-yielding pathways (Melis et al. 2000). Because C. reinhardtii [Fe]-hydrogenase enzymes are inactivated by O2, light-catalyzed water-splitting and subsequent release of O2 by the Photosystem II complex inhibits H2 production (Melis et al. 2000). Under anaerobic conditions, light can stimulate H2 production due to excitation of Photosystem I (PSI), which donates electrons to chloroplastic ferredoxin, the only known reductant of [Fe]-hydrogenase enzymes (Melis et al. 2000). Since the discovery of algal H2 production, it has been theorized that a strictly fermentative pathway exists. However, complete characterization of the pathway(s) has not yet been accomplished (Gaffron 1939; Gaffron and Rubin 1942; Healy 1970; Klein and Betz 1978; Bamberger et al. 1982; Geffler and Gibbs 1984; Ohta et al. 1987). Hydrogen production in complete darkness, first witnessed by Gaffron, demonstrates that ferredoxin, the only known electron donor to the C. reinhardtii [Fe]-hydrogenase enzymes, must be reduced independently of PSI activity.

Many anaerobic eukaryotes produce H2 gas anaerobically. These amitochondriate anaerobes often possess a hydrogenosome, a specialized organelle that, like the mitochondria, compartmentalize carbon metabolism, but differ by functioning in the production of H2 gas (Hackstein et al. 1999; Dyal and Johnson 2000). PFOR is likely an ancient component of anaerobic metabolism, existing before the divergence of archaeabacteria and eukaryotes (Ragsdale 2003). PFOR generates low-potential electrons (E0’ = -540 mV), more negative than that of the NAD+/NADH couple (Nicotinamide Adenine Dinucleotide), and reduces ferredoxin and/or flavodoxin (Ragsdale 2003). The PFOR enzyme is a key component of amitochondriate eukaryote metabolism, linking the decarboxylation of pyruvate to terminal electron acceptors via the reduction of ferredoxin (Horner et al. 1999). In many hydrogenosomal eukaryotes, the electron acceptor is H+ via an [Fe]-hydrogenase with high similarity to algal [Fe]-hydrogenases (Hackstein et al. 1999; Forestier et al. 2003). This reaction maintains the redox balance through the dispensing of electrons as H2 gas (Hackstein et al. 1999). In C. reinhardtii, the [Fe]-hydrogenases, HydA1 and HydA2, which produce molecular H2 by the reduction of protons, receive electrons from ferredoxin, a common intermediate of electron transport chains (Semin et al. 2003). In the dark, algae produce H2 in the absence of photosynthetically reduced ferredoxin. Thus, an alternative electron-donor to ferredoxin (other than photolyzed water) must exist under fermentative conditions.

Here, we test the hypothesis that dark, fermentative H2 metabolism in C. reinhardtii involves a PFOR pathway, as demonstrated in H2-producing anaerobic eukaryotes (King, personal communication). We provide an initial study on identification of a PFOR gene and its role in algal fermentative H2 production.

 

Materials and methods

Putative Gene Identification

A putative PFOR was identified through a tBLASTn (protein vs. translated nucleotide) search of the DOE Joint Genome Institute Chlamydomonas reinhardtii genome (JGI Chlamy v1.0) using the protein sequence of the POR B subunit of the Pyrococcus abysii pyruvate:ferredoxin oxidoreductase (Protein Accession #Q9UYZ5, NCBI). Other domains and potential orthologs of the putative PFOR were found by a National Center for Biotechnological Information (NCBI) BLASTp (protein: protein). The entire putative C. reinhardtii gene sequence was located on contig 6 of Scaffold 548, encoding a 4449bp transcript and a 1315 amino acid protein.

Sequencing of Unresolved Repeat Region

An unresolved AAC repeat sequence in the N-terminal region of the PFOR was amplified by PCR with primer pairs designed using PrimerQuestSM (Integrated DNA Technologies, Coralville, IA). PCR reactions contained 1µL of KOD HOTSTART polymerase (Novagen, Madison WI), 5-7?L of EcoR1 digested genomic DNA as template, 125ng of each of forward (PFOR forward 5’-CTTTGTCTGCTTGCAACACGAGTC-3’), and reverse (PFOR reverse 5’-TGTTGGAACTCGCTCGGTGGATA-3’) primers, 1 mM MgSO4, 0.2mM each dNTP’s, and 2% DMSO in a 50?L volume. Resulting PCR products were purified by gel electrophoresis using a Qiaquick gel purification kit (Qiagen, Valencia, Ca). The PFOR N-terminal fragment was estimated by gel electrophoresis to be 0.7kb. Concentration and purity of the PCR products were verified by UV absorbance at 260nm and by gel electrophoresis. Sequencing was performed by SeqWright (Houston, Texas) using PFOR-specific oligos (forward 5’-CTTGCTTCACCAACATCACCAACG-3’ and reverse 5’-GATGATGTGA-TGTGATGTGCGCCT-3’).

