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Pseudomonas sp. strain MM223, Pseudomonas sp. strain MM227, and

The bacterial strains Pseudomonas sp. strain MM221 and

The bacterial strains Pseudomonas sp. strain MM221 and Pseudoarthrobacter sp. strain MM222 were isolated from a sandy soil sample. Here, we report on their complete genome sequences, including a circular plasmid for MM221, which were assembled after sequencing with an Oxford Nanopore Technologies flow cell.

Pseudomonas sp. strain MM223, Pseudomonas sp. strain MM227, and Rheinheimera sp. strain MM224 were isolated from a muddy soil sample from the edge of a pond. Here, we present whole-genome sequences and phylogenetic classifications for all three bacterial isolates.

Microalgae represent a potential solution to reduce CO2 emission exploiting their photosynthetic activity. Here, the physiologic and metabolic responses at the base of CO2 assimilation were investigated in conditions of high or low CO2 availability in two of the most promising algae species for industrial cultivation, Chlorella sorokiniana and Chlorella vulgaris. In both species, high CO2 availability increased biomass accumulation with specific increase of triacylglycerols in C. vulgaris and polar lipids and proteins in C. sorokiniana. Moreover, high CO2 availability caused only in C. vulgaris a reduced NAD(P)H/NADP+ ratio and reduced mitochondrial respiration, suggesting a CO2 dependent increase of reducing power consumption in the chloroplast, which in turn influences the redox state of the mitochondria. Several rearrangements of the photosynthetic machinery were observed in both species, differing from those described for the model organism Chlamydomonas reinhardtii, where adaptation to carbon availability is mainly controlled by the translational repressor NAB1. NAB1 homologous protein could be identified only in C. vulgaris but lacked the regulation mechanisms previously described in C. reinhardtii. Acclimation strategies to cope with a fluctuating inorganic carbon supply are thus diverse among green microalgae, and these results suggest new biotechnological strategies to boost CO2 fixation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Microalgae represent a potential solution to reduce CO2 emission exploiting their photosynthetic activity. Here, the physiologic and metabolic responses at the base of CO2 assimilation were investigated in conditions of high or low CO2 availability in two of the most promising algae species for industrial cultivation, Chlorella sorokiniana and Chlorella vulgaris. In both species, high CO2 availability increased biomass accumulation with specific increase of triacylglycerols in C. vulgaris and polar lipids and proteins in C. sorokiniana. Moreover, high CO2 availability caused only in C. vulgaris a reduced NAD(P)H/NADP+ ratio and reduced mitochondrial respiration, suggesting a CO2 dependent increase of reducing power consumption in the chloroplast, which in turn influences the redox state of the mitochondria. Several rearrangements of the photosynthetic machinery were observed in both species, differing from those described for the model organism Chlamydomonas reinhardtii, where adaptation to carbon availability is mainly controlled by the translational repressor NAB1. NAB1 homologous protein could be identified only in C. vulgaris but lacked the regulation mechanisms previously described in C. reinhardtii. Acclimation strategies to cope with a fluctuating inorganic carbon supply are thus diverse among green microalgae, and these results suggest new biotechnological strategies to boost CO2 fixation., High/low CO2 availability induces in Chlorella vulgaris and Chlorella sorokiniana specific cell responses as lipids remodelling, modulation of mitochondrial respiration and adaptations of the photosynthetic apparatus.

