The Clinical Promise of Microalgae in Rheumatoid Arthritis: From Natural Compounds to Recombinant Therapeutics.

Rheumatoid arthritis (RA) is an invalidating chronic autoimmune disorder characterized by joint inflammation and progressive bone damage. Dietary intervention is an important component in the treatment of RA to mitigate oxidative stress, a major pathogenic driver of the disease. Alongside traditional sources of antioxidants, microalgae-a diverse group of photosynthetic prokaryotes and eukaryotes-are emerging as anti-inflammatory and immunomodulatory food supplements. Several species accumulate therapeutic metabolites-mainly lipids and pigments-which interfere in the pro-inflammatory pathways involved in RA and other chronic inflammatory conditions. The advancement of the clinical uses of microalgae requires the continuous exploration of phytoplankton biodiversity and chemodiversity, followed by the domestication of wild strains into reliable producers of said metabolites. In addition, the tractability of microalgal genomes offers unprecedented possibilities to establish photosynthetic microbes as light-driven biofactories of heterologous immunotherapeutics. Here, we review the evidence-based anti-inflammatory mechanisms of microalgal metabolites and provide a detailed coverage of the genetic engineering strategies to enhance the yields of endogenous compounds and to develop innovative bioproducts.

Proteomic characterization of a lutein-hyperaccumulating Chlamydomonas reinhardtii mutant reveals photoprotection-related factors as targets for increasing cellular carotenoid content.

BACKGROUND: Microalgae are emerging hosts for the sustainable production of lutein, a high-value carotenoid; however, to be commercially competitive with existing systems, their capacity for lutein sequestration must be augmented. Previous attempts to boost microalgal lutein production have focussed on upregulating carotenoid biosynthetic enzymes, in part due to a lack of metabolic engineering targets for expanding lutein storage. RESULTS: Here, we isolated a lutein hyper-producing mutant of the model green microalga Chlamydomonas reinhardtii and characterized the metabolic mechanisms driving its enhanced lutein accumulation using label-free quantitative proteomics. Norflurazon- and high light-resistant C. reinhardtii mutants were screened to yield four mutant lines that produced significantly more lutein per cell compared to the CC-125 parental strain. Mutant 5 (Mut-5) exhibited a 5.4-fold increase in lutein content per cell, which to our knowledge is the highest fold increase of lutein in C. reinhardtii resulting from mutagenesis or metabolic engineering so far. Comparative proteomics of Mut-5 against its parental strain CC-125 revealed an increased abundance of light-harvesting complex-like proteins involved in photoprotection, among differences in pigment biosynthesis, central carbon metabolism, and translation. Further characterization of Mut-5 under varying light conditions revealed constitutive overexpression of the photoprotective proteins light-harvesting complex stress-related 1 (LHCSR1) and LHCSR3 and PSII subunit S regardless of light intensity, and increased accrual of total chlorophyll and carotenoids as light intensity increased. Although the photosynthetic efficiency of Mut-5 was comparatively lower than CC-125, the amplitude of non-photochemical quenching responses of Mut-5 was 4.5-fold higher than in CC-125 at low irradiance. CONCLUSIONS: We used C. reinhardtii as a model green alga and identified light-harvesting complex-like proteins (among others) as potential metabolic engineering targets to enhance lutein accumulation in microalgae. These have the added value of imparting resistance to high light, although partially compromising photosynthetic efficiency. Further genetic characterization and engineering of Mut-5 could lead to the discovery of unknown players in photoprotective mechanisms and the development of a potent microalgal lutein production system.

Analysis of state 1-state 2 transitions by genome editing and complementation reveals a quenching component independent from the formation of PSI-LHCI-LHCII supercomplex in Arabidopsis thaliana.

