A gene regulatory network for antenna size control in carbon dioxide-deprived Chlamydomonas reinhardtii cells.

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.

The Molecular Function of Plant mTERFs as Key Regulators of Organellar Gene Expression.

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.

CO2 supply modulates lipid remodelling, photosynthetic and respiratory activities in Chlorella species.

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.

Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii does not synthesize high-value ketocarotenoids like canthaxanthin and astaxanthin; however, a ß-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 redesign 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 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 4.3 mg/L/day. Astaxanthin productivity in engineered C. reinhardtii shown here might be 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 were 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.

Impaired Mitochondrial Transcription Termination Disrupts the Stromal Redox Poise in Chlamydomonas.

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.

A Light Switch Based on Protein S-Nitrosylation Fine-Tunes Photosynthetic Light Harvesting in Chlamydomonas.

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.

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.

Complete Genome Sequences of Pseudomonas sp. Strains MM223 and MM227 and Rheinheimera sp. Strain MM224, Isolated from a Pond Edge in Bielefeld, Germany.

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.

The Full Genome Sequences of Pseudomonas sp. Strain MM221 and Pseudoarthrobacter sp. Strain MM222, Isolated from a Meadow in Bielefeld, Germany.

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.

Protein arginine methylation modulates light-harvesting antenna translation in Chlamydomonas reinhardtii.

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.

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.

High cell density cultivation enables efficient and sustainable recombinant polyamine production in the microalga Chlamydomonas reinhardtii.

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 (20 g/L biomass dry weight, ~2·108 cells/mL). Application of high cell density cultivations in established photobioreactors resulted in a 10-fold increase of cadaverine yields, with up to 0.24 g/L after 9 days and maximal productivity of 0.1 g/L/d.

A novel, robust and mating-competent Chlamydomonas reinhardtii strain with an enhanced transgene expression capacity for algal biotechnology.

In the future, algae biotechnology could play an important role in sustainable development, especially with regard to the production of valuable chemicals. Among the established laboratory strains with efficient transgene expression, there are none that have demonstrated the required robustness for industrial applications, which generally require growth at larger scale. Here, we created a robust and mating-competent cell line of the green microalga Chlamydomonas reinhardtii, which also possesses a high transgene expression capacity. This strain shows a comparably high resistance to shear stress by accumulating increased amounts of biomass under these conditions. As a proof-of-concept, a high phototrophic productivity of cadaverine from CO2 and nitrate was demonstrated by efficiently expressing a bacterial l-lysine decarboxylase. In contrast to other established strains, this novel chassis strain for phototrophic production schemes is equipped with the traits required for industrial applications, by combining mating-competence, cell wall-mediated robustness and high level transgene expression.

Microfluidics for Biotechnology: Bridging Gaps to Foster Microfluidic Applications.

Microfluidics and novel lab-on-a-chip applications have the potential to boost biotechnological research in ways that are not possible using traditional methods. Although microfluidic tools were increasingly used for different applications within biotechnology in recent years, a systematic and routine use in academic and industrial labs is still not established. For many years, absent innovative, ground-breaking and "out-of-the-box" applications have been made responsible for the missing drive to integrate microfluidic technologies into fundamental and applied biotechnological research. In this review, we highlight microfluidics' offers and compare them to the most important demands of the biotechnologists. Furthermore, a detailed analysis in the state-of-the-art use of microfluidics within biotechnology was conducted exemplarily for four emerging biotechnological fields that can substantially benefit from the application of microfluidic systems, namely the phenotypic screening of cells, the analysis of microbial population heterogeneity, organ-on-a-chip approaches and the characterisation of synthetic co-cultures. The analysis resulted in a discussion of potential "gaps" that can be responsible for the rare integration of microfluidics into biotechnological studies. Our analysis revealed six major gaps, concerning the lack of interdisciplinary communication, mutual knowledge and motivation, methodological compatibility, technological readiness and missing commercialisation, which need to be bridged in the future. We conclude that connecting microfluidics and biotechnology is not an impossible challenge and made seven suggestions to bridge the gaps between those disciplines. This lays the foundation for routine integration of microfluidic systems into biotechnology research procedures.