Microalgae represent potential solutions to reduce the atmospheric CO2 level through photosynthesis. To boost CO2 fixation by microalgae it is essential to understand physiologic and metabolic responses at the base of CO2 assimilation and carbon flow. In this work two Trebouxiophyceae species, Chlorella sorokiniana and Chlorella vulgaris, were investigated for their metabolic responses to high and low CO2 (air level) availability. High CO2 availability resulted in an increase in biomass accumulation in both species but with a different chloroplast and mitochondrial responses. In C. sorokiniana we observed increased polar lipids and protein amount and a balanced NADPH redox state and a similar total respiration in the two conditions analysed. In contrast, in C. vulgaris high CO2 level caused an increase in TAG accumulation and a higher NADPH consumption suggesting a CO2 dependent increase of reducing power consumption in the chloroplast, which in turn influences the redox state of the mitochondria by lowering total dark respiration. Several rearrangements of the photosynthetic machinery were observed in both species, which differ from those described for the model organism Chlamydomonas reinhardtii. In the case of C. reinhardtii, adaptation of the photosynthetic apparatus to different CO2 availability relies on the translational repressor NAB1. NAB1 homologous protein could be identified only in C. vulgaris but lacked the regulation mechanisms previously described in C. reinhardtii. These findings highlight that the acclimation strategies to cope with a fluctuating inorganic carbon supply are diverse among green microalgae and point to new biotechnological strategies to boost CO2 fixation.One sentence summaryHigh/low CO2 availability induces cell responses as lipids remodelling, adaptations of the photosynthetic apparatus and modulation of mitochondrial respiration not conserved among green algae

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Modern chemical industry calls for new resource-efficient and sustainable value chains for production of key base chemicals such as polyamines. The green microalga Chlamydomonas reinhardtii offers great potential as an innovative green-cell factory by combining fast and inexpensive, phototrophic growth with mature genetic engineering. Here, overexpression of recombinant lysine decarboxylases in C. reinhardtii enabled the robust accumulation of the non-native polyamine cadaverine, which serves as building block for bio-polyamides. The issue of low cell densities, limiting most microalgal cultivation processes was resolved by systematically optimizing cultivation parameters. A new, easy-to-apply and fully phototrophic medium enables high cell density cultivations of C. reinhardtii with a 6-fold increase in biomass and cell count (20g/L biomass dry weight, ~2·108cells/mL). Application of high cell density cultivations in established photobioreactors resulted in a 10-fold increase of cadaverine yields, with up to 0.24g/L after 9days and maximal productivity of 0.1g/L/d. Copyright © 2020 Elsevier Ltd. All rights reserved.

In green microalgae, prolonged exposure to inorganic carbon depletion requires long-term acclimation responses, involving modulated gene expression and the adjustment of photosynthetic activity to the prevailing supply of carbon dioxide. Here, we describe a microalgal regulatory cycle that adjusts the light-harvesting capacity at photosystem II (PSII) to the prevailing supply of carbon dioxide in Chlamydomonas (Chlamydomonas reinhardtii). It engages low carbon dioxide response factor (LCRF), a member of the squamosa promoter-binding protein (SBP) family of transcription factors, and the previously characterized cytosolic translation repressor nucleic acid-binding protein 1 (NAB1). LCRF combines a DNA-binding SBP domain with a conserved domain for protein–protein interaction. LCRF transcription is rapidly induced by carbon dioxide depletion. LCRF activates NAB1 transcription by specifically binding to tetranucleotide motifs present in its promoter. Accumulation of the NAB1 protein enhances translational repression of its prime target mRNA, encoding the PSII-associated major light-harvesting protein LHCBM6. The resulting truncation of the PSII antenna size helps maintaining a low excitation during carbon dioxide limitation. Analyses of low carbon dioxide acclimation in nuclear insertion mutants devoid of a functional LCRF gene confirm the essentiality of this novel transcription factor for the regulatory circuit.

Survival under excess light conditions requires the light-induced accumulation of protein LHCSR3 and other photoprotection factors, to enable efficient energy-dependent quenching in the green microalga Chlamydomonas reinhardtii. Here, we demonstrate that the high light-tolerant phenotype of mutant hit1 is caused by a de-repression of promoters belonging to photoprotection genes, which in turn results from an inactivation of the E3 ubiquitin ligase substrate adaptor LRS1. Transcriptome analyses of hit1 revealed massive alterations of gene expression modulation as a consequence of perturbed LRS1 function, indicating its role as a crown regulator. In conjunction with random forest-based network modeling, these transcriptome analyses predicted that LRS1 controls photoprotection gene expression via an algal HY5 homolog as its prime transcription factor target. CrHY5 binds to T-box elements present in the promoters of these genes and its inactivation in the hit1 mutant via CRISPR-Cas9 genome editing, confirmed the regulatory connection between LRS1 and CrHY5, predicted by the network analysis.

