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Photosynthetic organisms frequently experience abiotic stress that restricts their growth and development. Under such circumstances, most absorbed solar energy cannot be used for CO2 fixation and can cause the photoproduction of reactive oxygen species (ROS) that can damage the photosynthetic reaction centers of PSI and PSII, resulting in a decline in primary productivity. This work describes a biological “switch” in the green alga Chlamydomonas reinhardtii that reversibly restricts photosynthetic electron transport (PET) at the cytochrome b6f (Cyt b6f) complex when the capacity for accepting electrons downstream of PSI is severely limited. We specifically show this restriction in STARCHLESS6 (sta6) mutant cells, which cannot synthesize starch when they are limited for nitrogen (growth inhibition) and subjected to a dark-to-light transition. This restriction represents a form of photosynthetic control that causes diminished electron flow to PSI and thereby prevents PSI photodamage but does not appear to rely on a ΔpH. Furthermore, when electron flow is restricted, the plastid alternative oxidase (PTOX) becomes active, functioning as an electron valve that dissipates some excitation energy absorbed by PSII and allows the formation of a proton motive force (PMF) that would drive some ATP production (potentially sustaining PSII repair and nonphotochemical quenching [NPQ]). The restriction at the Cyt b6f complex can be gradually relieved with continued illumination. This study provides insights into how PET responds to a marked reduction in availability of downstream electron acceptors and the protective mechanisms involved. more...

Modulation of photoassimilate export from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study, we identified chloroplast TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR 2 (CreTPT2) and CreTPT3 in the green alga Chlamydomonas (Chlamydomonas reinhardtii), which exhibit similar substrate specificities but whose encoding genes are differentially expressed over the diurnal cycle. We focused mostly on CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that affected growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilates. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing cells from experiencing oxidative stress and accumulating reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR (CreTPT) transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants. more...

With fast growth rates, broad halotolerance and the ability to thrive at high temperatures, algae in the genus Picochlorum are emerging as promising biomass producers. Recently, we isolated a remarkably productive strain, Picochlorum celeri, that attains > 40 g m−2 day−1 productivities using simulated outdoor light. To test outdoor productivities, Picochlorum celeri was cultivated in 820 L raceway ponds at the Arizona Center for Algae Technology and Innovation. Picochlorum celeri demonstrated the highest outdoor biomass productivities reported to date at this testbed averaging ~ 31 g m−2 day−1 over four months with a monthly (August) high of ~ 36 g m−2 day−1. Several single day productivities were > 40 g m−2 day−1. Importantly for sustainability, Picochlorum celeri achieved these productivities in saline water ranging from seawater to 50 parts per thousand sea salts, without any biocides or pond crashes, for over 143 days. Lastly, we report robust genetic engineering tools for future strain improvements. more...

Microalgae are promising biocatalysts for applications in sustainable fuel, food, and chemical production. Here, we describe culture collection screening, down-selection, and development of a high-productivity, halophilic, thermotolerant microalga, Picochlorum renovo. This microalga displays a rapid growth rate and high diel biomass productivity (34 g m−2 day−1), with a composition well-suited for downstream processing. P. renovo exhibits broad salinity tolerance (growth at 107.5 g L−1 salinity) and thermotolerance (growth up to 40 °C), beneficial traits for outdoor cultivation. We report complete genome sequencing and analysis, and genetic tool development suitable for expression of transgenes inserted into the nuclear or chloroplast genomes. We further evaluate mechanisms of halotolerance via comparative transcriptomics, identifying novel genes differentially regulated in response to high salinity cultivation. These findings will enable basic science inquiries into control mechanisms governing Picochlorum biology and lay the foundation for development of a microalga with industrially relevant traits as a model photobiology platform. Lukas Dahlin et al. report the development of Picochlorum renovo, a high-productivity, halophilic, thermotolerant microalga. They report the nuclear and chloroplast genomes, and develop a system for inserting transgenes in both organelles. more...