Orthologous Protein Identification

Comparisons of the predicted C. reinhardtii PFOR conserved subunits to other organisms showed similarity using the NCBI CDART: Conserved Domain Architecture Retrieval Tool. The inferred PFOR amino acid sequence and the selected orthologs were aligned using ClustalW1.8 (Jeanmougin et al. 1998) and characterized with Genedoc (Nicholas and Nicholas 1997).

Cell growth, anaerobic induction, and H2 assay

Chlamydomonas reinhardtii strain cc849 (cell wall-less) was grown in 1-liter Roux flasks photoheterotropically in TAP liquid medium (Harris 1989) supplemented with sterile 5% CO2 in air. Cool white fluorescent light (150 μE m-2s-1, PAR) illuminated the culture. Cells were harvested and anaerobically induced as previously described (Melis et al. 2000; Forestier et al. 2004). Hydrogenase activity of anaerobically induced cultures was quantified as methyl-viologen-dependent H2 gas production measured by gas chromatography (Flynn et al. 2002).

Northern blots and hybridization

A probe was designed to the 3’ end of the PFOR gene and specificity tested by an NCBI BLAST. PCR was used to amplify the probe region; the product was purified by gel electrophoresis using a Qiaquick gel purification kit (Qiagen, Valencia, Ca). Total RNA was isolated using the Micro to Midi kit (Invitrogen, Carlsbad, Ca). Ten ?g of RNA were separated by gel electrophoresis on denaturing 1.1% agarose, 0.22 M formaldehyde gels and blotted onto a Nytran N+ nylon membrane with 10X SSC transfer buffer (Forestier et al. 2004). The Rediprime random primer labeling kit (Amersham, Piscataway, NJ) was used to generate radiolabeled probes, which were hybridized in buffer (6x SSC, 5x Denhardt’s solution, and 0.1% SDS) overnight at 65? C. The membranes were washed the following day and exposed to Kodak Bio Max X-ray film at -80? C.

 

Results

Genetic analysis of the putative PFOR

Based on bioinformatics, a predicted a PFOR gene was identified as a 7 kb region on Scaffold 548. The predicted PFOR transcript is 4.5 kb, encoding a 1316 amino acid protein. Purification and sequencing of a poorly resolved repeat region resolved the sequence of the N-terminus coding region, which contains nine consecutive ACC repeats (Figure 1).

Figure 1 .N-terminal region of putative Chlamydomonas PFOR with verified repeat region boxed.



The putative C. reinhardtii PFOR protein exhibits highest similarity to a putative PFOR of Heliobacillus mobilis. Also, extensive similarity exists between C. reinhardtii PFOR and other PFOR enzymes found in a range of prokaryotes and eukaryotes (Table 1, Figure 2) (Ragsdale 2000; Pieulle 1997; Jeanmougin 1998). The C. reinhardtii putative PFOR gene sequence and the sequence of five orthologous organisms show high levels of similarity covering ~50-68% of the total protein sequence (Figure 3).

Table 1.Orthology of Other PFORs to C. reinhardtii’s Predicted Protein

*Similarity refers to a BLAST positive matrix score, whereas identity refers to the extent to which two amino acid sequences are invariant.
Source: National Center for Biotechnology Information (NCBI)

Figure 2. Predicted subunits of C. reinhardtii putative PFOR based on amino acid sequence homology to other known orthologous domains.

Prokaryotic orthologs showed little similarity in the N-terminal 60 amino acids of the putative C. reinhardtii PFOR. However, a green alga, Euglena gracilis, pyruvate: NADP+ oxidoreductase (PNO), (PFOR:CPR fusion) with a known mitochondrial transit peptide sequence (Rotte et al. 2001), aligns strongly with the corresponding C. reinhardtii predicted PFOR (Figure 4). The first four domains of the Euglena oxidoreductase are highly homologous to the putative C. reinhardtii PFOR (Figure 3). The similarity between the N-terminus of the Euglena PNO and the predicted N-terminus of the Chlamydomonas. PFOR were based on 12 groups representing three overlapping hierarchies (Figure 4). The three hierarchies are based on size, the electric charge of polar amino acids and aromaticity (Nicholas and Nicholas 1997).