SummaryThe green alga Chlamydomonas reinhardtii does not synthesize high-value ketocarotenoids like canthaxanthin and astaxanthin, however, a β-carotene ketolase (CrBKT) can be found in its genome. CrBKT is poorly expressed, contains a long C-terminal extension not found in homologues and likely represents a pseudogene in this alga. Here, we used synthetic re-design of this gene to enable its constitutive overexpression from the nuclear genome of C. reinhardtii. Overexpression of the optimized CrBKT extended native carotenoid biosynthesis to generate ketocarotenoids in the algal host causing noticeable changes the green algal colour to a reddish-brown. We found that up to 50% of native carotenoids could be converted into astaxanthin and more than 70% into other ketocarotenoids by robust CrBKT overexpression. Modification of the carotenoid metabolism did not impair growth or biomass productivity of C. reinhardtii, even at high light intensities. Under different growth conditions, the best performing CrBKT overexpression strain was found to reach ketocarotenoid productivities up to 4.5 mg L-1 day-1. Astaxanthin productivity in engineered C. reinhardtii shown here is competitive with that reported for Haematococcus lacustris (formerly pluvialis) which is currently the main organism cultivated for industrial astaxanthin production. In addition, the extractability and bio-accessibility of these pigments was much higher in cell wall deficient C. reinhardtii than the resting cysts of H. lacustris. Engineered C. reinhardtii strains could thus be a promising alternative to natural astaxanthin producing algal strains and may open the possibility of other tailor-made pigments from this host.

The protein family of mTERFs (mitochondrial transcription termination factors) was initially studied in mammalian and insect mitochondria before the first Arabidopsis mTERF mutant was characterized. More than 10 years of research on the function of plant mTERFs in the flowering plants Arabidopsis thaliana, Zea mays and the green microalga Chlamydomonas reinhardtii has since highlighted that mTERFs are key regulators of organellar gene expression (OGE) in mitochondria and in chloroplasts. Additional functions to be fulfilled by plant mTERFs (e.g. splicing) and the fact that the expression of two organellar genomes had to be facilitated have led to a massive expansion of the plant mTERF portfolio compared to that found in mammals. Plant mTERFs are implicated in all steps of OGE ranging from the modulation of transcription to the maturation of tRNAs and hence translation. Furthermore, being regulators of OGE, mTERFs are required for a successful long-term acclimation to abiotic stress, retrograde signaling and interorganellar communication. Here, I review the recent progress in the elucidation of molecular mTERF functions.

In green microalgae, prolonged exposure to inorganic carbon depletion requires long-term acclimation responses, based on a modulated expression of genes and adjusting photosynthetic activity to the prevailing supply of carbon dioxide. Here, we depict a microalgal regulatory cycle, adjusting the light-harvesting capacity at PSII to the prevailing supply of carbon dioxide inChlamydomonas reinhardtii. It engages a newly identified low carbon dioxide response factor (LCRF), which belongs to the Squamosa promoter binding protein (SBP) family of transcription factors, and the previously characterized cytosolic translation repressor NAB1. LCRF combines a DNA-binding SBP domain with a conserved domain for protein-protein interactions and transcription of theLCRFgene is rapidly induced by carbon dioxide depletion. LCRF activates transcription of theNAB1gene by specifically binding to tetranucleotide motifs present in its promoter. Accumulation of the NAB1 protein enhances translational repression of its prime target mRNA, encoding the PSII-associated major light-harvesting protein LHCBM6. The resulting reduction of the PSII antenna size helps maintaining a low excitation during the prevailing carbon dioxide limitation. Analyses of low carbon dioxide acclimation in nuclear insertion mutants devoid of a functionalLCRFgene confirm the essentiality of this novel transcription factor for the regulatory circuit.