Significance Most forms of life on Earth cannot exist without photosynthesis. Our food and atmosphere depend on it. To obtain high photosynthetic yields, light energy must be efficiently coupled to the fixation of CO2 into organic molecules. Suboptimal environmental conditions can severely impact the conversion of light energy to biomass and lead to reactive oxygen production, which in turn can cause cellular damage and loss of productivity. Hence, plants, algae, and photosynthetic bacteria have evolved a network of alternative outlets to sustain the flow of photosynthetically derived electrons. Our work is focused on the nature and integration of these outlets, which will inform a rational engineering of crop plants and algae to optimize photosynthesis and meet the increased global demand for food., Environmental stresses dramatically impact the balance between the production of photosynthetically derived energetic electrons and Calvin–Benson–Bassham cycle (CBBC) activity; an imbalance promotes accumulation of reactive oxygen species and causes cell damage. Hence, photosynthetic organisms have developed several strategies to route electrons toward alternative outlets that allow for storage or harmless dissipation of their energy. In this work, we explore the activities of three essential outlets associated with Chlamydomonas reinhardtii photosynthetic electron transport: (i) reduction of O2 to H2O through flavodiiron proteins (FLVs) and (ii) plastid terminal oxidases (PTOX) and (iii) the synthesis of starch. Real-time measurements of O2 exchange have demonstrated that FLVs immediately engage during dark-to-light transitions, allowing electron transport when the CBBC is not fully activated. Under these conditions, we quantified maximal FLV activity and its overall capacity to direct photosynthetic electrons toward O2 reduction. However, when starch synthesis is compromised, a greater proportion of the electrons is directed toward O2 reduction through both the FLVs and PTOX, suggesting an important role for starch synthesis in priming/regulating CBBC and electron transport. Moreover, partitioning energized electrons between sustainable (starch; energetic electrons are recaptured) and nonsustainable (H2O; energetic electrons are not recaptured) outlets is part of the energy management strategy of photosynthetic organisms that allows them to cope with the fluctuating conditions encountered in nature. Finally, unmasking the repertoire and control of such energetic reactions offers new directions for rational redesign and optimization of photosynthesis to satisfy global demands for food and other resources. more...

The complete mitochondrial and plastid genomes of the microalga Pavlova lutheri strain NIVA-4/92 are reported. The circular-mapping mitogenome is 36,202 bp in length, contains 22 protein-coding genes, 24 tRNAs, and has a GC content of 37.5%. Like other haptophytes the mitogenome contains a single large, complex repeat region of approximately 5.4 kbp. The plastome is 95,281 bp in length and has a GC content of 35.6%. It contains 111 protein-coding genes and 27 tRNAs. more...

Haptophytes are biogeochemically and industrially important protists with underexplored genomic diversity. We present a nuclear genome assembly for the class Pavlovales, which was assembled with PacBio long-read data into highly contiguous sequences. We sequenced strain Diacronema lutheri NIVA-4/92, formerly known as Pavlova lutheri, because it has established roles in aquaculture and has been a key organism for studying microalgal lipid biosynthesis. Our data show that D. lutheri has the smallest and most streamlined haptophycean genome assembled to date, with an assembly size of 43.503 Mb and 14,446 protein-coding genes. Together with its high nuclear GC content, Diacronema is an important genus for investigating selective pressures on haptophyte genome evolution, contrasting with the much larger and more repetitive genome of the coccolithophore Emiliania huxleyi. The D. lutheri genome will be a valuable resource for resolving the genetic basis of algal lipid biosynthesis and metabolic remodeling that takes place during adaptation and stress response in natural and engineered environments. more...

Picochlorum celeri is a fast-growing marine microalga with high biomass productivity. Here, we report the use of PacBio sequencing to assemble the phased diploid genome of P. celeri.

Picochlorum celeri is a fast-growing marine microalga with high biomass productivity. Here, we report the use of PacBio sequencing to assemble the phased diploid genome of P. celeri .

As a result of a secondary endosymbiosis of a red alga, stramenopiles retained β-1,3 glucans as the primary soluble storage carbohydrate, in contrast to the starch and glycogen of green plants and animals. This storage carbohydrate has been identified and characterized in representative diatoms, brown algae, and oomycetes, but has not been biochemically characterized in species of Nannochloropsis, an industrially relevant genus of algae favored for its lipid content and productivity. In this study, the soluble storage carbohydrate of Nannochloropsis is characterized by 1H NMR, linkage analysis, and size exclusion chromatography. The putative genes encoding enzymes required for synthesis of the carbohydrate oligosaccharide from glucose 6-phosphate are identified. A glycogenin-like glycosyltransferase family 8 (GT8) protein was also identified to be conserved among all species of Nannochloropsis, despite the lack of glycogen in the genus. Homologs were identified in available genomes of brown algae, diatoms, and oomycetes, all documented to utilize β-1,3 glucans for carbon storage. Finally, a set of three likely laminarinases are highlighted from the glycosyl hydrolases by phylogenetic analyses. more...