Type II flavoprotein monooxygenase PsFMO_A from the bacterium Pimelobacter sp. Bb-B catalyzes enantioselective Baeyer-Villiger oxidations with a relaxed cofactor specificity.

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.

Extremely robust photocurrent generation of titanium dioxide photoanodes bio-sensitized with recombinant microalgal light-harvesting proteins.

Bio-dyes for light harvesting in dye-sensitized solar cells (DSSC) have the advantage of being environmentally-friendly, non-toxic alternatives, which can be produced in a sustainable fashion. Free photosynthetic pigments are unstable in the presence of light and oxygen, a situation which can hardly be avoided during the operation of DSSCs, especially in large-scale applications. We therefore investigated the recombinant light-harvesting protein LHCBM6, which naturally occurs in the photosynthetic apparatus of the green microalga Chlamydomonas reinhardtii as a bio-dye in DSSCs. Photocurrent densities of up to 0.87 and 0.94 mA·cm-2 were determined for the DSSCs and solar energy to electricity conversion efficiencies (η) reached about 0.3% (100 mW·cm-2; AM 1.5 G filter applied). Importantly, we observed an unprecedented stability of LHCII-based DSSCs within long DSSC operation times of at least 7 days in continuous light and show that operation times are restricted by electrolyte decomposition rather than reduced dye performance, as could be demonstrated by DSSC reactivation following re-supplementation with fresh electrolyte. To the best of our knowledge, this is the first study analysing bio-dye sensitized DSSCs over such long periods, which revealed that during illumination an activation of the DSSCs occurs.

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.

Providing reducing power by microalgal photosynthesis: a novel perspective towards sustainable biocatalytic production of bulk chemicals exemplified for aliphatic amines.

A biotechnological process is reported, which enables an enzymatic reduction without the need for addition of an organic co-substrate for in situ-cofactor recycling. The process is based on merging the fields of enzymatic reductive amination with formate dehydrogenase-based in situ-cofactor recycling and algae biotechnology by means of the photoautotrophic microorganism Chlamydomonas reinhardtii, providing the needed formate in situ by formation from carbon dioxide, water and light. This biotransformation has been exemplified for the synthesis of various aliphatic amines known as bulk chemicals.

Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii.

The protein superfamily of short-chain dehydrogenases/reductases (SDR), including members of the atypical type (aSDR), covers a huge range of catalyzed reactions and in vivo substrates. This superfamily also comprises isoflavone reductase-like (IRL) proteins, which are aSDRs highly homologous to isoflavone reductases from leguminous plants. The molecular function of IRLs in non-leguminous plants and green microalgae has not been identified as yet, but several lines of evidence point at their implication in reactive oxygen species homeostasis. The Chlamydomonas reinhardtii IRL protein IFR1 was identified in a previous study, analyzing the transcriptomic changes occurring during the acclimation to sulfur deprivation and anaerobiosis, a condition that triggers photobiological hydrogen production in this microalgae. Accumulation of the cytosolic IFR1 protein is induced by sulfur limitation as well as by the exposure of C. reinhardtii cells to reactive electrophile species (RES) such as reactive carbonyls. The latter has not been described for IRL proteins before. Over-accumulation of IFR1 in the singlet oxygen response 1 (sor1) mutant together with the presence of an electrophile response element, known to be required for SOR1-dependent gene activation as a response to RES, in the promoter of IFR1, indicate that IFR1 expression is controlled by the SOR1-dependent pathway. An implication of IFR1 into RES homeostasis, is further implied by a knock-down of IFR1, which results in a diminished tolerance toward RES. Intriguingly, IFR1 knock-down has a positive effect on photosystem II (PSII) stability under sulfur-deprived conditions used to trigger photobiological hydrogen production, by reducing PSII-dependent oxygen evolution, in C. reinhardtii. Reduced PSII photoinhibition in IFR1 knock-down strains prolongs the hydrogen production phase resulting in an almost doubled final hydrogen yield compared to the parental strain. Finally, IFR1 knock-down could be successfully used to further increase hydrogen yields of the high hydrogen-producing mutant stm6, demonstrating that IFR1 is a promising target for genetic engineering approaches aiming at an increased hydrogen production capacity of C. reinhardtii cells.

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.