Microbial consortia, which degrade branched, long-chain hydrocarbons, can be regarded as a promising source of novel enzymes for the stereo- and regio-selective oxyfunctionalization of hydrocarbons. The hydrocarbon-degrading bacterium Pimelobacter sp. Bb-B was isolated from the consortium associated with the colonial hydrocarbon-excreting microalga Botryococcus braunii. Three new type II flavoprotein monooxygenases (type II FMOs) from this bacterium have been made available in recombinant form through cloning and overexpression in an E. coli host organism. These enzymes (PsFMO_A-C) were characterized in terms of their capability of catalyzing Baeyer-Villiger oxidations with distinct substrates. The highest activity was detected when utilizing camphor and bicyclo[3.2.0]hept-2-en-6-one as substrate in combination with PsFMO_A as the most promising enzyme. Furthermore, synthetic biotransformations with 5 mM of the substrate bicyclo[3.2.0]hept-2-en-6-one, formate and formate dehydrogenase for in situ-cofactor recycling were conducted with this enzyme, leading to a substrate consumption of 85% after 66 h and excellent enantioselectivity of 99% ee for the (1R,5S)-enantiomer. Additionally, an alternative in situ-cofactor recycling based on the use of microalgae for in situ-production of formate from carbon dioxide, water and light together with a formate dehydrogenase was combined successfully with the enzyme PsFMO_A, leading to a substrate consumption of 94% and an enantioselectivity of >99% ee for the so-called “normal lactone”-enantiomer with the absolute configuration 1R,5S.

Microalgae represent promising organisms for the sustainable production of commodities, chemicals or fuels. Future use of such systems, however, requires increased productivity of microalgal mass cultures in order to reach an economic viability for microalgae-based production schemes. The efficiency of sunlight-to-biomass conversion that can be observed in bulk cultures is generally far lower (35-80%) than the theoretical maximum, because energy losses occur at multiple steps during the light-driven conversion of carbon dioxide to organic carbon. The light-harvesting system is a major source of energy losses and thus a prime target for strain engineering. Truncation of the light-harvesting antenna in the algal model organism Chlamydomonas reinhardtii was shown to be an effective way of increasing culture productivity at least under saturating light conditions. Furthermore engineering of the Calvin-Benson cycle or the creation of photorespiratory bypasses in A. thaliana proved to be successful in terms of achieving higher biomass productivities. An efficient generation of novel microalgal strains with improved sunlight conversion efficiencies by targeted engineering in the future will require an expanded molecular toolkit. In the meantime random mutagenesis coupled to high-throughput screening for desired phenotypes can be used to provide engineered microalgae.

A deregulation of mitochondrial respiration in a Chlamydomonas mutant devoid of the mTERF protein MOC1 disrupts the chloroplast redox poise with detrimental effects on photoprotective mechanisms.

The molecular function of mTERFs (mitochondrial transcription termination factors) has so far only been described for metazoan members of the protein family and in animals they control mitochondrial replication, transcription and translation. Cells of photosynthetic eukaryotes harbour chloroplasts and mitochondria, which are in an intense cross-talk that is vital for photosynthesis. Chlamydomonas reinhardtii is a unicellular green alga widely used as a model organism for photosynthesis research and green biotechnology. Among the six nuclear C. reinhardtii mTERF genes is mTERF-like gene of Chlamydomonas (MOC1), whose inactivation alters mitorespiration and interestingly also light-acclimation processes in the chloroplast that favour the enhanced production of biohydrogen. We show here from in vitro studies that MOC1 binds specifically to a sequence within the mitochondrial rRNA-coding module S3, and that a knockout of MOC1 in the mutant stm6 increases read-through transcription at this site, indicating that MOC1 acts as a transcription terminator in vivo. Whereas the level of certain antisense RNA species is higher in stm6, the amount of unprocessed mitochondrial sense transcripts is strongly reduced, demonstrating that a loss of MOC1 causes perturbed mitochondrial DNA (mtDNA) expression. Overall, we provide evidence for the existence of mitochondrial antisense RNAs in C. reinhardtii and show that mTERF-mediated transcription termination is an evolutionary-conserved mechanism occurring in phototrophic protists and metazoans.