Microalgae are promising biocatalysts for applications in sustainable fuel, food, and chemical production. Here, we describe culture collection screening, down-selection, and development of a high-productivity, halophilic, thermotolerant microalga, Picochlorum renovo. This microalga displays a rapid growth rate and high diel biomass productivity (34 g m−2 day−1), with a composition well-suited for downstream processing. P. renovo exhibits broad salinity tolerance (growth at 107.5 g L−1 salinity) and thermotolerance (growth up to 40 °C), beneficial traits for outdoor cultivation. We report complete genome sequencing and analysis, and genetic tool development suitable for expression of transgenes inserted into the nuclear or chloroplast genomes. We further evaluate mechanisms of halotolerance via comparative transcriptomics, identifying novel genes differentially regulated in response to high salinity cultivation. These findings will enable basic science inquiries into control mechanisms governing Picochlorum biology and lay the foundation for development of a microalga with industrially relevant traits as a model photobiology platform., Lukas Dahlin et al. report the development of Picochlorum renovo, a high-productivity, halophilic, thermotolerant microalga. They report the nuclear and chloroplast genomes, and develop a system for inserting transgenes in both organelles. more...

The high oxygen (O2) sensitivity of green algal [FeFe]-hydrogenases is a significant limitation for the sustained production of hydrogen gas (H2) from photosynthetic water splitting. To address this limitation we replaced the native [FeFe]-hydrogenases with a more O2-tolerant clostridial [FeFe]-hydrogenase CaI in Chlamydomonas reinhardtii strain D66ΔHYD (hydA1− hydA2−) that contains insertionally inactivated [FeFe]-hydrogenases genes. Expression and translocation of CaI in D66ΔHYD led to the recovery of H2 photoproduction at ~ 20% of the rates of the wild-type parent strain D66. We show for the first time that a bacterial [FeFe]-hydrogenase can be expressed, localized and matured to a catalytically active form that couples to photosynthetic electron transport in the green alga C. reinhardtii. The lower rates of O2 inactivation of CaI led to more sustained H2 photoproduction when cultures were challenged with O2 or kept under prolonged illumination at solar intensities. These results provide new insights into the requisites for attaining photobiological H2 production from water splitting using a more O2-tolerant hydrogenase. more...

Partitioning of carbon to lipids is engineered to improve oil accumulation in an industrial alga.

In this study, the reuse of nitrogen from fuel-extracted algal residues was investigated. The alga Scenedesmus acutus was found to be able to assimilate nitrogen contained in amino acids, yeast extracts, and proteinaceous alga residuals. Moreover, these alternative nitrogen resources could replace nitrate in culturing media. The ability of S. acutus to utilize the nitrogen remaining in processed algal biomass was unique among the promising biofuel strains tested. This alga was leveraged in a recycling approach where nitrogen is recovered from algal biomass residuals that remain after lipids are extracted and carbohydrates are fermented to ethanol. The protein-rich residuals not only provided an effective nitrogen resource, but also contributed to a carbon “heterotrophic boost” in subsequent culturing, improving overall biomass and lipid yields relative to the control medium with only nitrate. Prior treatment of the algal residues with Diaion HP20 resin was required to remove compounds inhibitory to algal growth. more...

The use of [FeFe]-hydrogenase enzymes for the biotechnological production of H2 or other reduced products has been limited by their sensitivity to oxygen (O2). Here, we apply a PCR-directed approach to determine the distribution, abundance, and diversity of hydA gene fragments along co-varying salinity and O2 gradients in a vertical water column of Great Salt Lake (GSL), UT. The distribution of hydA was constrained to water column transects that had high salt and relatively low O2 concentrations. Recovered HydA deduced amino acid sequences were enriched in hydrophilic amino acids relative to HydA from less saline environments. In addition, they harbored interesting variations in the amino acid environment of the complex H-cluster metalloenzyme active site and putative gas transfer channels that may be important for both H2 transfer and O2 susceptibility. A phylogenetic framework was created to infer the accessory cluster composition and quaternary structure of recovered HydA protein sequences based on phylogenetic relationships and the gene contexts of known complete HydA sequences. Numerous recovered HydA are predicted to harbor multiple N- and C-terminal accessory iron-sulfur cluster binding domains and are likely to exist as multisubunit complexes. This study indicates an important role for [FeFe]-hydrogenases in the functioning of the GSL ecosystem and provides new target genes and variants for use in identifying O2 tolerant enzymes for biotechnological applications. more...

Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (∼75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes. more...

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. more...

Nannochloropsis species have emerged as leading phototrophic microorganisms for the production of biofuels. Several isolates produce large quantities of triacylglycerols, grow rapidly, and can be cultivated at industrial scales. Recently, the mitochondrial, plastid and nuclear genomes of Nannochloropsis gaditana were sequenced. Genomic interrogation revealed several key features that likely facilitate the oleaginous phenotype observed in Nannochloropsis, including an over-representation of genes involved in lipid biosynthesis. Here we present additional analyses on gene orientation, vitamin B12 requiring enzymes, the acetyl-CoA metabolic node, and codon usage in N. gaditana. Nuclear genome transformation methods are established with exogenous DNA integration occurring via either random incorporation or by homologous recombination, making Nannochloropsis amenable to both forward and reverse genetic engineering. Completion of a draft genomic sequence, establishment of transformation techniques, and robust outdoor growth properties have positioned Nannochloropsis as a new model alga with significant potential for further development into an integrated photons-to-fuel production platform. more...

Harnessing the metabolic potential of photosynthetic microbes for next-generation biotechnology objectives requires detailed scientific understanding of the physiological constraints and regulatory controls affecting carbon partitioning between biomass, metabolite storage pools, and bioproduct synthesis. We dissected the cellular mechanisms underlying the remarkable physiological robustness of the euryhaline unicellular cyanobacterium Synechococcus sp. strain PCC 7002 (Synechococcus 7002) and identify key mechanisms that allow cyanobacteria to achieve unprecedented photoautotrophic productivities (~2.5-h doubling time). Ultrafast growth of Synechococcus 7002 was supported by high rates of photosynthetic electron transfer and linked to significantly elevated transcription of precursor biosynthesis and protein translation machinery. Notably, no growth or photosynthesis inhibition signatures were observed under any of the tested experimental conditions. Finally, the ultrafast growth in Synechococcus 7002 was also linked to a 300% expansion of average cell volume. We hypothesize that this cellular adaptation is required at high irradiances to support higher cell division rates and reduce deleterious effects, corresponding to high light, through increased carbon and reductant sequestration., IMPORTANCE Efficient coupling between photosynthesis and productivity is central to the development of biotechnology based on solar energy. Therefore, understanding the factors constraining maximum rates of carbon processing is necessary to identify regulatory mechanisms and devise strategies to overcome productivity constraints. Here, we interrogate the molecular mechanisms that operate at a systems level to allow cyanobacteria to achieve ultrafast growth. This was done by considering growth and photosynthetic kinetics with global transcription patterns. We have delineated putative biological principles that allow unicellular cyanobacteria to achieve ultrahigh growth rates through photophysiological acclimation and effective management of cellular resource under different growth regimes. more...

The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novo assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase III increased MCFA synthesis up to fivefold. The level of increase is dependent on promoter strength and culturing conditions. more...

To improve bioenergy production from photosynthetic microorganisms it is necessary to optimize an extensive network of highly integrated biological processes. Systematic advances in pathway engineering and culture modification have resulted in strains with increased yields of biohydrogen, lipids, and carbohydrates, three bioenergy foci. However, additional improvements in photosynthetic efficiency are necessary to establish a viable system for biofuel production. Advances in optimizing light capture, energy transfer, and carbon fixation are essential, as the efficiencies of these processes are the principal determinants of productivity. However, owing to their regulatory, catalytic, and structural complexities, manipulating these pathways poses considerable challenges. This review covers novel developments in the optimization of photosynthesis, carbon fixation, and metabolic pathways for the synthesis of targeted bioenergy carriers. more...

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. more...