Based on PSORT (51.7% likelihood) and IPSORT (Nakai 1992), a putative mitochondrial localization sequence and processing site have been identified in the C. reinhardtii PFOR. However, the ChloroP (Emanuelsson et al. 1992) transit peptide prediction program predicted a 52.5% chance of chloroplast localization for the C. reinhardtii PFOR.

Figure 4. Physio-chemical alignment based on three hierarchies of size, charge of polar amino acids, and aromaticity (Genedoc) of C. reinhardtii and Euglena gracilis protein sequences. Alignment shows similarity of C. reinhardtii predicted protein to the known transit motif of E. gracilis to predict potential mitochondrial localization and cleavage site (predicted cleavage site is shown by the arrow).

Anaerobic expression of PFOR

C. reinhardtii PFOR transcript can be detected by Northern blot analysis of total RNA isolated from anaerobically induced cells in a position close to its predicted transcript size (4.5 kb), demonstrating that PFOR is expressed in C. reinhardtii under anaerobic conditions (Figure 5). Anaerobic induction was also verified by measurement of H2 gas production of anaerobically-induced cells exposed to reduced methyl viologen, which can donate electrons to [Fe]-hydrogenase enzymes. The HydA1 and HydA2 hydrogenases were shown to be co-expressed with PFOR. Northern blot of RNA sample used to detect PFOR expression also produced positive signals using HydA1 or HydA2 probes (Figure 5).

Figure 5. Northern blot showing C. reinhardtii HydA1 (lane 1), HydA2 (lane 2), and the putative C. reinhardtii PFOR (lane 3) transcript accumulation after five hours of anaerobiosis in the dark (non-specific ribosomal hybridization omitted). “MV activity” refers to amount of hydrogen gas produced in anaerobically induced cells exposed to reduced methyl viologen, an electon donor to [Fe]-hydrogenase enzymes.

Discussion

The PFOR gene is an important part of anaerobic metabolism in many H2-producing amitochondriate eukaryotes (Hackstein et al. 1999; Forestier et al. 2003). Thus we hypothesized that C. reinhardtii, which anaerobically produces H2 in the dark, might also possess a PFOR ortholog (King, personal communication). In this work, a number of approaches were employed to initially identify and characterize PFOR in Chlamydomonas. First, we used bioinformatics to identify the PFOR ortholog (Table 1). Sequencing of the PFOR gene was then done to resolve the gene sequence (Figure 2). A phylogenetic study verified the predicted protein sequence and identified potential functional domains and a potential transit peptide cleavage site (Figure 3, Figure 4). Lastly, Northern blot confirmed expression of PFOR using gene specific probes on total RNA from anaerobically induced cells (Figure 5). Thus, the present communication provides an initial study on the role of a new PFOR in algal fermentative H2 production.

In aerobic metabolism the oxidative decarboxylation of pyruvate to acetyl-coA is catalyzed by the pyruvate dehydrogenase (PDH) system, a multi-enzyme complex that reduces a molecule of NAD+ to NADH for each pyruvate (Lehinger 1970). Whereas the mitochondrial electron transport chain recycles NADH to NAD+ in aerobic metabolism, the regeneration of NAD+, an essential cofactor in the glycolytic pathway, becomes paramount in fermentative metabolism due to the inactivation of the mitochondrial NADH oxidoreductase (Lehinger 1970). Under anaerobic conditions the dynamic metabolism of Chlamydomonas can adapt to the needs of the cell, activating endogenous fermentative pathways similar to those found in anaerobic prokaryotes (Happe et al. 2002). Enzymes that catalyze the regeneration of NAD+ such as alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) are upregulated in C. reinhardtii under anaerobic conditions (Figure 6). Likewise, pyruvate:formate lyase (PFL), a non-NAD+ reducing alternative to PDH, is upregulated. (Lehinger 1970; Hackstein et al. 1999).

Figure 6. Hypothetical fermentative metabolism of C. rheinhardtii showing the proposed role of the putative PFOR in dark fermentative hydrogen production. Other enzymes: pyruvate:formate lyase (PFL); acetylaldehyde dehydrogenase (ACDH); alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH). Other pathways in figure adapted from Hackstein et al. (1999).

We propose that, in C. reinhardtii, an alternative to PDH, PFOR, contributes to redox state balance during anaerobiosis by coupling the conversion of pyruvate to acetyl CoA with the production of H2 gas. In Trichomonas vaginalis, a eukaryotic containing a PFOR (Table 1, Figure 3) and [Fe]-hydrogenase, this pathway has been well characterized (Rasoloson et al. 2002). Because PFOR has the potential to reduce ferredoxin, the source of electrons for C. reinhardtii hydrogenases, we theorize that PFOR contributes to the reduction of hydrogenase(s) in C. reinhardtii (Figure 6).