Photosynthetic organisms like plants and algae can harvest, convert, and store solar energy and thus represent readily available sources for renewable biofuels production on a domestic or industrial scale. Anaerobic digestion (AD) of the organic biomass yields biogas, containing methane and carbon dioxide as major constituents. Combustion of the biogas or purification of the energy-rich methane fraction can be applied to provide electricity or fuel. AD procedures have been applied for several decades with organic waste, animal products, or higher plants and more recently, utilization of photosynthetic algae as substrates have gained considerable research interest. To provide an overview of recent research efforts made to characterize the AD process of microalgal biomass, we present extended summaries of experimentally determined biochemical methane potentials (BMP), biomass pretreatment options and digestion strategies in this article. We conclude that cultivation options, biomass composition and time of harvesting, application of biomass pretreatment strategies, and parameters of the digestion process are all important factors, which can significantly affect the AD process efficiency. The transition from batch to continuous microalgal biomass digestion trials, accompanied by state-of-the-art analytical techniques, is now in demand to refine the assessments of the overall process feasibility.

Life on Earth relies on photosynthesis, and the ongoing depletion of fossil carbon fuels has renewed interest in phototrophic light-energy conversion processes as a blueprint for the conversion of atmospheric CO2 into various organic compounds. Light-harvesting systems have evolved in plants and green algae, which are adapted to the light intensity and spectral composition encountered in their habitats. These organisms are constantly challenged by a fluctuating light supply and other environmental cues affecting photosynthetic performance. Excess light can be especially harmful, but plants and microalgae are equipped with different acclimation mechanisms to control the processing of sunlight absorbed at both photosystems. We summarize the current knowledge and discuss the potential for optimization of phototrophic light-energy conversion.

Methylation of protein arginines represents an important post-translational modification mechanism, which has so far primarily been characterized in mammalian cells. In this work, we successfully identified and characterized arginine methylation as a crucial type of post-translational modification in the activity regulation of the cytosolic translation repressor protein NAB1 in the plant model organism Chlamydomonas reinhardtii. NAB1 represses the cytosolic translation of light-harvesting protein encoding mRNAs by sequestration into translationally silent messenger ribonucleoprotein complexes (mRNPs). Protein arginine methylation of NAB1 could be demonstrated by PRMT1 catalyzed methylation of recombinant NAB1 in vitro, and by immunodetection of methylated NAB1 arginines in vivo. Mass spectrometric analyses of NAB1 purified from C. reinhardtii revealed the asymmetric dimethylation of Arg90 and Arg92 within GAR motif I. Inhibition of arginine methylation by either adenosine-2'-3'-dialdehyde (AdOx) or 7,7'-carbonylbis(azanediyl)bis(4-hydroxynaphthalene-2-sulfonic acid) sodium salt hydrate (AMI-1) caused a dark-green phenotype characterized by the increased accumulation of light-harvesting complex proteins, and indicating a reduced translation repressor activity of NAB1. The extent of NAB1 arginine methylation depends on the growth conditions, with phototrophic growth causing a high methylation state and heterotrophic growth resulting in lowered methylation of the protein. In addition, we could show that NAB1 activity regulation by arginine methylation operates independently from cysteine-based redox control, which has previously been shown to control the activity of NAB1.