Chlamydomonas reinhardtii (Chlamydomonas throughout) encodes two [FeFe]-hydrogenases, designated HYDA1 and HYDA2. While HYDA1 is considered the dominant hydrogenase, the role of HYDA2 is unclear. To study the individual functions of each hydrogenase and provide a platform for future bioengineering, we isolated the Chlamydomonas hydA1-1, hydA2-1 single mutants and the hydA1-1 hydA2-1 double mutant. A reverse genetic screen was used to identify a mutant with an insertion in HYDA2, followed by mutagenesis of the hydA2-1 strain coupled with a H2 chemosensor phenotypic screen to isolate the hydA1-1 hydA2-1 mutant. Genetic crosses of the hydA1-1 hydA2-1 mutant to wild-type cells allowed us to also isolate the single hydA1-1 mutant. Fermentative, photosynthetic, and in vitro hydrogenase activities were assayed in each of the mutant genotypes. Surprisingly, analyses of the hydA1-1 and hydA2-1 single mutants, as well as the HYDA1 and HYDA2 rescued hydA1-1 hydA2-1 mutant demonstrated that both hydrogenases are able to catalyze H2 production from either fermentative or photosynthetic pathways. The physiology of both mutant and complemented strains indicate that the contribution of HYDA2 to H2 photoproduction is approximately 25% that of HYDA1, which corresponds to similarly low levels of in vitro hydrogenase activity measured in the hydA1-1 mutant. Interestingly, enhanced in vitro and fermentative H2 production activities were observed in the hydA1-1 hydA2-1 strain complemented with HYDA1, while maximal H2-photoproduction rates did not exceed those of wild-type cells. more...

Hydrogenases are metalloenzymes that are key to energy metabolism in a variety of microbial communities. Divided into three classes based on their metal content, the [Fe]-, [FeFe]-, and [NiFe]-hydrogenases are evolutionarily unrelated but share similar nonprotein ligand assemblies at their active site metal centers that are not observed elsewhere in biology. These nonprotein ligands are critical in tuning enzyme reactivity, and their synthesis and incorporation into the active site clusters require a number of specific maturation enzymes. The wealth of structural information on different classes and different states of hydrogenase enzymes, biosynthetic intermediates, and maturation enzymes has contributed significantly to understanding the biochemistry of hydrogen metabolism. This review highlights the unique structural features of hydrogenases and emphasizes the recent biochemical and structural work that has created a clearer picture of the [FeFe]-hydrogenase maturation pathway. more...

[FeFe]-hydrogenases (HYDA) link the production of molecular H(2) to anaerobic metabolism in many green algae. Similar to Chlamydomonas reinhardtii, Chlorella variabilis NC64A (Trebouxiophyceae, Chlorophyta) exhibits [FeFe]-hydrogenase (HYDA) activity during anoxia. In contrast to C. reinhardtii and other chlorophycean algae, which contain hydrogenases with only the HYDA active site (H-cluster), C. variabilis NC64A is the only known green alga containing HYDA genes encoding accessory FeS cluster-binding domains (F-cluster). cDNA sequencing confirmed the presence of F-cluster HYDA1 mRNA transcripts, and identified deviations from the in silico splicing models. We show that HYDA activity in C. variabilis NC64A is coupled to anoxic photosynthetic electron transport (PSII linked, as well as PSII-independent) and dark fermentation. We also show that the in vivo H(2)-photoproduction activity observed is as O(2) sensitive as in C. reinhardtii. The two C. variabilis NC64A HYDA sequences are similar to homologs found in more deeply branching bacteria (Thermotogales), diatoms, and heterotrophic flagellates, suggesting that an F-cluster HYDA is the ancestral enzyme in algae. Phylogenetic analysis indicates that the algal HYDA H-cluster domains are monophyletic, suggesting that they share a common origin, and evolved from a single ancestral F-cluster HYDA. Furthermore, phylogenetic reconstruction indicates that the multiple algal HYDA paralogs are the result of gene duplication events that occurred independently within each algal lineage. Collectively, comparative genomic, physiological, and phylogenetic analyses of the C. variabilis NC64A hydrogenase has provided new insights into the molecular evolution and diversity of algal [FeFe]-hydrogenases. more...

Many microbes in the soil environment experience micro-oxic or anoxic conditions for much of the late afternoon and night, which inhibit or prevent respiratory metabolism. To sustain the production of energy and maintain vital cellular processes during the night, organisms have developed numerous pathways for fermentative metabolism. This review discusses fermentation pathways identified for the soil-dwelling model alga Chlamydomonas reinhardtii, its ability to produce molecular hydrogen under anoxic conditions through the activity of hydrogenases, and the molecular flexibility associated with fermentative metabolism that has only recently been revealed through the analysis of specific mutant strains. more...