BACKGROUND: The light-harvesting antennae of photosystem (PS) I and PSII are pigment-protein complexes responsible of the initial steps of sunlight conversion into chemical energy. In natural environments plants are constantly confronted with the variability of the photosynthetically active light spectrum. PSII and PSI operate in series but have different optimal excitation wavelengths. The prompt adjustment of light absorption by photosystems is thus crucial to ensure efficient electron flow needed to sustain downstream carbon fixing reactions. Fast structural rearrangements equilibrate the partition of excitation pressure between PSII and PSI following the enrichment in the red (PSII-favoring) or far-red (PSI-favoring) spectra. Redox imbalances trigger state transitions (ST), a photoacclimation mechanism which involves the reversible phosphorylation/dephosphorylation of light harvesting complex II (LHCII) proteins by the antagonistic activities of the State Transition 7 (STN7) kinase/TAP38 phosphatase enzyme pair. During ST, a mobile PSII antenna pool associates with PSI increasing its absorption cross section. LHCII consists of assorted trimeric assemblies of Lhcb1, Lhcb2 and Lhcb3 protein isoforms (LHCII), several being substrates of STN7. However, the precise roles of Lhcb phosphorylation during ST remain largely elusive. RESULTS: We inactivated the complete Lhcb1 and Lhcb2 gene clades in Arabidopsis thaliana and reintroduced either wild type Lhcb1.3 and Lhcb2.1 isoforms, respectively, or versions lacking N-terminal phosphorylatable residues proposed to mediate state transitions. While the substitution of Lhcb2.1 Thr-40 prevented the formation of the PSI-LHCI-LHCII complex, replacement of Lhcb1.3 Thr-38 did not affect the formation of this supercomplex, nor did influence the amplitude or kinetics of PSII fluorescence quenching upon state 1-state 2 transition. CONCLUSIONS: Phosphorylation of Lhcb2 Thr-40 by STN7 alone accounts for ≈ 60% of PSII fluorescence quenching during state transitions. Instead, the presence of Thr-38 phosphosite in Lhcb1.3 was not required for the formation of the PSI-LHCI-LHCII supercomplex nor for re-equilibration of the plastoquinone redox state. The Lhcb2 phosphomutant was still capable of ≈ 40% residual fluorescence quenching, implying that a yet uncharacterized, STN7-dependent, component of state transitions, which is unrelated to Lhcb2 Thr-40 phosphorylation and to the formation of the PSI-LHCI-LHCII supercomplex, contributes to the equilibration of the PSI/PSII excitation pressure upon plastoquinone over-reduction.

A paler shade of green: engineering cellular chlorophyll content to enhance photosynthesis in crowded environments.

In natural ecosystems, plants compete for space, nutrients and light. The optically dense canopies limit the penetration of photosynthetically active radiation and light often becomes a growth-limiting factor for the understory. The reduced availability of photons in the lower leaf layers is also a major constraint for yield potential in canopies of crop monocultures. Traditionally, crop breeding has selected traits related to plant architecture and nutrient assimilation rather than light use efficiency. Leaf optical density is primarily determined by tissue morphology and by the foliar concentration of photosynthetic pigments (chlorophylls and carotenoids). Most pigment molecules are bound to light-harvesting antenna proteins in the chloroplast thylakoid membranes, where they serve photon capture and excitation energy transfer toward reaction centers of photosystems. Engineering the abundance and composition of antenna proteins has been suggested as a strategy to improve light distribution within canopies and reduce the gap between theoretical and field productivity. Since the assembly of the photosynthetic antennas relies on several coordinated biological processes, many genetic targets are available for modulating cellular chlorophyll levels. In this review, we outline the rationale behind the advantages of developing pale green phenotypes and describe possible approaches toward engineering light-harvesting systems.

Harnessing the Algal Chloroplast for Heterologous Protein Production.

Photosynthetic microbes are gaining increasing attention as heterologous hosts for the light-driven, low-cost production of high-value recombinant proteins. Recent advances in the manipulation of unicellular algal genomes offer the opportunity to establish engineered strains as safe and viable alternatives to conventional heterotrophic expression systems, including for their use in the feed, food, and biopharmaceutical industries. Due to the relatively small size of their genomes, algal chloroplasts are excellent targets for synthetic biology approaches, and are convenient subcellular sites for the compartmentalized accumulation and storage of products. Different classes of recombinant proteins, including enzymes and peptides with therapeutical applications, have been successfully expressed in the plastid of the model organism Chlamydomonas reinhardtii, and of a few other species, highlighting the emerging potential of transplastomic algal biotechnology. In this review, we provide a unified view on the state-of-the-art tools that are available to introduce protein-encoding transgenes in microalgal plastids, and discuss the main (bio)technological bottlenecks that still need to be addressed to develop robust and sustainable green cell biofactories.