The cytosolic RNA-binding protein NAB1 represses translation of LHCII (light-harvesting complex of photosystem II) encoding mRNAs by sequestration into translationally silent mRNP complexes in the green alga Chlamydomonas reinhardtii . NAB1 contains 2 cysteine residues, Cys-181 and Cys-226, within its C-terminal RRM motif. Modification of these cysteines either by oxidation or by alkylation in vitro was accompanied by a decrease in RNA-binding affinity for the target mRNA sequence. To confirm the relevance of reversible NAB1 cysteine oxidation for the regulation of its activity in vivo, we replaced both cysteines with serines. All examined cysteine single and double mutants exhibited a reduced antenna at PSII caused by a perturbed NAB1 deactivation mechanism, with double mutations and Cys-226 single mutations causing a stronger and more distinctive phenotype compared with the Cys-181 mutation. Our data indicated that the responsible redox control mechanism is mediated by modification of single cysteines. Polysome analyses and RNA co-immunoprecipitation experiments demonstrated the interconnection of the NAB1 thiol state and its activity as a translation repressor in vivo. NAB1 is fully active in its dithiol state and is reversibly deactivated by modification of its cysteines. In summary, this work is an example that cytosolic translation of nucleus encoded photosynthetic genes is regulated via a reversible cysteine-based redox switch in a RNA-binding translation repressor protein.

Novel efficient and robust affinity chromatography material: There are several strategies known for the purification of integrins by affinity chromatography, but the disadvantages of common strategies like insufficient selectivity or compelling conditions for the elution still require alternatives. A new strategy, based on the immobilized C-terminally modified peptide Ac-Gly-Ala-c-(CysSS-Arg-Arg-Glu-Thr-Ala-Trp-Ala-CysSS)-Gly-Ala-O(CH2CH2O)2CH2CH2-NH2 allows for the affinity purification of the integrin α5β1. While RGD peptides have been proven in the past to be inappropriate for selective purification of integrins by affinity chromatography, the new peptide can be efficiently used for selective enrichment of the integrin α5β1. It is a specific ligand of the target protein, but does not contain an RGD sequence. The application of well-characterized affinity chromatography material with a site-specifically immobilized peptide allows to obtain integrin α5β1 in a single chromatography step without contamination by other integrins. This process combines the advantages of a selective and monospecific protein-ligand recognition with mild elution conditions and a low sensitivity of the immobilized ligand with respect to column regeneration.

It is getting more and more evident that photosynthetic gene expression is fine-tuned by translation regulation factors encoded in the nucleus of photosynthetic cells. The research of the past decades led to the identification of several nucleus-encoded protein factors that recognize cis-acting elements in plastid transcripts, thereby modulating the stoichiometry and abundance of photosynthetic multisubunit complexes. Despite of its importance for photoacclimatory processes, the investigation of pathways that regulate translation of nuclear-encoded photosynthetic genes is still in its infancy. This review summarizes the yet known paradigms of translation control in chloroplast and cytosol of photosynthetic eukaryotes.

Photosynthetic organisms respond to changes in ambient light by modulating the size and composition of their light-harvesting complexes, which in the case of the green alga Chlamydomonas reinhardtii consists of >15 members of a large extended family of chlorophyll binding subunits. How their expression is coordinated is unclear. Here, we describe the analysis of an insertion mutant, state transitions mutant3 (stm3), which we show has increased levels of LHCBM subunits associated with the light-harvesting antenna of photosystem II. The mutated nuclear gene in stm3 encodes the RNA binding protein NAB1 (for putative nucleic acid binding protein). In vitro and in vivo RNA binding and protein expression studies have confirmed that NAB1 differentially binds to LHCBM mRNA in a subpolysomal high molecular weight RNA-protein complex. Binding of NAB1 stabilizes LHCBM mRNA at the preinitiation level via sequestration and thereby represses translation. The specificity and affinity of binding are determined by an RNA sequence motif similar to that used by the Xenopus laevis translation repressor FRGY2, which is conserved to varying degrees in the LHCBM gene family. We conclude from our results that NAB1 plays an important role in controlling the expression of the light-harvesting antenna of photosystem II at the posttranscriptional level. The similarity of NAB1 and FRGY2 of Xenopus implies the existence of similar RNA-masking systems in animals and plants.