There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H 2 yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H 2 production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes. more...

There is currently substantial interest in utilizing eukaryotic algae for the renewable production of several bioenergy carriers, including starches for alcohols, lipids for diesel fuel surrogates, and H2 for fuel cells. Relative to terrestrial biofuel feedstocks, algae can convert solar energy into fuels at higher photosynthetic efficiencies, and can thrive in salt water systems. Recently, there has been considerable progress in identifying relevant bioenergy genes and pathways in microalgae, and powerful genetic techniques have been developed to engineer some strains via the targeted disruption of endogenous genes and/or transgene expression. Collectively, the progress that has been realized in these areas is rapidly advancing our ability to genetically optimize the production of targeted biofuels. more...

The green alga Chlamydomonas reinhardtii has a network of fermentation pathways that become active when cells acclimate to anoxia. Hydrogenase activity is an important component of this metabolism, and we have compared metabolic and regulatory responses that accompany anaerobiosis in wild-type C. reinhardtii cells and a null mutant strain for the HYDEF gene (hydEF-1 mutant), which encodes an [FeFe] hydrogenase maturation protein. This mutant has no hydrogenase activity and exhibits elevated accumulation of succinate and diminished production of CO2 relative to the parental strain during dark, anaerobic metabolism. In the absence of hydrogenase activity, increased succinate accumulation suggests that the cells activate alternative pathways for pyruvate metabolism, which contribute to NAD(P)H reoxidation, and continued glycolysis and fermentation in the absence of O2. Fermentative succinate production potentially proceeds via the formation of malate, and increases in the abundance of mRNAs encoding two malateforming enzymes, pyruvate carboxylase and malic enzyme, are observed in the mutant relative to the parental strain following transfer of cells from oxic to anoxic conditions. Although C. reinhardtii has a single gene encoding pyruvate carboxylase, it has six genes encoding putative malic enzymes. Only one of the malic enzyme genes, MME4, shows a dramatic increase in expression (mRNA abundance) in the hydEF-1 mutant during anaerobiosis. Furthermore, there are marked increases in transcripts encoding fumarase and fumarate reductase, enzymes putatively required to convert malate to succinate. These results illustrate the marked metabolic flexibility of C. reinhardtii and contribute to the development of an informed model of anaerobic metabolism in this and potentially other algae. more...

Maturation of the [FeFe]-hydrogenase active site depends on at least the expression of three gene products called HydE, HydF, and HydG. We have solved the high resolution structure of recombinant, reconstituted S-adenosine-L-methionine-dependent HydE from Thermotoga maritima. Besides the conserved [Fe(4)S(4)] cluster involved in the radical-based reaction, this HydE was reported to have a second [Fe(4)S(4)] cluster coordinated by three Cys residues. However, in our crystals, depending on the reconstitution and soaking conditions, this second cluster is either a [Fe(2)S(2)] center, with water occupying the fourth ligand site or is absent. We have carried out site-directed mutagenesis studies on the related HydE from Clostridium acetobutylicum, along with in silico docking and crystal soaking experiments, to define the active site region and three anion-binding sites inside a large, positive cavity, one of which binds SCN(-) with high affinity. Although the overall triose-phosphate isomerase-barrel structure of HydE is very similar to that of biotin synthase, the residues that line the internal cavity are significantly different in the two enzymes. more...

To mitigate some of the potentially deleterious environmental and agricultural consequences associated with current land-based-biofuel feedstocks, we propose the use of biofuels derived from aquatic microbial oxygenic photoautotrophs (AMOPs), more commonly known as cyanobacteria, algae, and diatoms. Herein we review their demonstrated productivity in mass culturing and aspects of their physiology that are particularly attractive for integration into renewable biofuel applications. Compared with terrestrial crops, AMOPs are inherently more efficient solar collectors, use less or no land, can be converted to liquid fuels using simpler technologies than cellulose, and offer secondary uses that fossil fuels do not provide. AMOPs pose a new set of technological challenges if they are to contribute as biofuel feedstocks. more...