If the PFOR gene in C. reinhardtii links fermentative metabolism to fermentative H2 evolution, this association helps to explain the final link of electrons to H2 under dark fermentative growth conditions. In contrast to photohydrogen production, dark fermentative metabolism produces CO2 at an average rate of 2.2 ? 0.46 moles per mole of H2 (Healy 1970; Bamberger et al. 1982). Because ferredoxin is a single electron carrier, a mole of H2 gas could be produced for every two moles of pyruvate decarboxylated by the PFOR enzyme. Gaffron and Rubin (1942) showed that glucose had a stimulatory effect on algal H2 production and concluded the existence of distinct light and dark H2 pathways. The presence of PFOR helps to explain how glucose could cause this effect. Experimental evidence that a larger percentage of pyruvate is decarboxylated to acetyl-coA in C. reinhardtii fermentative metabolism led Healey (1970) to theorize that the oxidative decarboxylation of pyruvate to acetyl-coA produces hydrogenase reductant (Bamberger et al. 1982; Healey 1970). Low lipoic acid levels in a variety of organisms, including C. reinhardtii, also suggested the presence of PFOR (Bothe and Nolteernsting 1975), though these results were never independently validated. Current work lends support to these theories.

The predicted amino-acid sequence of the C. reinhardtii PFOR contains four highly conserved PFOR motifs; the ?, ?, ?, and NAPF subunits (Figure 2) (Horner 1999; Rotte et al. 2001). The NAPF ferredoxin-binding domain contains two conserved 4Fe-4S (CxxCxxCxxxC) binding domains, which have been shown to participate in electron transfer to ferredoxin (Pieulle 1997; Rotte et al. 2001).

A preliminary survey of organisms with orthologous proteins elucidates two commonalities. First, they share the ability to grow in an anaerobic environment. Secondly, most PFOR-possessing organisms produce H2 gas and more importantly, they do so with an [Fe]-only hydrogenase like that found in Chlamydomonas (Nakos et al. 1971; Holo and Sirevåg 1986; Meyer et al. 1991; Blankenship et al. 1995; Bui and Johnson 1995; Nicholas and Nicholas 1997; Horner et al. 2000; Tamagnini et al. 2002; Pohorelic et al. 2002; Ragsdale 2003; Lin et al. 2003). Thus, the suspected role of PFOR in C. reinhardtii has been documented in other anaerobic eukaryotes.

It is likely that the PFOR gene in C. reinhardtii PFOR is localized in the mitochondria. Physio-chemical alignment of the Euglena transit peptide with known mitochondrial targeting lends support to this hypothesis (Figure 4) (Rotte et al. 2001). Based on the hydrogenosomal localization of PFOR in other organisms (Hackstein 1999) and its likely protomitochondrion origin (Horner 1999), mitochondrial localization in C. reinhardtii would be expected. Further work is necessary, however, to definitively show PFOR localization in C. reinhardtii.

Expression studies provide the most convincing evidence of the role of PFOR in C. reinhardtii fermentative H2 production (Figure 5). Northern blot studies showed that the C. reinhardtii PFOR is expressed under anaerobic conditions (Figure 5). Northern blot data also showed PFOR to be expressed in parallel with HydA1 and HydA2 expression (Figure 5). The co-expression of PFOR, HydA1 and HydA2 is evidence that PFOR may donate electrons via ferredoxin to hydrogenase resulting in H2 gas production (Figure 6). Future biochemical work is needed to definitively show that a connection between C. reinhardtii PFOR, ferredoxin and hydrogenase.

By better understanding algal H2 evolution, we come closer to a direct biological system of producing a clean, renewable fuel. Though fermentative H2 evolution was the first mechanism of biological H2 production discovered in algae, the source of electrons was unknown. Here, we show that PFOR is the likely source of ferredoxin reductant in dark H2 production in C. reinhardtii, though much work is left to definitively characterize this biochemical pathway.

 

Acknowledgements

My appreciation goes to the Department of Energy Office of Science and National Renewable Energy Lab for my acceptance into the SULI program. I thank Dr. Paul King and Dr. Maria Ghirardi, who deserve special recognition for their outstanding mentorship. I also thank Dr. Mike Seibert for his mentorship and review. Lastly, my thanks goes to everyone in basic sciences at NREL for their support in this endeavor.
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