The unicellular green alga Chlamydomonas reinhardtii is capable of using organic and inorganic carbon sources simultaneously, which requires the adjustment of photosynthetic activity to the prevailing mode of carbon assimilation. We obtained novel insights into the regulation of light-harvesting at photosystem II (PSII) following altered carbon source availability. In C. reinhardtii, synthesis of PSII-associated light-harvesting proteins (LHCBMs) is controlled by the cytosolic RNA-binding protein NAB1, which represses translation of particular LHCBM isoform transcripts. This mechanism is fine-tuned via regulation of the nuclear NAB1 promoter, which is activated when linear photosynthetic electron flow is restricted by CO(2)-limitation in a photoheterotrophic context. In the wild-type, accumulation of NAB1 reduces the functional PSII antenna size, thus preventing a harmful overexcited state of PSII, as observed in a NAB1-less mutant. We further demonstrate that translation control as a newly identified long-term response to prolonged CO(2)-limitation replaces LHCII state transitions as a fast response to PSII over-excitation. Intriguingly, activation of the long-term response is perturbed in state transition mutant stt7, suggesting a regulatory link between the long- and short-term response. We depict a regulatory circuit operating on distinct timescales and in different cellular compartments to fine-tune light-harvesting in photoheterotrophic eukaryotes.

Plants convert sunlight to biomass, which is primarily composed of lignocellulose, the most abundant natural biopolymer and a potential feedstock for fuel and chemical production. Cellulose assimilation has so far only been described for heterotrophic organisms that rely on photosynthetically active primary producers of organic compounds. Among phototrophs, the unicellular green microalga Chlamydomonas reinhardtii is widely known as one of the best established model organisms. It occupies many habitats, including aquatic and soil ecosystems. This ubiquity underscores the versatile metabolic properties of this microorganism. Here we present yet another paradigm of adaptation for C. reinhardtii, highlighting its photoheterotrophic ability to utilize cellulose for growth in the absence of other carbon sources. When grown under CO(2)-limiting conditions in the light, secretion of endo-β-1,4-glucanases by the cell causes digestion of exogenous cellulose, followed by cellobiose uptake and assimilation. Phototrophic microbes like C. reinhardtii may thus serve as biocatalysts for cellulosic biofuel production.

The efficient use of microalgae to convert sun light energy into biomass is limited by losses during high light illumination of dense cell cultures in closed bioreactors. Uneven light distribution can be overcome by using cell cultures with smaller antenna sizes packed to high cell density cultures, thus allowing good light penetration into the inner sections of the reactor. We engineered a new small PSII antenna size Chlamydomonas reinhardtii strain with improved photon conversion efficiency and increased growth rates under high light conditions. We achieved this goal by transformation of a permanently active variant NAB1* of the LHC translation repressor NAB1* to reduce antenna size via translation repression. NAB1* expression was demonstrated in Stm6Glc477(T7), leading to a reduction of LHC antenna size by 10-17%. T7 showed a similar to 50% increase of photosynthetic efficiency (Phi PSII) at saturating light intensity compared to the parental strain. T7 converted light to biomass with much higher efficiencies with a similar to 50% improved mid log growth phase. Moreover, T7 cultures reached higher densities when grown in large-scale bioreactors. Thus, the phenotype of strain T7 may have important implications for biotechnological applications in which photosynthetic microalgae are used for large-scale culturing as an alternative plant biomass source. (C) 2009 Elsevier B.V. All rights reserved.

Breaking up is easy. Integrin a5b1 is a cell-surface receptor involved in many physiological and pathological processes. Small cyclic peptides can influence the interaction between this receptor and its natural ligand, fibronectin. The peptide shown, c-(-Arg-Gly-Asp-d-Phe- Val-b-Ala-), can bind a5b1 with submicromolar affinity. A conformational analysis of the peptide provided information on the structural properties responsible for binding to a5b1.