Chloroplast-derived photo-oxidative stress causes changes in H2O2 and EGSH in other subcellular compartments.

Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen species (ROS), which can operate protein redox switches by oxidation of specific cysteine residues. Redox buffers, such as the highly reduced glutathione pool, serve as reservoirs of reducing power for several ROS-scavenging and ROS-induced damage repair pathways. Formation of glutathione disulfide and a shift of the glutathione redox potential (EGSH) toward less negative values is considered as hallmark of several stress conditions. Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of intact Arabidopsis (Arabidopsis thaliana) seedlings and recorded dynamic changes in EGSH and H2O2 levels with the genetically encoded biosensors Grx1-roGFP2 (for EGSH) and roGFP2-Orp1 (for H2O2) targeted to chloroplasts, the cytosol, or mitochondria. Treatment of seedlings with MV caused rapid oxidation in chloroplasts and, subsequently, in the cytosol and mitochondria. MV-induced oxidation was significantly boosted by illumination with actinic light, and largely abolished by inhibitors of photosynthetic electron transport. MV also induced autonomous oxidation in the mitochondrial matrix in an electron transport chain activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provides a basis for understanding how compartment-specific redox dynamics might operate in retrograde signaling and stress acclimation in plants.

A Phosphite Dehydrogenase Variant with Promiscuous Access to Nicotinamide Cofactor Pools Sustains Fast Phosphite-Dependent Growth of Transplastomic Chlamydomonas reinhardtii.

Heterologous expression of the NAD+-dependent phosphite dehydrogenase (PTXD) bacterial enzyme from Pseudomonas stutzerii enables selective growth of transgenic organisms by using phosphite as sole phosphorous source. Combining phosphite fertilization with nuclear expression of the ptxD transgene was shown to be an alternative to herbicides in controlling weeds and contamination of algal cultures. Chloroplast expression of ptxD in Chlamydomonas reinhardtii was proposed as an environmentally friendly alternative to antibiotic resistance genes for plastid transformation. However, PTXD activity in the chloroplast is low, possibly due to the low NAD+/NADP+ ratio, limiting the efficiency of phosphite assimilation. We addressed the intrinsic constraints of the PTXD activity in the chloroplast and improved its catalytic efficiency in vivo via rational mutagenesis of key residues involved in cofactor binding. Transplastomic lines carrying a mutagenized PTXD version promiscuously used NADP+ and NAD+ for converting phosphite into phosphate and grew faster compared to those expressing the wild type protein. The modified PTXD enzyme also enabled faster and reproducible selection of transplastomic colonies by directly plating on phosphite-containing medium. These results allow using phosphite as selective agent for chloroplast transformation and for controlling biological contaminants when expressing heterologous proteins in algal chloroplasts, without compromising on culture performance.

The High Light Response in Arabidopsis Requires the Calcium Sensor Protein CAS, a Target of STN7- and STN8-Mediated Phosphorylation.

Reversible phosphorylation of thylakoid proteins contributes to photoacclimation responses in photosynthetic organisms, enabling the fine-tuning of light harvesting under changing light conditions and promoting the onset of photoprotective processes. However, the precise functional role of many of the described phosphorylation events on thylakoid proteins remains elusive. The calcium sensor receptor protein (CAS) has previously been indicated as one of the targets of the state transition kinase 8 (STN8). Here we show that in Arabidopsis thaliana, CAS is also phosphorylated by the state transition kinase 7 (STN7), as well as by another, so-far unknown, Ca2+-dependent kinase. Phosphoproteomics analysis and in vitro phosphorylation assays on CAS variants identified the phylogenetically conserved residues Thr-376, Ser-378, and Thr-380 as the major phosphorylation sites of the STN kinases. Spectroscopic analyses of chlorophyll fluorescence emission at 77K further showed that, while the cas mutant is not affected in state transition, it displays a persistent strong excitation of PSI under high light exposure, similar to the phenotype previously observed in other mutants defective in photoacclimation mechanisms. Together with the observation of a strong concomitant phosphorylation of light harvesting complex II (LHCII) and photosynthetic core proteins under high irradiance in the cas mutant this suggests a role for CAS in the STN7/STN8/TAP38 network of phosphorylation-mediated photoacclimation processes in Arabidopsis.