Microalgae represent an exceptionally diverse but highly specialized group of micro-organisms adapted to various ecological habitats. Many microalgae have the ability to produce substantial amounts (e.g. 20-50% dry cell weight) of triacylglycerols (TAG) as a storage lipid under photo-oxidative stress or other adverse environmental conditions. Fatty acids, the building blocks for TAGs and all other cellular lipids, are synthesized in the chloroplast using a single set of enzymes, of which acetyl CoA carboxylase (ACCase) is key in regulating fatty acid synthesis rates. However, the expression of genes involved in fatty acid synthesis is poorly understood in microalgae. Synthesis and sequestration of TAG into cytosolic lipid bodies appear to be a protective mechanism by which algal cells cope with stress conditions, but little is known about regulation of TAG formation at the molecular and cellular level. While the concept of using microalgae as an alternative and renewable source of lipid-rich biomass feedstock for biofuels has been explored over the past few decades, a scalable, commercially viable system has yet to emerge. Today, the production of algal oil is primarily confined to high-value specialty oils with nutritional value, rather than commodity oils for biofuel. This review provides a brief summary of the current knowledge on oleaginous algae and their fatty acid and TAG biosynthesis, algal model systems and genomic approaches to a better understanding of TAG production, and a historical perspective and path forward for microalgae-based biofuel research and commercialization. more...

Both prokaryotic and eukaryotic photosynthetic microbes experience conditions of anoxia, especially during the night when photosynthetic activity ceases. In Chlamydomonas reinhardtii, dark anoxia is characterized by the activation of an extensive set of fermentation pathways that act in concert to provide cellular energy, while limiting the accumulation of potentially toxic fermentative products. Metabolite analyses, quantitative PCR, and high density Chlamydomonas DNA microarrays were used to monitor changes in metabolite accumulation and gene expression during acclimation of the cells to anoxia. Elevated levels of transcripts encoding proteins associated with the production of H2, organic acids, and ethanol were observed in congruence with the accumulation of fermentation products. The levels of over 500 transcripts increased significantly during acclimation of the cells to anoxic conditions. Among these were transcripts encoding transcription/translation regulators, prolyl hydroxylases, hybrid cluster proteins, proteases, transhydrogenase, catalase, and several putative proteins of unknown function. Overall, this study uses metabolite, genomic, and transcriptome data to provide genome-wide insights into the regulation of the complex metabolic networks utilized by Chlamydomonas under the anaerobic conditions associated with H2 production. more...

International audience; 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. more...

Mercuric ion reductase (MerA), a mercury detoxification enzyme, has been tuned by evolution to have high specificity for mercuric ions (Hg2+) and to catalyze their reduction to a more volatile, less toxic elemental form. Here, we present a biochemical and structural characterization of MerA from the thermophilic crenarchaeon Metallosphaera sedula. MerA from M. sedula is a thermostable enzyme, and remains active after extended incubation at 97 °C. At 37 ᵒC, the NADPH oxidation-linked Hg2+ reduction specific activity was found to be 1.9 µmol/min•mg, increasing to 3.1µmol/min•mg at 70 °C. M. sedula MerA crystals were obtained and the structure was solved to 1.6 Å, representing the first solved crystal structure of a thermophilic MerA. Comparison of both the crystal structure and amino acid sequence of MerA from M. sedula to mesophillic counterparts provides new insights into the structural determinants that underpin the thermal stability of the enzyme. more...

Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate during both glycolysis and gluconeogenesis and is required by all three domains of life. Here, we report the purification and biochemical and structural characterization of enolase from Chloroflexus aurantiacus, a thermophilic andanoxygenic phototroph affiliated with the green non-sulfur bacteria. The protein was purified as a homodimer with a subunit molecular weight of 46 kDa. The temperature optimum for enolase catalysis was 80°C, close to the measured thermal stability of the protein which was determined to be 75°C, while the pH optimum for enzyme activity was 6.5. The specific activities of purified enolase determined at 25°C and 80°C were 147 and 300 units/mg of protein, respectively. Km values for the 2-phosphoglycerate/phosphoenolpyruvate reaction determined at 25°C and 80°C were 0.16 mM and 0.03 mM, respectively. The Km values for Mg2+ binding at these temperatures were 2.5 mM and 1.9 mM, respectively. When compared to enolase from mesophiles, the biochemical and structural properties of enolase from C. aurantiacus are consistent with this being thermally adapted. This data is consistent with the results of our phylogenetic analysis of enolase, which reveal that enolase has a thermophilic origin. more...

