Lutz Wobbe

Improvement of light to biomass conversion by de-regulation of light-harvesting protein translation in Chlamydomonas reinhardtii

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 Stm6Glc4T7 (T7), leading to a reduction of LHC antenna size by 10-17%. T7 showed a approximately 50% increase of photosynthetic efficiency (PhiPSII) at saturating light intensity compared to the parental strain. T7 converted light to biomass with much higher efficiencies with a approximately 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.

NAB1 Is an RNA Binding Protein Involved in the Light-Regulated Differential Expression of the Light-Harvesting Antenna of Chlamydomonas reinhardtii

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 Nucleus-encoded Protein MOC1 Is Essential for Mitochondrial Light Acclimation in Chlamydomonas reinhardtii

Mitochondrial respiration plays an important role in optimizing photosynthetic efficiency in plants. As yet, the mechanisms by which plant mitochondria sense and respond to changes in the environment are unclear, particularly when exposed to light. Here we describe the characterization of the Chlamydomonas reinhardtii mutant stm6, which was identified on the basis of impaired state transitions, a mechanism that regulates light harvesting in the chloroplast. The gene disrupted in stm6, termed Moc1, encodes a homologue of the human mitochondrial transcription termination factor (mTERF). MOC1 is targeted to the mitochondrion, and its expression is up-regulated in response to light. Loss of MOC1 causes a high light-sensitive phenotype and disrupts the transcription and expression profiles of the mitochondrial respiratory complexes causing, as compared with wild type, light-mediated changes in the expression levels of nuclear and mitochondrial encoded cytochrome c oxidase subunits and ubiquinone-NAD subunits. The absence of MOC1 leads to a reduction in the levels of cytochrome c oxidase and of rotenone-insensitive external NADPH dehydrogenase activities of the mitochondrial respiratory electron transfer chain. Overall, we have identified a novel mitochondrial factor that regulates the composition of the mitochondrial respiratory chain in the light so that it can act as an effective sink for reductant produced by the chloroplast.

Cysteine modification of a specific repressor protein controls the translational status of nucleus-encoded LHCII mRNAs in Chlamydomonas

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.

Multi-Level Light Capture Control in Plants and Green Algae

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.

Translational control of photosynthetic gene expression in phototrophic eukaryotes

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.

Integrin α5β1 Ligands: Biological Evaluation and Conformational Analysis

Integrins are heterodimeric glycoprotein receptors that are involved in many physiological and pathological processes. They mediate cell–cell and cell–matrix adhesion. Integrin a5b1 is the classical fibronectin receptor. Together with integrin a4b1, it has an important function in the migration of activated lymphocytes during the immune response and is also involved in cancer, diabetes and inflammatory diseases like rheumatoid arthritis. The tripeptide sequence -Arg-Gly-Asp(RGD) present in the extracellular-matrix protein, fibronectin, is essential for binding to integrin a5b1. This sequence is found in the tenth type III repeat of fibronectin, which is part of the major cellbinding domain. Small molecules that are able to interfere with the fibronectin–a5b1 binding event might be of interest in the therapy of cancer and inflammatory diseases. Selective a5b1 ligands have been proposed as lead structures for anticancer agents. Synthetic peptides containing the RGD sequence emerged as an excellent starting point for the identification, synthesis and development of selective integrin ligands, as was shown, for example, for integrin avb3. [2, 4] As the three-dimensional structure of integrin a5b1 is not yet available, cyclic peptides are optimally suited to explore the structural requirements with respect to the three-dimensional arrangement of pharmacophoric groups (e.g. , the guanidino group of Arg and the carboxylate of Asp) on a rational basis. For this approach the term “spatial screening” was coined by Kessler et al. In order to analyze the integrin a5b1–fibronectin interaction in the frame of the spatial screening procedure, it is necessary to induce different conformations within the RGD sequence. We synthesized a library of cyclic RGD peptides that contain b-amino acids as

Cellulose degradation and assimilation by the unicellular phototrophic eukaryote Chlamydomonas reinhardtii.

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.

Time-Course Global Expression Profiles of Chlamydomonas reinhardtii during Photo-Biological H2 Production

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.

The mTERF protein MOC1 terminates mitochondrial DNA transcription in the unicellular green alga Chlamydomonas reinhardtii

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.