We used a microarray study in order to compare the time course expression profiles of two Chlamydomonas reinhardtii strains, namely the high H2 producing mutant stm6glc4 and its parental WT strain during H2 production induced by sulfur starvation. Major cellular reorganizations in photosynthetic apparatus, sulfur and carbon metabolism upon H2 production were confirmed as common to both strains. More importantly, our results pointed out factors which lead to the higher H2 production in the mutant including a higher starch accumulation in the aerobic phase and a lower competition between the H2ase pathway and alternative electron sinks within the H2 production phase. Key candidate genes of interest with differential expression pattern include LHCSR3, essential for efficient energy quenching (qE). The reduced LHCSR3 protein expression in mutant stm6glc4 could be closely related to the high-light sensitive phenotype. H2 measurements carried out with the LHCSR3 knock-out mutant npq4 however clearly demonstrated that a complete loss of this protein has almost no impact on H2 yields under moderate light conditions. The nuclear gene disrupted in the high H2 producing mutant stm6glc4 encodes for the mitochondrial transcription termination factor (mTERF) MOC1, whose expression strongly increases during –S-induced H2 production in WT strains. Studies under phototrophic high-light conditions demonstrated that the presence of functional MOC1 is a prerequisite for proper LHCSR3 expression. Furthermore knock-down of MOC1 in a WT strain was shown to improve the total H2 yield significantly suggesting that this strategy could be applied to further enhance H2 production in other strains already displaying a high H2 production capacity. By combining our array data with previously published metabolomics data we can now explain some of the phenotypic characteristics which lead to an elevated H2 production in stm6glc4.

In photosynthetic eukaryotes, the metabolite exchange between chloroplast and mitochondria ensures efficient photosynthesis under saturating light conditions. The Chlamydomonas reinhardtii mutant stm6 is devoid of the mitochondrial transcription termination factor MOC1 and aberrantly expresses the mitochondrial genome, resulting in enhanced photosynthetic hydrogen production and diminished light tolerance. We analyzed the modulation of mitochondrial and chlororespiration during the acclimation of stm6 and the MOC1-complemented strain to excess light. Although light stress stimulated mitochondrial respiration via the energy-conserving cytochrome c pathway in both strains, the mutant was unable to fine-tune the expression and activity of oxidative phosphorylation complex I in excess light, which was accompanied by an increased mitochondrial respiration via the alternative oxidase pathway. Furthermore, stm6 failed to fully activate chlororespiration and cyclic electron flow due to a more oxidized state of the chloroplast stroma, which is caused by an increased mitochondrial electron sink capacity. Increased susceptibility to photoinhibition of PSII in stm6 demonstrates that the MOC1-dependent modulation of mitochondrial respiration helps control the stromal redox poise as a crucial part of high-light acclimation in C. reinhardtii.

International audience; Photosynthetic eukaryotes are challenged by a fluctuating light supply, demanding for a modulated expression of nucleus-encoded light-harvesting proteins associated with photosystem II (LHCII) to adjust light-harvesting capacity to the prevailing light conditions. Here, we provide clear evidence for a regulatory circuit that controls cytosolic LHCII translation in response to light quantity changes. In the green unicellular alga Chlamydomonas reinhardtii, the cytosolic RNA-binding protein NAB1 represses translation of certain LHCII isoform mRNAs. Specific nitrosylation of Cys-226 decreases NAB1 activity and could be demonstrated in vitro and in vivo. The less active, nitrosylated form of NAB1 is found in cells acclimated to limiting light supply, which permits accumulation of light-harvesting proteins and efficient light capture. In contrast, elevated light supply causes its denitrosylation, thereby activating the repression of light-harvesting protein synthesis, which is needed to control excitation pressure at photosystem II. Denitrosylation of recombinant NAB1 is efficiently performed by the cytosolic thioredoxin system in vitro. To our knowledge, NAB1 is the first example of stimulus-induced denitrosylation in the context of photosynthetic acclimation. By identifying this novel redox cross-talk pathway between chloroplast and cytosol, we add a new key element required for drawing a precise blue print of the regulatory network of light harvesting.