Cyanobacterial glycogen-deficient mutants display impaired degradation of light-harvesting phycobilisomes under nitrogen-limiting growth conditions and secrete a suite of organic acids as a putative reductant-spilling mechanism. This genetic background, therefore, represents an important platform to better understand the complex relationships between light harvesting, photosynthetic electron transport, carbon fixation, and carbon/nitrogen metabolisms. In this study, we conducted a comprehensive analysis of the dynamics of photosynthesis as a function of reductant sink manipulation in a glycogen-deficient glgC mutant of Synechococcus sp. strain PCC 7002. The glgC mutant showed increased susceptibility to photoinhibition during the initial phase of nitrogen deprivation. However, after extended periods of nitrogen deprivation, glgC mutant cells maintained higher levels of photosynthetic activity than the wild type, supporting continuous organic acid secretion in the absence of biomass accumulation. In contrast to the wild type, the glgC mutant maintained efficient energy transfer from phycobilisomes to photosystem II (PSII) reaction centers, had an elevated PSII/PSI ratio as a result of reduced PSII degradation, and retained a nitrogen-replete-type ultrastructure, including an extensive thylakoid membrane network, after prolonged nitrogen deprivation. Together, these results suggest that multiple global signals for nitrogen deprivation are not activated in the glgC mutant, allowing the maintenance of active photosynthetic complexes under conditions where photosynthesis would normally be abolished. more...

To identify genes necessary for the photoproduction of H(2) in Chlamydomonas reinhardtii, random insertional mutants were screened for clones unable to produce H(2). One of the identified mutants, denoted hydEF-1, is incapable of assembling an active [Fe] hydrogenase. Although the hydEF-1 mutant transcribes both hydrogenase genes and accumulates full-length hydrogenase protein, H(2) production activity is not observed. The HydEF protein contains two unique domains that are homologous to two distinct prokaryotic proteins, HydE and HydF, which are found exclusively in organisms containing [Fe] hydrogenase. In the C. reinhardtii genome, the HydEF gene is adjacent to another hydrogenase-related gene, HydG. All organisms with [Fe] hydrogenase and sequenced genomes contain homologues of HydE, HydF, and HydG, which, prior to this study, were of unknown function. Within several prokaryotic genomes HydE, HydF, and HydG are found in putative operons with [Fe] hydrogenase structural genes. Both HydE and HydG belong to the emerging radical S-adenosylmethionine (commonly designated "Radical SAM") superfamily of proteins. We demonstrate here that HydEF and HydG function in the assembly of [Fe] hydrogenase. Northern blot analysis indicates that mRNA transcripts for both the HydEF gene and the HydG gene are anaerobically induced concomitantly with the two C. reinhardtii [Fe] hydrogenase genes, HydA1 and HydA2. Complementation of the bx;1C. reinhardtii hydEF-1 mutant with genomic DNA corresponding to a functional copy of the HydEF gene restores hydrogenase activity. Moreover, co-expression of the C. reinhardtii HydEF, HydG, and HydA1 genes in Escherichia coli results in the formation of an active HydA1 enzyme. This represents the first report on the nature of the accessory genes required for the maturation of an active [Fe] hydrogenase. more...

We have isolated and characterized a second [Fe]-hydrogenase gene from the green alga, Chlamydomonas reinhardtii. The HydA2 gene encodes a protein of 505 amino acids that is 74% similar and 68% identical to the known HydA1 hydrogenase from C. reinhardtii. HydA2 contains all the conserved residues and motifs found in the catalytic core of the family of [Fe]-hydrogenases. We demonstrate that both the HydA1 and the HydA2 transcripts are expressed upon anaerobic induction, achieved either by neutral gas purging or by sulfur deprivation of the cultures. Furthermore, the expression levels of both transcripts are regulated (in some cases differently) by incubation conditions, such as the length of anaerobiosis, the readdition of O2, the presence of acetate, and/or the absence of nutrients such as sulfate during growth. Antibodies specific for HydA2 recognized a protein of about 49 kDa in extracts from anaerobically induced C. reinhardtii cells, strongly suggesting that HydA2 encodes for an expressed protein. Homology-based 3D modeling of the HydA2 hydrogenase shows that its catalytic site models well to the known structure of Clostridium pasteurianum CpI, including the H2-gas channel. The major differences between HydA1, HydA2 and CpI are the absence of the N-terminal Fe-S centers and the existence of extra sequences in the algal enzymes. To our knowledge, this work represents the first systematic study of expression of two algal [Fe]-hydrogenases in the same organism. more...